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BACKGROUND The National Heart, Lung, and Blood Institute-sponsored IPF Clinical Research Network (IPFnet) studies enrolled subjects with idiopathic pulmonary fibrosis (IPF) to evaluate drug therapies in treatment trials. An adjudication committee (AC) provided a structured review of cases in which there was uncertainty or disagreement regarding diagnosis or clinical event classification. This article describes the diagnosis and adjudication processes. METHODS The diagnostic process was based on review of clinical data and high-resolution CT scans with central review of lung biopsies when available. The AC worked closely with the data coordinating center to obtain clinical, radiologic, and histologic data and to communicate with the clinical centers. The AC used a multidisciplinary discussion model with four clinicians, one radiologist, and one pathologist to adjudicate diagnosis and outcome measures. RESULTS The IPFnet trials screened 1,015 subjects; of these, 23 cases required review by the AC to establish eligibility. The most common diagnosis for exclusion was suspected chronic hypersensitivity pneumonitis. The AC reviewed 88 suspected acute exacerbations (AExs), 93 nonelective hospitalizations, and 16 cases of bleeding. Determination of AEx presented practical challenges to adjudicators, as necessary clinical data were often not collected, particularly when subjects were evaluated outside of the primary study site. CONCLUSIONS The IPFnet diagnostic process was generally efficient, but a multidisciplinary adjudication committee was critical to assure correct phenotype for study enrollment. The AC was key in adjudicating all adverse outcomes in two IPFnet studies terminated early because of safety issues. Future clinical trials in IPF should consider logistical and cost issues as they incorporate AExs and hospitalizations as outcome measures. TRIAL REGISTRY ClinicalTrials.gov; No.: NCT00517933, NCT00650091, NCT00957242; URL: www.clinicaltrials.gov

Background: Inflammatory eye disease has been reported as a side effect with Nivolumab. Case presentation: We report a case of a 92-year-old woman presenting with bilateral and simultaneous keratitis and uveitis in the setting of recurring infusions of nivolumab for metastatic melanoma. The patient underwent successful coordinated treatment of both eyes coinciding with ongoing systemic infusion treatments with ophthalmic topical medications alone. Conclusion: The interest of this case resides in the simultaneous nature of presentation of eye inflammation both internally and of the ocular surface. Prior case reports have cited uveitis or ocular surface disease, however not in simultaneous fashion. Clinicians should raise their index of suspicion of side effects of nivolumab systemic infusion for any vision or eye symptom changes around the timing of treatment.

Cohesion between sister chromatids in eukaryotes is mediated by the evolutionarily conserved cohesin complex. Cohesin forms a proteinaceous ring, large enough to trap pairs of replicated sister chromatids. The circumference consists of the Smc1 and Smc3 subunits, while Scc1 is thought to close the ring by bridging the Smc (structural maintenance of chromosomes) ATPase head domains. Little is known about two additional subunits, Scc3 and Pds5, and about possible conformational changes of the complex during the cell cycle. We have employed fluorescence resonance energy transfer (FRET) to analyse interactions within the cohesin complex in live budding yeast. These experiments reveal an unexpected geometry of Scc1 at the Smc heads, and suggest that Pds5 plays a role at the Smc hinge on the opposite side of the ring. Key subunit interactions, including close proximity of the two ATPase heads, are constitutive throughout the cell cycle. This depicts cohesin as a stable molecular machine undergoing only transient conformational changes during binding and dissociation from chromosomes. Using FRET, we did not observe interactions between more than one cohesin complex in vivo.

The spindle-pole body (SPB), the yeast analog of the centrosome, serves as the major microtubule (MT) organizing center in the yeast cell. In addition to this central function, the SPB organizes and concentrates proteins required for proper coordination between the nuclear-division cycle and cytokinesis. For example, the Schizosaccharomyces pombe septation-initiation network (SIN), which is responsible for initiating actomyosin ring constriction and septation, is assembled at the SPB through its two scaffolding components, Sid4 and Cdc11. In an effort to identify novel SIN interactors, we purified Cdc11 and identified by mass spectrometry a previously uncharacterized protein associated with it, Ppc89. Ppc89 localizes constitutively to the SPB and interacts directly with Sid4. A fusion between the N-terminal 300 amino acids of Sid4 and a SPB targeting domain of Ppc89 supplies the essential function of Sid4 in anchoring the SIN. ppc89Δ cells are inviable and exhibit defects in SPB integrity, and hence in spindle formation, chromosome segregation, and SIN localization. Ppc89 overproduction is lethal, resulting primarily in a G2 arrest accompanied by massive enlargement of the SPB and increased SPB MT nucleation. These results suggest a fundamental role for Ppc89 in organization of the S. pombe SPB.

Interpreting genome sequences requires the functional analysis of thousands of predicted proteins, many of which are uncharacterized and without obvious homologs. To assess whether the roles of large sets of uncharacterized genes can be assigned by targeted application of a suite of technologies, we used four complementary protein-based methods to analyze a set of 100 uncharacterized but essential open reading frames (ORFs) of the yeast Saccharomyces cerevisiae. These proteins were subjected to affinity purification and mass spectrometry analysis to identify copurifying proteins, two-hybrid analysis to identify interacting proteins, fluorescence microscopy to localize the proteins, and structure prediction methodology to predict structural domains or identify remote homologies. Integration of the data assigned function to 48 ORFs using at least two of the Gene Ontology (GO) categories of biological process, molecular function, and cellular component; 77 ORFs were annotated by at least one method. This combination of technologies, coupled with annotation using GO, is a powerful approach to classifying genes.

Kinetochore proteins contribute to the fidelity of chromosome transmission by mediating the attachment of a specialized chromosomal region, the centromere, to the mitotic spindle during mitosis. In budding yeast, a subset of kinetochore proteins, referred to as the outer kinetochore, provides a link between centromere DNA-binding proteins of the inner kinetochore and microtubule-binding proteins. Using a combination of chromatin immunoprecipitation, in vivo localization, and protein coimmunoprecipitation, we have established that yeast Chl4p and Iml3p are outer kinetochore proteins that localize to the kinetochore in a Ctf19p-dependent manner. Chl4p interacts with the outer kinetochore proteins Ctf19p and Ctf3p, and Iml3p interacts with Chl4p and Ctf19p. In addition, Chl4p is required for the Ctf19p-Ctf3p and Ctf19p-Iml3p interactions, indicating that Chl4p is an important structural component of the outer kinetochore. These physical interaction dependencies provide insights into the molecular architecture and centromere DNA loading requirements of the outer kinetochore complex.

Cytoprotective functions of a 20S proteasome activator were investigated. Saccharomyces cerevisiae [Blm10][1] and human 20S proteasome activator 200 (PA200) are homologs. Comparative genome-wide analyses of untreated diploid cells lacking [Blm10][1] and growing at steady state at defined growth rates revealed downregulation of numerous genes required for accurate chromosome structure, assembly and repair, and upregulation of a specific subset of genes encoding protein-folding chaperones. [Blm10][1] loss or truncation of the [Ubp3][2]/[Blm3][2] deubiquitinating enzyme caused massive chromosomal damage and cell death in homozygous diploids after phleomycin treatments, indicating that [Blm10][1] and [Ubp3][2]/[Blm3][2] function to stabilize the genome and protect against cell death. Diploids lacking [Blm10][1] also were sensitized to doxorubicin, hydroxyurea, 5-fluorouracil, rapamycin, hydrogen peroxide, methyl methanesulfonate, and calcofluor. Fluorescently tagged [Blm10][1] localized in nuclei, with enhanced fluorescence after DNA replication. After DNA damage that caused a classic G2/M arrest, fluorescence remained diffuse, with evidence of nuclear fragmentation in some cells. Protective functions of [Blm10][1] did not require the carboxyl-terminal region that makes close contact with 20S proteasomes, indicating that protection does not require this contact or the truncated [Blm10][1] can interact with the proteasome apart from this region. Without its carboxyl-terminus, [Blm10][1](−339aa) localized to nuclei in untreated, nonproliferating (G) cells, but not during G1 S, G2, and M. The results indicate [Blm10][1] functions in protective mechanisms that include the machinery that assures proper assembly of chromosomes. These essential guardian functions have implications for ubiquitin-independent targeting in anticancer therapy. Targeting [Blm10][1]/PA200 together with one or more of the upregulated chaperones or a conventional treatment could be efficacious. [1]: http://www.yeastgenome.org/cgi-bin/locus.fpl?dbid=S000001887 [2]: http://www.yeastgenome.org/cgi-bin/locus.fpl?dbid=S000000953

The meiosis II outer plaque (MOP) acts a vesicle tethering complex that is a site for de novo membrane formation. Novel mutants in a MOP protein reveal that interaction of vesicles with the MOP surface is required to recruit a second tethering complex, the exocyst, to the vesicles, suggesting a mechanism by which the MOP promotes vesicle fusion., During meiosis II in Saccharomyces cerevisiae, the cytoplasmic face of the spindle pole body, referred to as the meiosis II outer plaque (MOP), is modified in both composition and structure to become the initiation site for de novo formation of a membrane called the prospore membrane. The MOP serves as a docking complex for precursor vesicles that are targeted to its surface. Using fluorescence resonance energy transfer analysis, the orientation of coiled-coil proteins within the MOP has been determined. The N-termini of two proteins, Mpc54p and Spo21p, were oriented toward the outer surface of the structure. Mutations in the N-terminus of Mpc54p resulted in a unique phenotype: precursor vesicles loosely tethered to the MOP but did not contact its surface. Thus, these mpc54 mutants separate the steps of vesicle association and docking. Using these mpc54 mutants, we determined that recruitment of the Rab GTPase Sec4p, as well as the exocyst components Sec3p and Sec8p, to the precursor vesicles requires vesicle docking to the MOP. This suggests that the MOP promotes membrane formation both by localization of precursor vesicles to a particular site and by recruitment of a second tethering complex, the exocyst, that stimulates downstream events of fusion.

The spindle pole body (SPB) is the microtubule organizing center of Saccharomyces cerevisiae. Its core includes the proteins Spc42, Spc110 (kendrin/pericentrin ortholog), calmodulin (Cmd1), Spc29, and Cnm67. Each was tagged with CFP and YFP and their proximity to each other was determined by fluorescence resonance energy transfer (FRET). FRET was measured by a new metric that accurately reflected the relative extent of energy transfer. The FRET values established the topology of the core proteins within the architecture of SPB. The N-termini of Spc42 and Spc29, and the C-termini of all the core proteins face the gap between the IL2 layer and the central plaque. Spc110 traverses the central plaque and Cnm67 spans the IL2 layer. Spc42 is a central component of the central plaque where its N-terminus is closely associated with the C-termini of Spc29, Cmd1, and Spc110. When the donor-acceptor pairs were ordered into five broad categories of increasing FRET, the ranking of the pairs specified a unique geometry for the positions of the core proteins, as shown by a mathematical proof. The geometry was integrated with prior cryoelectron tomography to create a model of the interwoven network of proteins within the central plaque. One prediction of the model, the dimerization of the calmodulin-binding domains of Spc110, was confirmed by in vitro analysis.

As the quantity of seismic refraction data in the oceans increases it is becoming apparent1–3 that there is a systematic increase in crustal thickness with age, at least out to about 60 Myr (refs 1,2). To explain this phenomenon more precisely the detailed structure of the lower crust is required. We present seismic refraction data from the Northern Cocos plate which shows that thickening occurs by the formation of a low velocity zone (LVZ) at the base of the crust with a velocity < 6.8 km s−1. One possible explanation of this in terms of these relatively low seismic velocities for the lower crust4, is that upper mantle material becomes serpentinised by a hydration reaction as it cools. If so, this implies that the Moho boundary, except at very young ages, is a phase change (hydration) boundary. This supports the ideas of Hess5.

From seismic refraction data on the Northern Cocos Plate, it is found that the crust thickens with age. Assuming that processes of crustal formation have remained constant over at least the past 10 m.y., the seismic data indicate that the thickening is caused by a gradual transformation of the top 2 km of the upper mantle into material having crustal-like velocities. Serpentinization is a possible mechanism. The data also suggest that the crust—mantle interface may be laterally variable, in some places being relatively sharp and in others gradational. Upper mantle anisotropy appears to decrease from about 0.6 km/sec near the rise axis to 0.3 km/sec at 10 m.y.

Refraction lines on the Northern Cocos Plate between the Orozco and Clipperton Fracture Zones have been used to determine upper mantle velocities over the plate. The velocities range from 7.50 to 8.43 km/sec. Azimuthal variations are found near the rise crest with low velocities parallel to the rise crest. The low velocities increase with age, lessening the observed azimuthal dependence away from the rise crest. A low-velocity zone is found in the mantle and may extend over a considerable portion of the plate near the rise crest. A 7.1-km/sec basal crustal layer is also observed and makes up a substantial portion of the crustal thickness.

Image processing and pattern recognition techniques are used in many aspects of seismic data processing and interpretation. These tools can effectively complement and simplify the task of the geophysicist and geologist in detecting and mapping potential hydrocarbon reservoirs. In the past, seismic interpretation was concerned mainly with mapping subsurface structures. With the advent of digital field recording and improved signal processing techniques, it became clear that the seismic section contains more than just structural information. The data contain details about attenuation and other attributes of the seismic signal that can be used to indirectly determine rock properties. The geophysicist now has the capability not only to map structure but to determine subsurface lithology. Image processing and pattern recognition techniques are being used to classify and sort the myriad of data available into an easily understood depiction of subsurface structure and lithology. This paper consists of an overview of the seismic technique and recent applications of pattern recognition to seismic exploration. A case history is presented showing how pattern recognition techniques were used to locate the termination of a gas-bearing sandstone between a producing well and a dry well.

It is recommended that patients with acute myocardial infarction be able to perform activities of daily living at approximately 3 METs at the time of hospital discharge. Implementation of this recommendation requires that the hemodynamic responses at the 3 METs level be assessed prior to discharge. Symptoms, blood pressures, heart rates, and electrocardiographic responses of 41 AMI patients (eight women and 33 men, mean age, 60 years) during a low-level treadmill test were studied 11 days after acute myocardial infarction. Twenty-nine of 41 patients (71 per cent) completed the test. Fatigue was the most common reason for stopping the test early. Between rest and maximum exercise there were increases of 13 per cent in systolic blood pressure, 25 per cent in heart rate, and 40 per cent in pressure-rate product. The resting systolic blood pressures, heart rates, and pressure-rate products were significantly higher (p less than or equal to 0.05) in the patients who were unable to finish the test. ST-segment elevation or depression larger than or equal to 1 mm. was seen in 14 patients. This low-level treadmill test was safe under well supervised conditions; it provided objective information about the patient's readiness for discharge. This type of information can be used for patient teaching and discharge planning.