David S. McClintock

Reactive Oxygen Species Generated at Mitochondrial Complex III Stabilize Hypoxia-inducible Factor-1α during Hypoxia A MECHANISM OF O2 SENSING

During hypoxia, hypoxia-inducible factor-1α (HIF-1α) is required for induction of a variety of genes including erythropoietin and vascular endothelial growth factor. Hypoxia increases mitochondrial reactive oxygen species (ROS) generation at Complex III, which causes accumulation of HIF-1α protein responsible for initiating expression of a luciferase reporter construct under the control of a hypoxic response element. This response is lost in cells depleted of mitochondrial DNA (ρ0 cells). Overexpression of catalase abolishes hypoxic response element-luciferase expression during hypoxia. Exogenous H2O2 stabilizes HIF-1α protein during normoxia and activates luciferase expression in wild-type and ρ0 cells. Isolated mitochondria increase ROS generation during hypoxia, as does the bacterium Paracoccus denitrificans. These findings reveal that mitochondria-derived ROS are both required and sufficient to initiate HIF-1α stabilization during hypoxia.

Role of Oxidants in NF-κB Activation and TNF-α Gene Transcription Induced by Hypoxia and Endotoxin

The transcription factor NF-κB stimulates the transcription of proinflammatory cytokines including TNF-α. LPS (endotoxin) and hypoxia both induce NF-κB activation and TNF-α gene transcription. Furthermore, hypoxia augments LPS induction of TNF-α mRNA. Previous reports have indicated that antioxidants abolish NF-κB activation in response to LPS or hypoxia, which suggests that reactive oxygen species (ROS) are involved in NF-κB activation. This study tested whether mitochondrial ROS are required for both NF-κB activation and the increase in TNF-α mRNA levels during hypoxia and LPS. Our results indicate that hypoxia (1.5% O2) stimulates NF-κB and TNF-α gene transcription and increases ROS generation as measured by the oxidant sensitive dye 2′,7′-dichlorofluorescein diacetate in murine macrophage J774.1 cells. The antioxidants N-acetylcysteine and pyrrolidinedithiocarbamic acid abolished the hypoxic activation of NF-κB, TNF-α gene transcription, and increases in ROS levels. Rotenone, an inhibitor of mitochondrial complex I, abolished the increase in ROS signal, the activation of NF-κB, and TNF-α gene transcription during hypoxia. LPS stimulated NF-κB and TNF-α gene transcription but not ROS generation in J774.1 cells. Rotenone, pyrrolidinedithiocarbamic acid, and N-acetylcysteine had no effect on the LPS stimulation of NF-κB and TNF-α gene transcription, indicating that LPS activates NF-κB and TNF-α gene transcription through a ROS-independent mechanism. These results indicate that mitochondrial ROS are required for the hypoxic activation of NF-κB and TNF-α gene transcription, but not for the LPS activation of NF-κB.

Bcl-2 Family Members and Functional Electron Transport Chain Regulate Oxygen Deprivation-Induced Cell Death

ABSTRACT The mechanisms underlying cell death during oxygen deprivation are unknown. We report here a model for oxygen deprivation-induced apoptosis. The death observed during oxygen deprivation involves a decrease in the mitochondrial membrane potential, followed by the release of cytochrome c and the activation of caspase-9. Bcl-XL prevented oxygen deprivation-induced cell death by inhibiting the release of cytochrome c and caspase-9 activation. The ability of Bcl-XL to prevent cell death was dependent on allowing the import of glycolytic ATP into the mitochondria to generate an inner mitochondrial membrane potential through the F1F0-ATP synthase. In contrast, although activated Akt has been shown to inhibit apoptosis induced by a variety of apoptotic stimuli, it did not prevent cell death during oxygen deprivation. In addition to Bcl-XL, cells devoid of mitochondrial DNA (ρ° cells) that lack a functional electron transport chain were resistant to oxygen deprivation. Further, murine embryonic fibroblasts from bax −/− bak −/− mice did not die in response to oxygen deprivation. These data suggest that when subjected to oxygen deprivation, cells die as a result of an inability to maintain a mitochondrial membrane potential through the import of glycolytic ATP. Proapoptotic Bcl-2 family members and a functional electron transport chain are required to initiate cell death in response to oxygen deprivation.

Using Computerized Workflow Simulations to Assess the Feasibility of Whole Slide Imaging Full Adoption in a High-Volume Histology Laboratory

Background: Whole slide Imaging (WSI) has been touted by many as the future of pathology, with estimates of full adoption occurring sometime in the next 5 to 15 years. While WSI devices have become increasingly capable since their inception, there has been little consideration of how WSI will be implemented and subsequently affect the workflow of high volume histology laboratories. Methods: Histology workflow process data was collected from a high-volume histology laboratory (Massachusetts General Hospital) and a process model developed using business process management software. Computerized workflow simulations were performed and total histology process time evaluated under a number of different WSI conditions. Results: Total histology process time increased approximately 10-fold to 20-fold over baseline with the presence of one WSI robot in the histology workflow. Depending on the specifications of the WSI robot, anywhere from 9 to 14 WSI robots were required within the histology workflow to minimize the effects of WSI. Conclusion: Placing a WSI robot into the current workflow of a high-volume histology laboratory with the intent of full adoption is not feasible. Implementing WSI without making significant changes to the current workflow of the histology laboratory would prove to be both disruptive and costly to surgical pathology.

Evaluation and optimization for liquid-based preparation cytology in whole slide imaging

Background: Cytology poses different obstacles in whole slide imaging compared to surgical pathology slides. A single focal plane suffices for most of the latter, but cytology slides are thicker, potentially requiring multiple focal planes for adequate diagnostic information. Multiple focal planes adversely impact scanning time per slide, evaluation times, and file sizes. In this pilot study, we evaluated and compared the multilayer stack method to the extended focus algorithm as an alternative which collapses multiple focal planes into a single image, retaining only focused areas from each plane. Materials and Methods: 10 SurePath; cervical cytology slides were scanned at three thickness settings: 18, 24, and 30 μm. Three scanners were used: (1) Hamamatsu Nanozoomer 2.0-HT, (2) 3DHISTECH Mirax scan, and (3) Bioimagene iScan Coreo Au. The Nanozoomer and iScan utilized multilayer stacking, while the Mirax files were composited by extended focus. Scan times and file sizes were recorded, and image quality compared. Results: The Nanozoomer stacks averaged 1.58 gb and around 25 min for each slide, while the iScan stacks ranged from 6.23 to 9.3 gb and took 34-50 min to scan. The Mirax images averaged 210 mb and took 13-20 min to scan. Multilayer stack image quality from both Nanozoomer and iScan was fairly comparable. The iScan revealed significant mechanical issues that did not correspond to user settings. The Mirax images showed worrisome loss of crisp focus detail, worsening with increasing focal planes and impacting assessment of nuclear contours and chromatin detail. Conclusions: The optimal number of focal planes remains unknown for cytology. Multilayer stacks require excessive scanning time, network bandwidth, and file storage. Extended focus was evaluated as an alternative, but significant image quality issues were revealed. Further large-scale studies are needed to assess their clinical impact.

A core curriculum for clinical fellowship training in pathology informatics

Background: In 2007, our healthcare system established a clinical fellowship program in Pathology Informatics. In 2010 a core didactic course was implemented to supplement the fellowship research and operational rotations. In 2011, the course was enhanced by a formal, structured core curriculum and reading list. We present and discuss our rationale and development process for the Core Curriculum and the role it plays in our Pathology Informatics Fellowship Training Program. Materials and Methods: The Core Curriculum for Pathology Informatics was developed, and is maintained, through the combined efforts of our Pathology Informatics Fellows and Faculty. The curriculum was created with a three-tiered structure, consisting of divisions, topics, and subtopics. Primary (required) and suggested readings were selected for each subtopic in the curriculum and incorporated into a curated reading list, which is reviewed and maintained on a regular basis. Results: Our Core Curriculum is composed of four major divisions, 22 topics, and 92 subtopics that cover the wide breadth of Pathology Informatics. The four major divisions include: (1) Information Fundamentals, (2) Information Systems, (3) Workflow and Process, and (4) Governance and Management. A detailed, comprehensive reading list for the curriculum is presented in the Appendix to the manuscript and contains 570 total readings (current as of March 2012). Discussion: The adoption of a formal, core curriculum in a Pathology Informatics fellowship has significant impacts on both fellowship training and the general field of Pathology Informatics itself. For a fellowship, a core curriculum defines a basic, common scope of knowledge that the fellowship expects all of its graduates will know, while at the same time enhancing and broadening the traditional fellowship experience of research and operational rotations. For the field of Pathology Informatics itself, a core curriculum defines to the outside world, including departments, companies, and health systems considering hiring a pathology informatician, the core knowledge set expected of a person trained in the field and, more fundamentally, it helps to define the scope of the field within Pathology and healthcare in general.

Pathology informatics fellowship retreats: The use of interactive scenarios and case studies as pathology informatics teaching tools

Background: Last year, our pathology informatics fellowship added informatics-based interactive case studies to its existing educational platform of operational and research rotations, clinical conferences, a common core curriculum with an accompanying didactic course, and national meetings. Methods: The structure of the informatics case studies was based on the traditional business school case study format. Three different formats were used, varying in length from short, 15-minute scenarios to more formal multiple hour-long case studies. Case studies were presented over the course of three retreats (Fall 2011, Winter 2012, and Spring 2012) and involved both local and visiting faculty and fellows. Results: Both faculty and fellows found the case studies and the retreats educational, valuable, and enjoyable. From this positive feedback, we plan to incorporate the retreats in future academic years as an educational component of our fellowship program. Conclusions: Interactive case studies appear to be valuable in teaching several aspects of pathology informatics that are difficult to teach in more traditional venues (rotations and didactic class sessions). Case studies have become an important component of our fellowship′s educational platform.

Pathology informatics fellowship training: Focus on molecular pathology

Background: Pathology informatics is both emerging as a distinct subspecialty and simultaneously becoming deeply integrated within the breadth of pathology practice. As specialists, pathology informaticians need a broad skill set, including aptitude with information fundamentals, information systems, workflow and process, and governance and management. Currently, many of those seeking training in pathology informatics additionally choose training in a second subspecialty. Combining pathology informatics training with molecular pathology is a natural extension, as molecular pathology is a subspecialty with high potential for application of modern biomedical informatics techniques. Methods and Results: Pathology informatics and molecular pathology fellows and faculty evaluated the current fellowship program′s core curriculum topics and subtopics for relevance to molecular pathology. By focusing on the overlap between the two disciplines, a structured curriculum consisting of didactics, operational rotations, and research projects was developed for those fellows interested in both pathology informatics and molecular pathology. Conclusions: The scope of molecular diagnostics is expanding dramatically as technology advances and our understanding of disease extends to the genetic level. Here, we highlight many of the informatics challenges facing molecular pathology today, and outline specific informatics principles necessary for the training of future molecular pathologists.