Author Correction: Determination of RNA structural diversity and its role in HIV-1 RNA splicing.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Determination of RNA structural diversity and its role in HIV-1 RNA splicing.

Human immunodeficiency virus 1 (HIV-1) is a retrovirus with a ten-kilobase single-stranded RNA genome. HIV-1 must express all of its gene products from a single primary transcript, which undergoes alternative splicing to produce diverse protein products that include structural proteins and regulatory factors1,2. Despite the critical role of alternative splicing, the mechanisms that drive the choice of splice site are poorly understood. Synonymous RNA mutations that lead to severe defects in splicing and viral replication indicate the presence of unknown cis-regulatory elements3. Here we use dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) to investigate the structure of HIV-1 RNA in cells, and develop an algorithm that we name 'detection of RNA folding ensembles using expectation-maximization' (DREEM), which reveals the alternative conformations that are assumed by the same RNA sequence. Contrary to previous models that have analysed population averages4, our results reveal heterogeneous regions of RNA structure across the entire HIV-1 genome. In addition to confirming that in vitro characterized5 alternative structures for the HIV-1 Rev responsive element also exist in cells, we discover alternative conformations at critical splice sites that influence the ratio of transcript isoforms. Our simultaneous measurement of splicing and intracellular RNA structure provides evidence for the long-standing hypothesis6-8 that heterogeneity in RNA conformation regulates splice-site use and viral gene expression.

The architecture and evolution of cancer neochromosomes.

We isolated and analyzed, at single-nucleotide resolution, cancer-associated neochromosomes from well- and/or dedifferentiated liposarcomas. Neochromosomes, which can exceed 600 Mb in size, initially arise as circular structures following chromothripsis involving chromosome 12. The core of the neochromosome is amplified, rearranged, and corroded through hundreds of breakage-fusion-bridge cycles. Under selective pressure, amplified oncogenes are overexpressed, while coamplified passenger genes may be silenced epigenetically. New material may be captured during punctuated chromothriptic events. Centromeric corrosion leads to crisis, which is resolved through neocentromere formation or native centromere capture. Finally, amplification terminates, and the neochromosome core is stabilized in linear form by telomere capture. This study investigates the dynamic mutational processes underlying the life history of a special form of cancer mutation.

IMGT/HighV QUEST paradigm for T cell receptor IMGT clonotype diversity and next generation repertoire immunoprofiling.

T cell repertoire diversity and clonotype follow-up in vaccination, cancer, infectious and immune diseases represent a major challenge owing to the enormous complexity of the data generated. Here we describe a next generation methodology, which combines 5'RACE PCR, 454 sequencing and, for analysis, IMGT, the international ImMunoGeneTics information system (IMGT), IMGT/HighV-QUEST web portal and IMGT-ONTOLOGY concepts. The approach is validated in a human case study of T cell receptor beta (TRB) repertoire, by chronologically tracking the effects of influenza vaccination on conventional and regulatory T cell subpopulations. The IMGT/HighV-QUEST paradigm defines standards for genotype/haplotype analysis and characterization of IMGT clonotypes for clonal diversity and expression and achieves a degree of resolution for next generation sequencing verifiable by the user at the sequence level, while providing a normalized reference immunoprofile for human TRB.

Transsphenoidal approaches for the extracapsular resection of midline suprasellar and anterior cranial base lesions.

OBJECTIVE: The transsphenoidal approach is an effective method for treating tumors contained within the sella or extending into the suprasellar cistern. The technique of tumor dissection is predicated on preservation of the integrity of the diaphragma, i.e., intracapsular removal. Gross total extracapsular dissection may, however, be accomplished either by using a standard approach to the pituitary fossa or by extending the exposure to include removal of a portion of the planum sphenoidale and division of the superior intercavernous sinus. METHODS: Included in this series were 14 patients with parasellar or sellar tumors with extension into the anterior fossa and/or suprasellar cistern. For 4 of 14 patients (29%), extracapsular access was gained by broaching the tumor capsule from within the pituitary fossa. For the remaining 10 of 14 patients (71%), the dura of the floor of the sella and the planum sphenoidale was exposed, using neuronavigation to verify the limits of bony dissection; extracapsular tumor resection was performed using the operating microscope and endoscopy as indicated. The dural defect was repaired with abdominal fat, the sellar floor and planum sphenoidale were reconstructed, and in selected cases a lumbar drain was placed. RESULTS: Seven of 14 tumors (50%) were craniopharyngiomas, 3 of 14 (21%) were pituitary adenomas, and 2 of 14 (14%) were meningiomas. There was one case of lymphocytic hypophysitis and one yolk sac tumor. Gross total resection was possible in 11 of 14 cases (79%). Immediate postoperative visual function worsened in 2 of 14 cases (14%), improved in 3 of 14 cases (21%), and was stable in the remainder of cases. Postoperatively, 2 of 14 patients (14%) developed bacterial meningitis. Overt postoperative cerebrospinal fluid rhinorrhea was not observed. CONCLUSION: Gross total extracapsular resection of midline suprasellar tumors via a transsphenoidal approach is possible but is associated with a higher risk of complications than is standard transsphenoidal surgery.

Cranial base reconstruction after transsphenoidal surgery with bioabsorbable implants.

OBJECTIVE: Reconstruction of the cranial base is often necessary after transsphenoidal surgery to prevent the occurrence of cerebrospinal fluid rhinorrhea and to maintain anatomic integrity. In most cases, sellar packing (fat, muscle, gelatin sponge) may be supported by bone or cartilage harvested at the time of surgery. The use of synthetic material, however, becomes desirable in cases in which an autograft is not available. Low-molecular-weight polylactide implants may serve as an effective alternative because they are immunologically inert, magnetic resonance imaging-compatible, and easily contoured to custom-fit a defect. METHODS AND RESULTS: MacroSorb (MacroPore, San Diego, CA) absorbable plates are made from amorphous 70:30 poly (L-lactide-co-D,L-lactide) polymers. Implants are malleable at temperatures of 70 degrees C and solidify at room or body temperature; plates are resorbed in 18 months. CONCLUSION: Polylactide polymer implants are effective adjuncts in transsphenoidal surgery when cranial base reconstruction is necessary and when an endogenous osseous or cartilaginous graft is unavailable.

Microsurgical and endoscopic approaches to the basilar bifurcation: quantitative comparison of combined pterional/anterior temporal and orbitozygomatic extended approaches.

To determine the most effective route to the upper third of the basilar artery, 10 cadaver dissections were performed comparing the exposure gained using the combined pterional/anterior temporal approach and the orbitozygomatic extended approach. Endoscopic evaluation through an eyebrow incision in each dissection helped determine whether any advantage was conferred by the cranial base approach. This also served to assess the benefit of using the endoscope as an adjunct to open procedures in evaluating the opticocarotid and retrocarotid routes to the basilar artery. The results of cadaveric dissections suggest that compared with the exposure afforded by the combined pterional/anterior temporal approach, the orbitozygomatic extended approach yields an increase of 8 cm in the anteroposterior axis and 10 cm in the superoinferior axis. Furthermore, as expected, the increases in visibility were directed anteriorly and posteriorly. When employed through the craniotomy site during aneurysm clipping, endoscopy was found to be potentially useful in assessing the contralateral elements of the upper basilar complex.

The proximal probe in esophageal pH monitoring: development of a normative database.

Healthy volunteers without symptoms of either gastroesophageal reflux or laryngopharyngeal reflux and without abnormalities on laryngologic examination were recruited for esophageal pH monitoring. Thirty subjects underwent ambulatory 24-hour double-channel pH probe monitoring to establish normative data for the upper probe, which was positioned just above the upper esophageal sphincter. Data were analyzed excluding meal periods plus 2 minutes of postprandial time. The mean, standard deviation, median, and 95th percentile were calculated for various reflux parameters for the following intervals: total study duration, upright time, supine time, and postprandial time. Normal subjects display physiologic reflux above the upper esophageal sphincter (median one event, 95th percentile 6.9 events), and 80.4% of these events occur in the upright position. The reflux area index (RAI) appears to be the most useful parameter to measure laryngopharyngeal reflux severity.

Experimental side-wall aneurysms: a natural history study.

We studied the natural history of canine side-wall experimental aneurysms to determine the incidence of spontaneous aneurysm thrombosis, to serve as control data for future studies focusing on development of aneurysm occlusion devices. Bilateral common carotid artery vein patch aneurysms were surgically created in eight mongrel dogs (20-25 kg). Duplex Doppler sonography was performed at 14 days and angiography between 30 and 210 days following aneurysm creation. Sonography demonstrated patency of 13 (81%) of 16 aneurysms. Patent aneurysms ranged in size from 8 x 10 mm to 14 x 16 mm. Conventional angiography was performed in four dogs approximately 30 days following aneurysm creation; in these four, all of 7 initially patent on sonography remained fully patent. One dog underwent conventional angiography at approximately 60 days following aneurysm creation; both aneurysms in this case remained widely patent. Three dogs underwent conventional angiography at approximately 200 days following aneurysm creation; all 4 aneurysms initially patent on sonography remained fully patent. None of the three aneurysms found to be occluded on sonographs demonstrated spontaneous recanalization. The canine side-wall aneurysm model is a valid tool for testing some aneurysm-occlusion devices, because control aneurysms remain patent indefinitely.

Effect of postmortem autolysis on Na,K-ATPase activity and antigenicity in the gerbil cochlea.

Alterations in the enzymatic activity and antigenicity of Na,K-ATPase as well as changes in cochlear morphology were assessed in gerbil inner ears harvested at selected time intervals up to 18 h postmortem. Na,K-ATPase activity was assayed biochemically in one cochlea from each animal and the other cochlea was fixed and embedded in paraffin for evaluation by light microscopy. Na,K-ATPase antigenicity was assessed by immunostaining with a broad-spectrum antiserum reactive with all known isoforms of the enzyme, and structural preservation was evaluated on adjacent sections stained with hematoxylin and eosin. The results showed a downward trend in enzymatic activity of Na,K-ATPase in lateral wall tissues within 1 h of death. In contrast, Na,K-ATPase immunoreactivity was fairly well preserved with postmortem fixation delays up to 12 h, despite the considerable structural degradation of cochlear tissues which began 2-3 h postmortem. It is concluded that under controlled environmental conditions, cochleas collected up to 4 h postmortem are suitable for morphological and immunohistochemical study of Na,K-ATPase by light microscopy. Cochleas collected more than 5 h postmortem were useful only for relatively gross immunohistochemical studies. It is suggested that cochleas intended for biochemical assays of Na,K-ATPase and probably most other enzymes should be collected within 1 h of death.

Reduced mastoidectomy complications with an open meatoplasty stent.

Open-tube stents were compared with standard petroleum gauze packs in meatoplasties after canal-wall-down mastoid surgery to determine whether any difference existed in postoperative complications. Tube stents were associated with much lower incidences of postoperative meatoplasty stenosis and postauricular wound complications in this series.

Na,K-ATPase activity decreases in the cochlear lateral wall of quiet-aged gerbils.

Alterations in the distribution and activity of Na,K-ATPase have been implicated in declining cell function with age. However, the location, size and anatomical complexity of the cochlea have limited study of this essential enzyme. Here we describe a micro-colorimetric assay which measures Na,K-ATPase activity in subregions of individual cochleae. Na,K-ATPase activity was determined in lateral wall and organ of Corti tissues by measuring liberation of inorganic phosphate (Pi) from ATP against a standard phosphate curve. Na,K-ATPase specific activity, expressed as mu mol Pi liberated/mg protein/h, was calculated as the difference between total Pi liberated versus Pi liberated in the presence of ouabain. Na,K-ATPase specific activity and total protein content in the lateral wall significantly exceeded those of the organ of Corti. Although lateral wall protein content remained constant with age, Na,K-ATPase specific activity declined in some older gerbils, suggesting a basis for age-related reductions in magnitude of the endocochlear potential and confirming previous histochemical results. This microassay offers a sensitive, reliable means to assay enzyme activity in subregions or single turns of the cochlea that unlike other methods does not rely on use of radioisotopes, enzymatic cycling or sample pooling.

Integrated systems: subspecialists as capitated primary care physicians?

The shortage of primary care physicians, the shifting roles of specialists and abundance of subspecialists are challenging hospital alliances. In the new world of managed care and managed competition, where providers assume financial risk for overutilization, management must discard traditional measures of profitability and focus on adding value. Hospitals are taking steps to become accountable health partners in smaller to mid-sized markets (two or three hospitals) by sponsoring community based direct contracting managed care networks. This type of system helps providers offer employers a local managed care alternative that is better able to demonstrate quality of care, and to control utilization through capitation and other pricing strategies.