ValiDichro: a website for validating and quality control of protein circular dichroism spectra

Circular dichroism (CD) spectroscopy is widely used in structural biology as a technique for examining the structure, folding and conformational changes of proteins. A new server, ValiDichro, has been developed for checking the quality and validity of CD spectral data and metadata, both as an aid to data collection and processing and as a validation procedure for spectra to be included in publications. ValiDichro currently includes 25 tests for data completeness, consistency and quality. For each test that is done, not only is a validation report produced, but the user is also provided with suggestions for correcting or improving the data. The ValiDichro server is freely available at http://valispec.cryst.bbk.ac.uk/circularDichroism/ValiDichro/upload.html

Single Exhaled Breath Metabolomic Analysis Identifies Unique Breathprint in Patients With Acute Decompensated Heart Failure

Acute decompensated heart failure (ADHF) is the most common indication for hospital admission, particularly in the elderly, yet the identification of those with impending decompensation using conventional clinical methods is unreliable and frequently leaves insufficient lag time for therapeutic interventions (1). Exhaled breath constitutes a complex mixture of hundreds of volatile organic compounds (VOCs) that could potentially be used as a safe and noninvasive method of diagnostic and therapeutic monitoring (2). Previous research studies have identified elevated acetone, pentane, and nitric oxide levels in exhaled breath in the setting of HF correlated with disease severity (3–5). Selected ion-flow tube mass-spectrometry (SIFT-MS) combines a fast flow tube technique with quantitative mass spectrometry that is ideally suited for exhaled breath analysis because it allows for the analysis of small and humid samples without the need for cumbersome sample preparation or calibration (6). Scan times are relatively brief, thus facilitating high throughput and serial comparisons. Using this technology, we conducted a prospective, single-center cohort study to assess the feasibility of exhaled breath analysis to identify patients admitted for ADHF. The study protocol was approved by the Cleveland Clinic Institutional Review Board. We recruited 25 consecutive patients admitted with ADHF as their primary diagnosis (mean left ventricular ejection fraction 27 ± 13%, median N-terminal pro–B-type natriuretic peptide level 954 pg/ml) and a control group of 16 subjects admitted with non-ADHF cardiovascular diagnoses and who had no clinical evidence of systemic or venous congestion at the time of enrollment. Indications for hospitalization in the control group included unstable angina or non–ST-segment elevation myocardial infarction (6 of 16), conduction disorders (3 of 16), hypertensive emergency (3 of 16), atrial tachyarrhythmia (2 of 16), or stable angina (2 of 16). All analyses were performed using JMP Pro 9.0 (SAS Institute, Cary, North Carolina). As expected, there were significant (p \u3c 0.01) baseline differences in the frequency of hypertension (54% vs. 100%) and baseline estimated glomerular filtration rate (68 ± 43 ml/min/1.73 m2 vs. 102 ± 44 ml/min/1.73 m2), which were significantly worse in the ADHF versus control group. Nevertheless, there were no significant differences between groups in age, body mass index, or several comorbidities (i.e., diabetes mellitus, chronic obstructive pulmonary disease, active smoking) theorized to result in alterations in the exhaled metabolome

Molecular basis of FIR-mediated c-myc transcriptional control

The far upstream element (FUSE) regulatory system promotes a peak in the concentration of c-Myc during cell cycle. First, the FBP transcriptional activator binds to the FUSE DNA element upstream of the c-myc promoter. Then, FBP recruits its specific repressor (FIR), which acts as an on/off transcriptional switch. Here we describe the molecular basis of FIR recruitment, showing that the tandem RNA recognition motifs of FIR provide a platform for independent FUSE DNA and FBP protein binding and explaining the structural basis of the reversibility of the FBP-FIR interaction. We also show that the physical coupling between FBP and FIR is modulated by a flexible linker positioned sequentially to the recruiting element. Our data explain how the FUSE system precisely regulates c-myc transcription and suggest that a small change in FBP-FIR affinity leads to a substantial effect on c-Myc concentration.MRC Grant-in-aid U11757455

High resolution crystal structures of piscine transthyretin reveal different binding modes for triiodothyronine and thyroxine

Transthyretin (TTR) is an extracellular transport protein involved in the distribution of thyroid hormones and vitamin A. So far, TTR has only been found in vertebrates, of which piscine TTR displays the lowest sequence identity with human TTR (47%). Human and piscine TTR bind both thyroid hormones 3,5,3 -triiodo- L-thyronine (T3) and 3,5,3 ,5 -tetraiodo-L-thyronine (thyroxine, T4). Human TTR has higher affinity for T4 than T3, whereas the reverse holds for piscine TTR. X-ray structures of Sparus aurata (sea bream) TTR have been determined as the apo-protein at 1.75 Å resolution and bound to ligands T3 and T4, both at 1.9 Å resolution. The apo structure is similar to human TTR with structural changes only at -strand D. This strand forms an extended loop conformation similar to the one in chicken TTR. The piscine TTR T4 complex shows the T4-binding site to be similar but not identical to human TTR, whereas the TTR T3 complex shows the I3 halogen situated at the site normally occupied by the hydroxyl group of T4. The significantly wider entrance of the hormone- binding channel in sea bream TTR, in combination with its narrower cavity, provides a structural explanation for the different binding affinities of human and piscine TTR to T3 and T4.We thank Anders Olofsson, Uwe H. Sauer, Andreas Ho¨rnberg, and Terese Bergfors for valuable discussions and critical reading of the manuscript

Lectin-like bacteriocins from pseudomonas spp. utilise D-rhamnose containing lipopolysaccharide as a cellular receptor

Lectin-like bacteriocins consist of tandem monocot mannose-binding domains and display a genus-specific killing activity. Here we show that pyocin L1, a novel member of this family from Pseudomonas aeruginosa, targets susceptible strains of this species through recognition of the common polysaccharide antigen (CPA) of P. aeruginosa lipopolysaccharide that is predominantly a homopolymer of d-rhamnose. Structural and biophysical analyses show that recognition of CPA occurs through the C-terminal carbohydrate-binding domain of pyocin L1 and that this interaction is a prerequisite for bactericidal activity. Further to this, we show that the previously described lectin-like bacteriocin putidacin L1 shows a similar carbohydrate-binding specificity, indicating that oligosaccharides containing d-rhamnose and not d-mannose, as was previously thought, are the physiologically relevant ligands for this group of bacteriocins. The widespread inclusion of d-rhamnose in the lipopolysaccharide of members of the genus Pseudomonas explains the unusual genus-specific activity of the lectin-like bacteriocins

Laparoscopic Sleeve Gastrectomy is a Safe and Effective Bariatric Procedure for the Lower BMI (35.0–43.0 kg/m2) Population

# The Author(s) 2010. This article is published with open access at Springerlink.com Background The laparoscopic vertical sleeve gastrectomy (LSG) is derived from the biliopancreatic diversion with duodenal switch operation (Marceau et al., Obes Surg 3:29

Orientation of the central domains of KSRP and its implications for the interaction with the RNA targets

KSRP is a multi-domain RNA-binding protein that recruits the exosome-containing mRNA degradation complex to mRNAs coding for cellular proliferation and inflammatory response factors. The selectivity of this mRNA degradation mechanism relies on KSRP recognition of AU-rich elements in the mRNA 3′UTR, that is mediated by KSRP’s KH domains. Our structural analysis shows that the inter-domain linker orients the two central KH domains of KSRP—and their RNA-binding surfaces—creating a two-domain unit. We also show that this inter-domain arrangement is important to the interaction with KSRP’s RNA targets

Functional and structural basis of the nuclear localization signal in the ZIC3 zinc finger domain

Disruptions in ZIC3 cause heterotaxy, a congenital anomaly of the left–right axis. ZIC3 encodes a nuclear protein with a zinc finger (ZF) domain that contains five tandem C2H2 ZF motifs. Missense mutations in the first ZF motif (ZF1) result in defective nuclear localization, which may underlie the pathogenesis of heterotaxy. Here we revealed the structural and functional basis of the nuclear localization signal (NLS) of ZIC3 and investigated its relationship to the defect caused by ZF1 mutation. The ZIC3 NLS was located in the ZF2 and ZF3 regions, rather than ZF1. Several basic residues interspersed throughout these regions were responsible for the nuclear localization, but R320, K337 and R350 were particularly important. NMR structure analysis revealed that ZF1–4 had a similar structure to GLI ZF, and the basic side chains of the NLS clustered together in two regions on the protein surface, similar to classical bipartite NLSs. Among the residues for the ZF1 mutations, C253 and H286 were positioned for the metal chelation, whereas W255 was positioned in the hydrophobic core formed by ZF1 and ZF2. Tryptophan 255 was a highly conserved inter-finger connector and formed part of a structural motif (tandem CXW-C-H-H) that is shared with GLI, Glis and some fungal ZF proteins. Furthermore, we found that knockdown of Karyopherin α1/α6 impaired ZIC3 nuclear localization, and physical interactions between the NLS and the nuclear import adapter proteins were disturbed by mutations in the NLS but not by W255G. These results indicate that ZIC3 is imported into the cell nucleus by the Karyopherin (Importin) system and that the impaired nuclear localization by the ZF1 mutation is not due to a direct influence on the NLS

Effect of Long-Term Zinc Pollution on Soil Microbial Community Resistance to Repeated Contamination

The aim of the study was to compare the effects of stress (contamination trials) on the microorganisms in zinc-polluted soil (5,018 mg Zn kg−1 soil dry weight) and unpolluted soil (141 mg Zn kg−1 soil dw), measured as soil respiration rate. In the laboratory, soils were subjected to copper contamination (0, 500, 1,500 and 4,500 mg kg−1 soil dw), and then a bactericide (oxytetracycline) combined with a fungicide (captan) along with glucose (10 mg g−1 soil dw each) were added. There was a highly significant effect of soil type, copper treatment and oxytetracycline/captan treatment. The initial respiration rate of chronically zinc-polluted soil was higher than that of unpolluted soil, but in the copper treatment it showed a greater decline. Microorganisms in copper-treated soil were more susceptible to oxytetracycline/captan contamination. After the successive soil contamination trials the decline of soil respiration was greater in zinc-polluted soil than in unpolluted soil

Functional Role of Glutamine 28 and Arginine 39 in Double Stranded RNA Cleavage by Human Pancreatic Ribonuclease

Human pancreatic ribonuclease (HPR), a member of RNase A superfamily, has a high activity on double stranded (ds) RNA. By virtue of this activity HPR appears to be involved in the host-defense against pathogenic viruses. To delineate the mechanism of dsRNA cleavage by HPR, we have investigated the role of glutamine 28 and arginine 39 of HPR in its activity on dsRNA. A non-basic residue glycine 38, earlier shown to be important for dsRNA cleavage by HPR was also included in the study in the context of glutamine 28 and arginine 39. Nine variants of HPR respectively containing Q28A, Q28L, R39A, G38D, Q28A/R39A, Q28L/R39A, Q28A/G38D, R39A/G38D and Q28A/G38D/R39A mutations were generated and functionally characterized. The far-UV CD-spectral analysis revealed all variants, except R39A, to have structures similar to that of HPR. The catalytic activity of all HPR variants on single stranded RNA substrate was similar to that of HPR, whereas on dsRNA, the catalytic efficiency of all single residue variants, except for the Q28L, was significantly reduced. The dsRNA cleavage activity of R39A/G38D and Q28A/G38D/R39A variants was most drastically reduced to 4% of that of HPR. The variants having reduced dsRNA cleavage activity also had reduction in their dsDNA melting activity and thermal stability. Our results indicate that in HPR both glutamine 28 and arginine 39 are important for the cleavage of dsRNA. Although these residues are not directly involved in catalysis, both arginine 39 and glutamine 28 appear to be facilitating a productive substrate-enzyme interaction during the dsRNA cleavage by HPR