Assigning NMR spectra of RNA, peptides and small organic molecules using molecular network visualization software.

NMR assignment typically involves analysis of peaks across multiple NMR spectra. Chemical shifts of peaks are measured before being assigned to atoms using a variety of methods. These approaches quickly become complicated by overlap, ambiguity, and the complexity of correlating assignments among multiple spectra. Here we propose an alternative approach in which a network of linked peak-boxes is generated at the predicted positions of peaks across all spectra. These peak-boxes correlate known relationships and can be matched to the observed spectra. The method is illustrated with RNA, but a variety of molecular types should be readily tractable with this approach.

Rescue alemtuzumab for refractory acute cellular rejection and bronchiolitis obliterans syndrome after lung transplantation.

Refractory acute cellular rejection (rACR) is associated with death and bronchiolitis obliterans syndrome (BOS) post-lung transplantation. We report the largest cohort of lung transplant recipients (LTRs) treated with rescue alemtuzumab for rACR or BOS. RACR outcomes included burden of ACR 30 days before and 180 days after rescue assessed by a novel composite rejection standardized score (CRSS, range 0-6) and freedom from ≥A2 ACR. BOS outcomes included freedom from BOS progression and FEV1 decline >10%. Univariate parametric and nonparametric statistical approaches were used to assess treatment response. Kaplan-Meier method with log rank conversion was used to assess freedom from events. Fifty-seven alemtuzumab doses (ACR 40 and BOS 17) given to 51 patients were included. Median time to rescue was 722 (IQR 42-1403) days. CRSS declined significantly (3 vs 0.67, P<0.001) after rescue. Freedom from ≥A2 was 62.5% in rACR. Freedom from BOS progression was 52.9% at 180 days in the BOS cohort. Freedom from FEV1 decline >10% was 70% in BOS grade 1 and 14.3% in advanced BOS grades 2-3. Infections developed in 72.5% and 76.5% of rACR and BOS groups. Rescue alemtuzumab appears useful for rACR. Patients with BOS 1 may have transient benefit, and patients with advanced BOS seem not to respond to alemtuzumab.

Chemo-enzymatic synthesis of site-specific isotopically labeled nucleotides for use in NMR resonance assignment, dynamics and structural characterizations.

Stable isotope labeling is central to NMR studies of nucleic acids. Development of methods that incorporate labels at specific atomic positions within each nucleotide promises to expand the size range of RNAs that can be studied by NMR. Using recombinantly expressed enzymes and chemically synthesized ribose and nucleobase, we have developed an inexpensive, rapid chemo-enzymatic method to label ATP and GTP site specifically and in high yields of up to 90%. We incorporated these nucleotides into RNAs with sizes ranging from 27 to 59 nucleotides using in vitro transcription: A-Site (27 nt), the iron responsive elements (29 nt), a fluoride riboswitch from Bacillus anthracis(48 nt), and a frame-shifting element from a human corona virus (59 nt). Finally, we showcase the improvement in spectral quality arising from reduced crowding and narrowed linewidths, and accurate analysis of NMR relaxation dispersion (CPMG) and TROSY-based CEST experiments to measure µs-ms time scale motions, and an improved NOESY strategy for resonance assignment. Applications of this selective labeling technology promises to reduce difficulties associated with chemical shift overlap and rapid signal decay that have made it challenging to study the structure and dynamics of large RNAs beyond the 50 nt median size found in the PDB.

Changing practice paradigms: negotiating your future.

There are many recent and ongoing changes in the practice of medicine from a business standpoint as well as in overall practice management. Economic and lifestyle desires have pushed many physicians to a decision point of whether or not to join a large multispecialty group or to sell their practice and become an employee of a hospital system. There are advantages and disadvantages to both options; however, deciding on the most appropriate path for each individual can be a daunting task. At our recent breakfast session at the vascular annual meeting in Chicago, Illinois, in June 2011, we brought to light these topics to try and help enlighten physicians on which option may be right for them. There is no single answer/option that will fit every practice, but discussion for various practice management designs are outlined and critiqued. This article cannot fully discuss each view in the allotted space, but it is designed to encourage thought and discussion among the vascular surgical community as a whole.

Use of endobronchial valves for native lung hyperinflation associated with respiratory failure in a single-lung transplant recipient for emphysema.

Emphysema is a common indication for adult pulmonary transplantation. Double-lung transplantation is increasingly the preferred approach because severe posttransplant native lung hyperinflation (NLH) following single-lung transplantation may compromise allograft lung function. We describe successful emergency use of bronchoscopic lung volume reduction using endobronchial valves (EBVs) [Zephyr; Emphasys Medical; Redwood, CA] in a single-lung transplant recipient who was critically ill with ventilator dependence from complications of NLH and at excessive risk for lung volume reduction surgery or pneumonectomy. Following placement of 17 valves in all segments of the native lung, atelectasis of the native lung was accompanied by volume expansion of the allograft. Immediately following valve placement, peak airway pressure decreased and alveolar ventilation increased. The patient was subsequently weaned from mechanical ventilation. This report suggests the need for clinical trials to evaluate the effectiveness of EBVs in single-lung transplant recipients with less critical functional impairment associated with NLH.

Statin use is associated with improved function and survival of lung allografts.

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) are widely used antilipidemic agents that are also immunomodulatory. We evaluated possible effects of these agents after lung transplantation by comparing outcomes of 39 allograft recipients, who were prescribed statins for hyperlipidemia, with those of 161 contemporaneous control recipients who did not receive these drugs. Acute rejection (>or= Grade II) was less frequently found in the statin group (15.1 versus 25.6% of biopsies, p < 0.01). None of 15 recipients started on statins during postoperative Year 1 developed obliterative bronchiolitis, whereas the cumulative incidence of this complication among control subjects was 37% (p < 0.01). Total cellularity, as well as proportions of inflammatory neutrophils and lymphocytes, were significantly lower in bronchoalveolar lavages of statin recipients. Among double lung recipients, those taking statins had significantly better spirometry: FVC (80 +/- 2 versus 70 +/- 1%) and FEV1 (87 +/- 2 versus 70 +/- 1%), as percentages of predicted values, and absolute FEV1/FVC (83.4 +/- 1.2 versus 78.6 +/- 0.5) (all p < 0.01). The 6-year survival of recipients taking statins (91%) was much greater than that of control subjects (54%) (p < 0.01). These data suggest statin use may have substantial clinical benefits after pulmonary transplantation.