Blur Quantification of Medical Images: Dicom Media, Whole Slide Images, Generic Images and Videos

Introduction/ Background We have designed a quality assurance tool to quantify blur by quantifying the sharpness of custom-sized tiles composing an image, generating global results, detailed exchangeable logs and sharpness-maps regardless of their dimensions, quantity or acquisition rate [1]. We have now integrated the programming libraries and the standalone program we built to existing workflows and software, in order to improve quality assurance procedures. Aims When integrated in an acquisition workflow, the ability to map the quality of local areas inside an image allows to re-acquire parts of the image that were not scanned properly, or discard and re-acquire images when the global results fall under the thresholds set by the users. When integrated in an image analysis software, the regions of interest can be automatically chosen or suggested to the user according to the quality-map of the image to analyze, reducing the amount of incoherent analysis results. When integrated as a library in an image management platform, or as a standalone program in a storage server, a systematic quality check can detect de-calibration of the acquisition software, failed acquisitions that may be automatically deleted and users can be notified to re-acquire the images when below their quality-thresholds profiles. A quantified quality score can be inserted in each image’s metadata for traceability purposes. When integrated as a library in a local or remote image viewer, the best quality tiles can be sent first to the viewer, and magnification levels can be dynamically resampled for a better render. Methods One notable implementation, using our Java library, is inside the FlexMIm project [2], which includes 27 pathology laboratories in the Paris area (coordinated by APHP), research laboratories from University Paris 6 Paris 7, as well as 3 companies: TRIBVN, PERTIMM and Orange. All Whole Slide Images are systematically analyzed and mapped as they enter the platform. The focus map may be displayed on the web interface next to the thumbnail link to the WSI, or in the viewer as a semi-transparent layer over the WSI, or over the WSI map. During the test phase and first integrations in laboratories and hospitals as well as in the FlexMIm project, more than 5000 whole slide images of multiple formats (Hamamatsu NDPI, Aperio SVS, Mirax MRXS, JPEG2000 …) as well as hundreds of thousands of images of various formats (DICOM, TIFF, PNG, JPEG ...) and videos (H264) have been analyzed using our standalone software or our C, C++, Java and Python libraries. Using default or customizable thresholds’ profiles, WSI are sorted as “accepted”, “to review”, “to rescan”. In order to target the samples contained inside each WSI, special attention was paid to detecting blank tiles. Dynamic blank tile detection based on statistical analysis of each WSI was built and successfully validated for all our samples. Results More than 20 trillion pixels have been analyzed at a 3.5 billion pixels per quad-core processor per minute speed rate. Quantified results can be stored in JSON formatted logs or inside a MySQL or MongoDB database) or converted to any chosen data structure to be interoperable with existing software, each tile’s result being accessible in addition to the quality map and the global quality results. This solution is easily scalable as images can be stored at different locations, analysis can be distributed amongst local or remote servers, and quantified results can be stored in remote databases

Evasion of cytotoxic T lymphocyte (CTL) responses by nef-dependent induction of Fas ligand (CD95L) expression on simian immunodeficiency virus-infected cells.

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Transmission traits of malaria parasites within the mosquito : Genetic variation, phenotypic plasticity, and consequences for control

Evaluating the risk of emergence and transmission of vector-borne diseases requires knowledge of the genetic and environmental contributions to pathogen transmission traits. Compared to the significant effort devoted to understanding the biology of malaria transmission from vertebrate hosts to mosquito vectors, the strategies that malaria parasites have evolved to maximize transmission from vectors to vertebrate hosts have been largely overlooked. While determinants of infection success within the mosquito host have recently received attention, the causes of variability for other key transmission traits of malaria, namely the duration of parasite development and its virulence within the vector, as well as its ability to alter mosquito behavior, remain largely unknown. This important gap in our knowledge needs to be bridged in order to obtain an integrative view of the ecology and evolution of malaria transmission strategies. Associations between transmission traits also need to be characterized, as they trade-offs and constraints could have important implications for understanding the evolution of parasite transmission. Finally, theoretical studies are required to evaluate how genetic and environmental influences on parasite transmission traits can shape malaria dynamics and evolution in response to disease control

Atmospheric-Pressure Plasma Jet Induces Apoptosis Involving Mitochondria via Generation of Free Radicals

The plasma jet has been proposed as a novel therapeutic method for anticancer treatment. However, its biological effects and mechanism of action remain elusive. Here, we investigated its cell death effects and underlying molecular mechanisms, using air and N2 plasma jets from a micro nozzle array. Treatment with air or N2 plasma jets caused apoptotic death in human cervical cancer HeLa cells, simultaneously with depolarization of mitochondrial membrane potential. In addition, the plasma jets were able to generate reactive oxygen species (ROS), which function as surrogate apoptotic signals by targeting the mitochondrial membrane potential. Antioxidants or caspase inhibitors ameliorated the apoptotic cell death induced by the air and N2 plasma jets, suggesting that the plasma jet may generate ROS as a proapoptotic cue, thus initiating mitochondria-mediated apoptosis. Taken together, our data suggest the potential employment of plasma jets as a novel therapy for cancer

Bax Function in the Absence of Mitochondria in the Primitive Protozoan Giardia lamblia

Bax-induced permeabilization of the mitochondrial outer membrane and release of cytochrome c are key events in apoptosis. Although Bax can compromise mitochondria in primitive unicellular organisms that lack a classical apoptotic machinery, it is still unclear if Bax alone is sufficient for this, or whether additional mitochondrial components are required. The protozoan parasite Giardia lamblia is one of the earliest branching eukaryotes and harbors highly degenerated mitochondrial remnant organelles (mitosomes) that lack a genome. Here we tested whether human Bax expressed in Giardia can be used to ablate mitosomes. We demonstrate that these organelles are neither targeted, nor compromised, by Bax. However, specialized compartments of the regulated secretory pathway are completely ablated by Bax. As a consequence, maturing cyst wall proteins that are sorted into these organelles are released into the cytoplasm, causing a developmental arrest and cell death. Interestingly, this ectopic cargo release is dependent on the carboxy-terminal 22 amino acids of Bax, and can be prevented by the Bax-inhibiting peptide Ku70. A C-terminally truncated Bax variant still localizes to secretory organelles, but is unable to permeabilize these membranes, uncoupling membrane targeting and cargo release. Even though mitosomes are too diverged to be recognized by Bax, off-target membrane permeabilization appears to be conserved and leads to cell death completely independently of mitochondria

Naturally Occurring Triggers that Induce Apoptosis-Like Programmed Cell Death in Plasmodium berghei Ookinetes

Several protozoan parasites have been shown to undergo a form of programmed cell death that exhibits morphological features associated with metazoan apoptosis. These include the rodent malaria parasite, Plasmodium berghei. Malaria zygotes develop in the mosquito midgut lumen, forming motile ookinetes. Up to 50% of these exhibit phenotypic markers of apoptosis; as do those grown in culture. We hypothesised that naturally occurring signals induce many ookinetes to undergo apoptosis before midgut traversal. To determine whether nitric oxide and reactive oxygen species act as such triggers, ookinetes were cultured with donors of these molecules. Exposure to the nitric oxide donor SNP induced a significant increase in ookinetes with condensed nuclear chromatin, activated caspase-like molecules and translocation of phosphatidylserine that was dose and time related. Results from an assay that detects the potential-dependent accumulation of aggregates of JC-1 in mitochondria suggested that nitric oxide does not operate via loss of mitochondrial membrane potential. L-DOPA (reactive oxygen species donor) also caused apoptosis in a dose and time dependent manner. Removal of white blood cells significantly decreased ookinetes exhibiting a marker of apoptosis in vitro. Inhibition of the activity of nitric oxide synthase in the mosquito midgut epithelium using L-NAME significantly decreased the proportion of apoptotic ookinetes and increased the number of oocysts that developed. Introduction of a nitric oxide donor into the blood meal had no effect on mosquito longevity but did reduce prevalence and intensity of infection. Thus, nitric oxide and reactive oxygen species are triggers of apoptosis in Plasmodium ookinetes. They occur naturally in the mosquito midgut lumen, sourced from infected blood and mosquito tissue. Up regulation of mosquito nitric oxide synthase activity has potential as a transmission blocking strategy

Apoptosis-Like Death in Bacteria Induced by HAMLET, a Human Milk Lipid-Protein Complex

Background: Apoptosis is the primary means for eliminating unwanted cells in multicellular organisms in order to preserve tissue homeostasis and function. It is characterized by distinct changes in the morphology of the dying cell that are orchestrated by a series of discrete biochemical events. Although there is evidence of primitive forms of programmed cell death also in prokaryotes, no information is available to suggest that prokaryotic death displays mechanistic similarities to the highly regulated programmed death of eukaryotic cells. In this study we compared the characteristics of tumor and bacterial cell death induced by HAMLET, a human milk complex of alpha-lactalbumin and oleic acid. Methodology/Principal Findings: We show that HAMLET-treated bacteria undergo cell death with mechanistic and morphologic similarities to apoptotic death of tumor cells. In Jurkat cells and Streptococcus pneumoniae death was accompanied by apoptosis-like morphology such as cell shrinkage, DNA condensation, and DNA degradation into high molecular weight fragments of similar sizes, detected by field inverse gel electrophoresis. HAMLET was internalized into tumor cells and associated with mitochondria, causing a rapid depolarization of the mitochondrial membrane and bound to and induced depolarization of the pneumococcal membrane with similar kinetic and magnitude as in mitochondria. Membrane depolarization in both systems required calcium transport, and both tumor cells and bacteria were found to require serine protease activity (but not caspase activity) to execute cell death. Conclusions/Significance: Our results suggest that many of the morphological changes and biochemical responses associated with apoptosis are present in prokaryotes. Identifying the mechanisms of bacterial cell death has the potential to reveal novel targets for future antimicrobial therapy and to further our understanding of core activation mechanisms of cell death in eukaryote cells

Evolution of apoptosis-like programmed cell death in unicellular protozoan parasites

Apoptosis-like programmed cell death (PCD) has recently been described in multiple taxa of unicellular protists, including the protozoan parasites Plasmodium, Trypanosoma and Leishmania. Apoptosis-like PCD in protozoan parasites shares a number of morphological features with programmed cell death in multicellular organisms. However, both the evolutionary explanations and mechanisms involved in parasite PCD are poorly understood. Explaining why unicellular organisms appear to undergo 'suicide' is a challenge for evolutionary biology and uncovering death executors and pathways is a challenge for molecular and cell biology. Bioinformatics has the potential to integrate these approaches by revealing homologies in the PCD machinery of diverse taxa and evaluating their evolutionary trajectories. As the molecular mechanisms of apoptosis in model organisms are well characterised, and recent data suggest similar mechanisms operate in protozoan parasites, key questions can now be addressed. These questions include: which elements of apoptosis machinery appear to be shared between protozoan parasites and multicellular taxa and, have these mechanisms arisen through convergent or divergent evolution? We use bioinformatics to address these questions and our analyses suggest that apoptosis mechanisms in protozoan parasites and other taxa have diverged during their evolution, that some apoptosis factors are shared across taxa whilst others have been replaced by proteins with similar biochemical activities

Cooperation between Apoptotic and Viable Metacyclics Enhances the Pathogenesis of Leishmaniasis

Mimicking mammalian apoptotic cells by exposing phosphatidylserine (PS) is a strategy used by virus and parasitic protozoa to escape host protective inflammatory responses. With Leishmania amazonensis (La), apoptotic mimicry is a prerogative of the intramacrophagic amastigote form of the parasite and is modulated by the host. Now we show that differently from what happens with amastigotes, promastigotes exposing PS are non-viable, non-infective cells, undergoing apoptotic death. As part of the normal metacyclogenic process occurring in axenic cultures and in the gut of sand fly vectors, a sub-population of metacyclic promastigotes exposes PS. Apoptotic death of the purified PS-positive (PSPOS) sub-population was confirmed by TUNEL staining and DNA laddering. Transmission electron microscopy revealed morphological alterations in PSPOS metacyclics such as DNA condensation, cytoplasm degradation and mitochondrion and kinetoplast destruction, both in in vitro cultures and in sand fly guts. TUNELPOS promastigotes were detected only in the anterior midgut to foregut boundary of infected sand flies. Interestingly, caspase inhibitors modulated parasite death and PS exposure, when added to parasite cultures in a specific time window. Efficient in vitro macrophage infections and in vivo lesions only occur when PSPOS and PS-negative (PSNEG) parasites were simultaneously added to the cell culture or inoculated in the mammalian host. The viable PSNEG promastigote was the infective form, as shown by following the fate of fluorescently labeled parasites, while the PSPOS apoptotic sub-population inhibited host macrophage inflammatory response. PS exposure and macrophage inhibition by a subpopulation of promastigotes is a different mechanism than the one previously described with amastigotes, where the entire population exposes PS. Both mechanisms co-exist and play a role in the transmission and development of the disease in case of infection by La. Since both processes confer selective advantages to the infective microorganism they justify the occurrence of apoptotic features in a unicellular pathogen

What does the structure-function relationship of the HIV-1 Tat protein teach us about developing an AIDS vaccine?

The human immunodeficiency virus type 1 (HIV-1) trans-activator of transcription protein Tat is an important factor in viral pathogenesis. In addition to its function as the key trans-activator of viral transcription, Tat is also secreted by the infected cell and taken up by neighboring cells where it has an effect both on infected and uninfected cells. In this review we will focus on the relationship between the structure of the Tat protein and its function as a secreted factor. To this end we will summarize some of the exogenous functions of Tat that have been implicated in HIV-1 pathogenesis and the impact of structural variations and viral subtype variants of Tat on those functions. Finally, since in some patients the presence of Tat-specific antibodies or CTL frequencies are associated with slow or non-progression to AIDS, we will also discuss the role of Tat as a potential vaccine candidate, the advances made in this field, and the importance of using a Tat protein capable of eliciting a protective or therapeutic immune response to viral challenge