Assessment of Advanced Diagnostic Bronchoscopy Outcomes for Peripheral Lung Lesions: A Delphi Consensus Definition of Diagnostic Yield and Recommendations for Patient-centered Study Designs. An Official American Thoracic Society/American College of Chest Physicians Research Statement.

Background: Advanced diagnostic bronchoscopy targeting the lung periphery has developed at an accelerated pace over the last two decades, whereas evidence to support introduction of innovative technologies has been variable and deficient. A major gap relates to variable reporting of diagnostic yield, in addition to limited comparative studies. Objectives: To develop a research framework to standardize the evaluation of advanced diagnostic bronchoscopy techniques for peripheral lung lesions. Specifically, we aimed for consensus on a robust definition of diagnostic yield, and we propose potential study designs at various stages of technology development. Methods: Panel members were selected for their diverse expertise. Workgroup meetings were conducted in virtual or hybrid format. The cochairs subsequently developed summary statements, with voting proceeding according to a modified Delphi process. The statement was cosponsored by the American Thoracic Society and the American College of Chest Physicians. Results: Consensus was reached on 15 statements on the definition of diagnostic outcomes and study designs. A strict definition of diagnostic yield should be used, and studies should be reported according to the STARD (Standards for Reporting Diagnostic Accuracy Studies) guidelines. Clinical or radiographic follow-up may be incorporated into the reference standard definition but should not be used to calculate diagnostic yield from the procedural encounter. Methodologically robust comparative studies, with incorporation of patient-reported outcomes, are needed to adequately assess and validate minimally invasive diagnostic technologies targeting the lung periphery. Conclusions: This American Thoracic Society/American College of Chest Physicians statement aims to provide a research framework that allows greater standardization of device validation efforts through clearly defined diagnostic outcomes and robust study designs. High-quality studies, both industry and publicly funded, can support subsequent health economic analyses and guide implementation decisions in various healthcare settings.

Proteomic profiling of human plasma identifies apolipoprotein E as being associated with smoking and a marker for squamous metaplasia of the lung.

Biomarkers to identify subjects at high-risk for developing lung cancer will revolutionize the disease outlook. Most biomarker studies have focused on patients already diagnosed with lung cancer and in most cases the disease is often advanced and incurable. The objective of this study was to use proteomics to identify a plasma biomarker for early detection of lung lesions that may subsequently be the harbinger for cancer. Plasma samples were obtained from subjects without lung cancer grouped as never, current, or ex-smokers. An iTRAQ-based proteomic analysis was performed on these pooled plasma samples. We identified 31 proteins differentially abundant in current smokers or ex-smokers relative to never smokers. Western blot and ELISA analyses confirmed the iTRAQ results that demonstrated an increase of apolipoprotein E (APOE) in current smokers as compared to both never and ex-smokers. There was a strong and significant correlation of the plasma APOE levels with development of premalignant squamous metaplasia. Additionally, we also showed that higher tissue levels of APOE are seen with squamous metaplasia, supporting a direct relationship. Our analysis reveals that elevated plasma APOE is associated with smoking, and APOE is a novel predictive protein biomarker for early morphological changes of squamous metaplasia in the lung.

Correlation of ultrasound findings with the Bethesda cytopathology classification for thyroid nodule fine-needle aspiration: a primer for radiologists.

OBJECTIVE: Ultrasound and ultrasound-guided fine-needle aspiration play a critical role in the evaluation of thyroid nodules. The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) was developed to facilitate communication among cytopathologists, radiologists, and referring physicians. The reporting scheme has rapidly become one of the most important contributions to thyroid nodule management. In this article, we review the significance of the TBSRTC categories and their implications in stratifying risk in the management of thyroid nodules. CONCLUSION: Knowledge of TBSRTC will allow the radiologist to better understand the criteria for thyroid nodule specimen adequacy, the components of risk stratification, and the standard terminology used for effective communication between patients and clinicians.

Training perspective: the impact of starting an endobronchial ultrasound program at a major academic center on fellows training of transbronchial needle aspiration.

The proliferation of endobronchial ultrasound as the standard of care in lymph node sampling has significantly impacted the way fellows are trained in transbronchial needle aspiration (TBNA). To assess the impact of starting an endobronchial ultrasound (EBUS) program on fellows training of conventional TBNA (cTBNA), we reviewed all TBNAs performed at the Johns Hopkins Hospital from September 2006 until December of 2009. The number of nodes sampled, specimen adequacy, diagnostic yield, and fellow involvement were recorded. We found that the initiation of an EBUS program was associated with a significantly increased number of cases performed, as well as a significantly higher diagnostic yield, when compared with cTBNA. There was an associated significant decline in the number of cTBNA procedures performed by the pulmonary fellows, as well as the diagnostic yield and accuracy, when compared with EBUS. As interventional pulmonology fellowships and the overall use of EBUS become more prevalent, institutions will need to consider how to train their fellows in lymph node sampling.

Observation of nanosecond laser induced fluorescence of in vitro seawater phytoplankton.

Seawater has been irradiated using a train of 70 ns flashes from a 440 nm laser source. This wavelength is on resonance with the blue absorption peak of Chlorophyll pigment associated with the photosystem of in vitro phytoplankton. The resulting fluorescence at 685 nm is instantaneously recorded during each laser pulse using a streak camera. Delayed fluorescence is observed, yielding clues about initiation of the photosynthetic process on a nanosecond time scale. Further data processing allows for determination of the functional absorption cross section, found to be 0.0095 A(2), which is the first reporting of this number for in vitro phytoplankton. Unlike other flash-pump studies of Chlorophyll, using a LED or flashlamp-based sources, the short laser pulse used here does not reveal any pulse-to-pulse hysteresis (i.e., variable fluorescence), indicating that the laser pulses used here are not able to drive the photosynthetic process to completion. This is attributed to competition from a back reaction between the photoexcited photosystem II and the intermediate electron acceptor. The significance of this work as a new type of deployable ocean fluorimeter is discussed, and it is believed the apparatus will have applications in thin-layer phytoplankton research.

Multiple approaches to study S. cerevisiae Rad9, a prototypical checkpoint protein.

The Saccharomyces cerevisiae RAD9 checkpoint gene is the prototypical checkpoint gene and is required for efficient checkpoint regulation in late G1, S, and at the G2/M cell cycle transition following DNA damage. Rad9 is required for the activation of Rad53 after damage and has been proposed to have roles in lesion recognition as well as DNA repair and the maintenance of genome stability. Here we describe methodology suitable for the study of G1, intra-S, and G2/M checkpoints in budding yeast, the analysis of Rad9/Rad53 phospho-forms, the biochemical analysis of Rad9 and Rad53, the fractionation of soluble and chromatin associated proteins, including Rad9, and the live cell imaging of GFP tagged Rad9.

RNA immunoprecipitation for determining RNA-protein associations in vivo.

Similar to chromatin immunoprecipitation (ChIP), RNA immunoprecipitation (RIP) can be used to detect the association of individual proteins with specific nucleic acid regions, in this case on RNA. Live cells are treated with formaldehyde to generate protein-RNA cross-links between molecules that are in close proximity in vivo. RNA sequences that cross-link with a given protein are isolated by immunoprecipitation of the protein, and reversal of the formaldehyde cross-linking permits recovery and quantitative analysis of the immunoprecipitated RNA by reverse transcription PCR. The basics of RIP are very similar to those of ChIP, but with some important caveats. This unit describes the RIP procedure for Saccharomyces cerevisiae. Although the corresponding steps for metazoan cells have not yet been worked out, it is likely that the yeast procedure can easily be adapted for use in other organisms.

A Rad26-Def1 complex coordinates repair and RNA pol II proteolysis in response to DNA damage.

Eukaryotic cells use multiple, highly conserved mechanisms to contend with ultraviolet-light-induced DNA damage. One important response mechanism is transcription-coupled repair (TCR), during which DNA lesions in the transcribed strand of an active gene are repaired much faster than in the genome overall. In mammalian cells, defective TCR gives rise to the severe human disorder Cockayne's syndrome (CS). The best-studied CS gene, CSB, codes for a Swi/Snf-like DNA-dependent ATPase, whose yeast homologue is called Rad26 (ref. 4). Here we identify a yeast protein, termed Def1, which forms a complex with Rad26 in chromatin. The phenotypes of cells lacking DEF1 are consistent with a role for this factor in the DNA damage response, but Def1 is not required for TCR. Rather, def1 cells are compromised for transcript elongation, and are unable to degrade RNA polymerase II (RNAPII) in response to DNA damage. Our data suggest that RNAPII stalled at a DNA lesion triggers a coordinated rescue mechanism that requires the Rad26-Def1 complex, and that Def1 enables ubiquitination and proteolysis of RNAPII when the lesion cannot be rapidly removed by Rad26-promoted DNA repair.