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-1␣ stabilization during hypoxia.Hypoxia initiates transcription of a number of gene products that help to sustain the supply of O 2 to tissues and to enhance cell survival during severe O 2 deprivation. Gene products that augment O 2 supply at the tissue level include erythropoietin (Epo) 1 which increases the proliferation of erythrocytes, tyrosine hydroxylase which is necessary for the synthesis of the neurotransmitter dopamine in the carotid bodies, and the angiogenic factor VEGF which stimulates growth of new capillaries (1-3). At the cellular level, gene products that enhance survival during hypoxia include the glycolytic enzymes and the glucose transporters Glut1 and Glut3 (4). The induction of these genes is mediated by hypoxia-inducible factor-1 (HIF-1) (5-7), a heterodimeric transcription factor consisting of HIF-1␣ and the aryl hydrocarbon nuclear translocator (ARNT or HIF-1␤) subunits (7-9). The significance of HIF-1 in transcriptional regulation was recently demonstrated by the marked decrease in mRNA expression of VEGF and glycolytic enzymes seen during hypoxia in HIF-1␣-or ARNT-deficient murine embryonic stem cells (10 -12).The mechanism by which HIF-1 activation is initiated during hypoxia remains unclear. Both HIF-1␣ and ARNT mRNAs are constitutively expressed, indicating that functional activity of the HIF-1␣⅐ARNT complex is regulated by post-transcriptional events. ARNT levels are not significantly affected by [O 2 ], whereas HIF-1␣ protein is rapidly degraded under normoxic conditions by the ubiquitin-proteasome system (13,14). Hypoxia enhances HIF-1␣ protein levels by inhibiting its degradation, thereby allowing it to accumulate, to dimerize with ARNT, and to bind to the hypoxia-responsive element (HRE) in the promoter or enhancer regions of various genes. Thus, the functional HIF-1␣⅐ARNT complex is primarily regulated by the abundance of the HIF-1␣ subunit.Although much has been learned about the role of HIF-1 in controlling the expression of hypoxia-responsive genes, the underlying mechanism by which cells detect the decrease in [O 2 ] and initiate the stabilization of HIF-1␣ is not known. Presently, four diverse O 2 -sensing mechanisms have been proposed to mediate the transcriptional response to hypoxia (15). Two of these models postulate the involvement of an iron-containing unit in the form of either a heme group or an iron/sulfur cluster, which undergoes a change in activity during hypoxia that triggers the transcriptional response. These models are supported by the observation that cobaltous ions, or alternatively the iron chelator desferrioxamine (DFO), stabilize HIF-1␣ under normoxic conditions (16). However, no specific proteins with this role have been identified in mammalian systems. Two other models involve the generation of reactive oxygen species (ROS) by a f...

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.

The molecular mechanisms by which cells detect hypoxia (1.5% O2), resulting in the stabilization of hypoxia-inducible factor 1alpha (HIF-1alpha) protein remain unclear. One model proposes that mitochondrial generation of reactive oxygen species is required to stabilize HIF-1alpha protein. Primary evidence for this model comes from the observation that cells treated with complex I inhibitors, such as rotenone, or cells that lack mitochondrial DNA (rho(0)-cells) fail to generate reactive oxygen species or stabilize HIF-1alpha protein in response to hypoxia. In the present study, we investigated the role of mitochondria in regulating HIF-1alpha protein stabilization under anoxia (0% O2). Wild-type A549 and HT1080 cells stabilized HIF-1alpha protein in response to hypoxia and anoxia. The rho(0)-A549 cells and rho(0)-HT1080 cells failed to accumulate HIF-1alpha protein in response to hypoxia. However, both rho(0)-A549 and rho(0)-HT1080 were able to stabilize HIF-1alpha protein levels in response to anoxia. Rotenone inhibited hypoxic, but not anoxic, stabilization of HIF-1alpha protein. These results indicate that a functional electron transport chain is required for hypoxic but not anoxic stabilization of HIF-1alpha protein.

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-X L prevented oxygen deprivation-induced cell death by inhibiting the release of cytochrome c and caspase-9 activation. The ability of Bcl-X L 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 F 1 F 0 -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-X L , cells devoid of mitochondrial DNA (°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.

Exposure of animals to hyperoxia results in lung injury that is characterized by apoptosis and necrosis of the alveolar epithelium and endothelium. The mechanism by which hyperoxia results in cell death, however, remains unclear. We sought to test the hypothesis that exposure to hyperoxia causes mitochondria-dependent apoptosis that requires the generation of reactive oxygen species from mitochondrial electron transport. Rat1a cells exposed to hyperoxia underwent apoptosis characterized by the release of cytochrome c, activation of caspase-9, and nuclear fragmentation that was prevented by the overexpression of Bcl-X L. Murine embryonic fibroblasts from bax ؊/؊ bak ؊/؊ mice were resistant to hyperoxia-induced cell death. The administration of the antioxidants manganese (III) tetrakis (4-benzoic acid) porphyrin, ebselen, and N-acetylcysteine failed to prevent cell death following exposure to hyperoxia. Human fibrosarcoma cells (HT1080) lacking mitochondrial DNA ( 0 cells) that failed to generate reactive oxygen species during exposure to hyperoxia were not protected against cell death following exposure to hyperoxia. We conclude that exposure to hyperoxia results in apoptosis that requires Bax or Bak and can be prevented by the overexpression of Bcl-X L . The mitochondrial generation of reactive oxygen species is not required for cell death following exposure to hyperoxia.

The intracellular signaling pathways that control O(2) deprivation (anoxia)-induced apoptosis have not been fully defined in lung epithelial cells. We show here that the lung epithelial cell line A549 releases cytochrome c and activates caspase-9 followed by DNA fragmentation and plasma membrane breakage in response to anoxia. The antiapoptotic protein Bcl-X(L) prevented the anoxia-induced cell death by inhibiting the release of cytochrome c and caspase-9 activation. A549 cells devoid of mitochondrial DNA (rho(o)-cells) and lacking a functional electron transport chain were resistant to anoxia-induced apoptosis. A549 cells preconditioned with either hypoxia (1.5% O(2)) or tumor necrosis factor-alpha, which activated the transcription factors hypoxia-inducible factor-1 or nuclear factor-kappaB, respectively, did not provide protection from anoxia-induced cell death. These results indicate that A549 cells require a functional electron transport chain and the release of cytochrome c for anoxia-induced apoptosis.

Significant advances in artificial intelligence (AI), deep learning, and other machine-learning approaches have been made in recent years, with applications found in almost every industry, including health care. AI is capable of completing a spectrum of mundane to complex medically oriented tasks previously performed only by boarded physicians, most recently assisting with the detection of cancers difficult to find on histopathology slides. Although computers will likely not replace pathologists any time soon, properly designed AI-based tools hold great potential for increasing workflow efficiency and diagnostic accuracy in pathology. Recent trends, such as data augmentation, crowdsourcing for generating annotated data sets, and unsupervised learning with molecular and/or clinical outcomes versus human diagnoses as a source of ground truth, are eliminating the direct role of pathologists in algorithm development. Proper integration of AI-based systems into anatomic-pathology practice will necessarily require fully digital imaging platforms, an overhaul of legacy information-technology infrastructures, modification of laboratory/ pathologist workflows, appropriate reimbursement/cost-offsetting models, and ultimately, the active participation of pathologists to encourage buy-in and oversight. Regulations tailored to the nature and limitations of AI are currently in development and, when instituted, are expected to promote safe and effective use. This review addresses the challenges in AI development, deployment, and regulation to be overcome prior to its widespread adoption in anatomic pathology.

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.