Scanning EM Research Articles

Deep learning, 3D ultrastructural analysis reveals quantitative differences in platelet and organelle packing in COVID-19/SARSCoV2 patient-derived platelets

Platelets contribute to COVID-19 clinical manifestations, of which microclotting in the pulmonary vasculature has been a prominent symptom. To investigate the potential diagnostic contributions of overall platelet morphology and their α-granules and mitochondria to the understanding of platelet hyperactivation and micro-clotting, we undertook a 3D ultrastructural approach. Because differences might be small, we used the high-contrast, high-resolution technique of focused ion beam scanning EM (FIB-SEM) and employed deep learning computational methods to evaluate nearly 600 individual platelets and 30 000 included organelles within three healthy controls and three severely ill COVID-19 patients. Statistical analysis reveals that the α-granule/mitochondrion-to-plateletvolume ratio is significantly greater in COVID-19 patient platelets indicating a denser packing of organelles, and a more compact platelet. The COVID-19 patient platelets were significantly smaller –by 35% in volume – with most of the difference in organelle packing density being due to decreased platelet size. There was little to no 3D ultrastructural evidence for differential activation of the platelets from COVID-19 patients. Though limited by sample size, our studies suggest that factors outside of the platelets themselves are likely responsible for COVID-19 complications. Our studies show how deep learning 3D methodology can become the gold standard for 3D ultrastructural studies of platelets.

Organ-on-a-Chip Microfluidic Systems for Tracking Exosomal Dynamic Communication

Organ-on-a-chip microfluid systems (OCMS) are miniaturized three-dimension models of human tissue and organ, designed to recapitulate the crucial physiological and biological parameters of their corresponding in vivo parts. They have emerged as a powerful multifunctional tool for various applications such as personalized medicine, drug screening, due to its ability to show biomimetic composition, designs, and functions. Recently, OCMS have been employed to model and decode inter-organ communication via exosomes. Exosomes are biological nanovesicles with approximately 30-200 nm diameter, released from most of the cell types and participate in various cellular functions via intracellular communication and by carrying different cargoes including protein, and nucleic acids. Under pathological conditions such as cancer, the release of exosomes enhances tremendously, which are either fused or internalized by the recipient cells to elicit specific biological responses. The research pertaining to the exosomal communication has employed different methods for characterizing their release by the donor cells and uptake by the recipient cells, such as nano tracking analyzer, protein quantification, transmission electron microscopy (TEM), scanning EM (SEM), and immunogold-EM, exosome labeling kits, microbead-based flow cytometry. However, the research associated with the regulation of exosomal release and uptake has been impeded by the dearth of advanced techniques for capturing dynamics of exosomes. Here in, we discuss the advances in biosensing for tracking exosomal dynamic communication in OCMS, which will open new avenues of exosomal research using microfluidic engineering for modeling intracellular communication in OCMS.

Astrocytic engagement of the corticostriatal synaptic cleft is disrupted in a mouse model of Huntington’s disease

Astroglial dysfunction contributes to the pathogenesis of Huntington's disease (HD), and glial replacement can ameliorate the disease course. To establish the topographic relationship of diseased astrocytes to medium spiny neuron (MSN) synapses in HD, we used 2-photon imaging to map the relationship of turboRFP-tagged striatal astrocytes and rabies-traced, EGFP-tagged coupled neuronal pairs in R6/2 HD and wild-type (WT) mice. The tagged, prospectively identified corticostriatal synapses were then studied by correlated light electron microscopy followed by serial block-face scanning EM, allowing nanometer-scale assessment of synaptic structure in 3D. By this means, we compared the astrocytic engagement of single striatal synapses in HD and WT brains. R6/2 HD astrocytes exhibited constricted domains, with significantly less coverage of mature dendritic spines than WT astrocytes, despite enhanced engagement of immature, thin spines. These data suggest that disease-dependent changes in the astroglial engagement and sequestration of MSN synapses enable the high synaptic and extrasynaptic levels of glutamate and K+ that underlie striatal hyperexcitability in HD. As such, these data suggest that astrocytic structural pathology may causally contribute to the synaptic dysfunction and disease phenotype of those neurodegenerative disorders characterized by network overexcitation.

Knema retusa is antibacterial and antibiofilm against antibiotic resistant Staphylococcus aureus and S. haemolyticus isolated in bovine mastitis.

This study aimed to assess antibacterial activity of Knema retusa wood extract (KRe) against antibiotic resistant staphylococci which are causative agents of bovine mastitis. From 75 cases of intramammary infections in dairy cows, 66 staphylococcal isolates were collected, including 11 Staphylococcus aureus isolates (17%) and 55 coagulase-negative staphylococci (83%). Sixty isolates (91%) formed strong biofilms. KRe had minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) against the isolates ranging 32-256 ug/mL and 64-512 ug/mL, respectively. Two-hour KRe exposures at 4×MIC, viabilities of S. aureus and S. haemolyticus decreased by 3 log10 compared to the control. Scanning EM (SEM) showed that KRe disrupted the bacterial cells of both species. KRe at 1/16×MIC significantly inhibited biofilm formation (P < 0.05) in both S. aureus and S. haemolyticus. At 1/2×MIC, S. aureus and S. haemolyticus biofilm inhibition ranged from 75 to 99%. Cells within established biofilms were disrupted 66-83% by KRe at 32×MIC. Moreover, 1/2×MIC KRe reduced bacterial adhesion to glass surfaces observed by SEM. According to GC-MS analysis, the major compound in KRe was endo-2-hydroxy-9,9-(ethylenedioxy)-1-carbethoxy bicyclo [3.3.1] nonane (E2N). Molecular docking analysis of E2N has a high affinity for staphylococcal accessory regulator A (SarA), binding free-energy - 6.40kcal/mol. The results suggested that KRe may have medicinal benefits by inhibiting the growth, biofilm, and adhesion of antibiotic resistant staphylococci isolated from bovine mastitis.

P14: Vibration-Mediated Endothelial Cell-Platelet Interaction: A Thrombotic Risk Factor in Obstructive Sleep Apnea and Beyond

Background: Obstructive sleep apnea (OSA) is an independent risk factor for thrombotic stroke. OSA stroke is felt to be driven by hypoxia and post apneacatecholamine surge, leading to platelet activation and thrombosis. OSA is also associated with significant snoring and related neck vibration. In our work related to thrombogenicity of mechanical circulatory support (MCS) devices we demonstrated that device vibration and related shear are biomechanical activators of platelets. Recently we showed that snore-associated vibration is platelet activating. As OSA snore, vibration is imparted to major neck blood vessels, i.e. carotid and jugular, and activated platelets interact with vessel endothelial cells, the effect of vibration on endothelial thrombogenicity warrants further definition. Methods: OSA snore recordings were obtained from the human sleep lab. A speaker-platform device, validated to generate snore neck vibrations, was fabricated (snore emulator). Human umbilical vein endothelial cells (HUVECs) grown on petri dishes, placed on the emulator and exposed to snore vibrations for 4,8,12, 24,or 36 hours at 37oC., non-vibration cultures serving as controls. HUVEC viability (LDH assay) and platelet adhesion (inactivated platelets bound, measured via scanning EM) were serially assessed. Results: HUVEC exposed to snore vibration remained viable, though with a progressive decline in viability vs control, i.e. a 28.9% and 46.5% reduction at 24h and 36h, respectively (p<0.05). Interestingly, vibration led to a disproportionally greater increase in thrombogenicity, with net platelet adhesion increasing by 64% and 86% at 24h and 36h respectively (p<0.05) Conclusion: Vibration, as a general biomechanical stimulus, previously demonstrated to increase platelet activation, also leads to endothelial dysfunction, including loss of viability, with an even greater increase in endothelial thrombogenicity. Further definition of vibration-mediated endothelial dysfunction has implication for developing translational therapeutics for OSA, as well as providing insight for endothelial-contacting therapeutic device design.

Preservation of Fluorescence Signal and Imaging Optimization for Integrated Light and Electron Microscopy.

Life science research often needs to define where molecules are located within the complex environment of a cell or tissue. Genetically encoded fluorescent proteins and or fluorescence affinity-labeling are the go-to methods. Although recent fluorescent microscopy methods can provide localization of fluorescent molecules with relatively high resolution, an ultrastructural context is missing. This is solved by imaging a region of interest with correlative light and electron microscopy (CLEM). We have adopted a protocol that preserves both genetically-encoded and antibody-derived fluorescent signals in resin-embedded cell and tissue samples and provides high-resolution electron microscopy imaging of the same thin section. This method is particularly suitable for dedicated CLEM instruments that combine fluorescence and electron microscopy optics. In addition, we optimized scanning EM imaging parameters for samples of varying thicknesses. These protocols will enable rapid acquisition of CLEM information from samples and can be adapted for three-dimensional EM.

Electron microscopy for imaging organelles in plants and algae.

Recent developments in both instrumentation and image analysis algorithms have allowed three-dimensional electron microscopy (3D-EM) to increase automated image collections through large tissue volumes using serial block-face scanning EM (SEM) and to achieve near-atomic resolution of macromolecular complexes using cryo-electron tomography (cryo-ET) and sub-tomogram averaging. In this review, we discuss applications of cryo-ET to cell biology research on plant and algal systems and the special opportunities they offer for understanding the organization of eukaryotic organelles with unprecedently resolution. However, one of the most challenging aspects for cryo-ET is sample preparation, especially for multicellular organisms. We also discuss correlative light and electron microscopy (CLEM) approaches that have been developed for ET at both room and cryogenic temperatures.

Preparation of Samples for Large-Scale Automated Electron Microscopy of Tissue and Cell Ultrastructure.

Manual selection of targets in experimental or diagnostic samples by transmission electron microscopy (TEM), based on single overview and detail micrographs, has been time-consuming and susceptible to bias. Substantial information and throughput gain may now be achieved by the automated acquisition of virtually all structures in a given EM section. Resulting datasets allow the convenient pan-and-zoom examination of tissue ultrastructure with preserved microanatomical orientation. The technique is, however, critically sensitive to artifacts in sample preparation. We, therefore, established a methodology to prepare large-scale digitization samples (LDS) designed to acquire entire sections free of obscuring flaws. For evaluation, we highlight the supreme performance of scanning EM in transmission mode compared with other EM technology. The use of LDS will substantially facilitate access to EM data for a broad range of applications.

Correlative Lightsheet and Electron Microscopy Applied to a Zebrafish Model of Fetal Bone Marrow: Resolving the Ultrastructure of a Single Hematopoietic Stem Cell in the Niche

Hematopoietic stem and progenitor cells (HSPC) emerge from the hemogenic endothelium of the dorsal aorta. Next, HSPC migrate to the fetal liver where they expand exponentially before migrating again to the fetal bone marrow. This early stage of bone marrow colonization is poorly understood. Current imaging technology does not allow direct visualization of the fetal bone marrow niche. Zebrafish provide a model to observe these events because they develop externally, are transparent, and ontogeny of the hematopoietic system is highly conserved. The adult marrow niche in zebrafish is in the kidney and is colonized 4 to 5 days post fertilization. To track HSPC during this dynamic process we used our previously characterized HSPC-specific transgenic reporter lines (Runx:GFP and Runx:mCherry). We demonstrated by transplantation these cells mark a highly pure population of long-term engrafting hematopoietic stem cells. To image the kidney marrow live we applied lightsheet microscopy. This technology uses three objective lenses: two that illuminate the sample in the XY plane, and one that takes images in the Z plane. This allows hundreds of optical sections to be captured in seconds. The temporal and spatial resolution of this technique produces a highly dynamic view of the entire developing marrow niche. By imaging Runx+ HSPC together with flk+ endothelial cells in a double transgenic embryo, we could track interactions between HSPC and the perivascular niche. Previously, we showed that HSPC arrival in the caudal hematopoietic tissue, the zebrafish equivalent of the fetal liver, triggers remodeling of endothelial cells into a surrounding pocket. In this study, we found a similar cellular structure forms in the kidney marrow niche. We wanted to resolve the ultrastructure of the HSPC and surrounding cells in the context of the larger developing kidney organ. To do this we applied Correlative Light and Electron Microscopy (CLEM) to match fluorescent cells tracked by lightsheet in the same embryo fixed and stained for EM. Micro-CT was used to orient and trim the embedded sample to isolate the kidney niche. Serial block-face scanning EM was used to capture high resolution serial sections (volume dimensions: x, y, z (um) = 236, 114, 105; pixel size: x, y, z (nm) = 9.5, 9.5, 70). Blood vessels observed in light and EM 3D data sets provided the anatomical markers needed to match the position of cells found using both techniques. As we previously found in the caudal hematopoietic tissue, a single round HSPC with a large nucleus, little cytoplasm, and a ruffled membrane, was lodged just under the vessel wall. This single HSPC was in a distinct anatomical location compared to larger clusters of HSPC in an abluminal space. This observation is suggestive of a marrow with distinct anatomical niche sites that may support different functions, such as proliferation versus quiescence. This unique system for analysis of the ultrastructure of an endogenous HSPC in an unperturbed niche will allow further functional and mechanistic studies at the subcellular level. Our goal is to gain novel insights into intercellular interactions between HSPC and the surrounding supportive cells in the perivascular niche. DisclosuresNo relevant conflicts of interest to declare.

High-precision targeting workflow for volume electron microscopy.

Cells are 3D objects. Therefore, volume EM (vEM) is often crucial for correct interpretation of ultrastructural data. Today, scanning EM (SEM) methods such as focused ion beam (FIB)-SEM are frequently used for vEM analyses. While they allow automated data acquisition, precise targeting of volumes of interest within a large sample remains challenging. Here, we provide a workflow to target FIB-SEM acquisition of fluorescently labeled cells or subcellular structures with micrometer precision. The strategy relies on fluorescence preservation during sample preparation and targeted trimming guided by confocal maps of the fluorescence signal in the resin block. Laser branding is used to create landmarks on the block surface to position the FIB-SEM acquisition. Using this method, we acquired volumes of specific single cells within large tissues such as 3D cultures of mouse mammary gland organoids, tracheal terminal cells in Drosophila melanogaster larvae, and ovarian follicular cells in adult Drosophila, discovering ultrastructural details that could not be appreciated before.

TEM, SEM, and STEM-based immuno-CLEM workflows offer complementary advantages

Identifying endogenous tissue stem cells remains a key challenge in developmental and regenerative biology. To distinguish and molecularly characterise stem cell populations in large heterogeneous tissues, the combination of cytochemical cell markers with ultrastructural morphology is highly beneficial. Here, we realise this through workflows of multi-resolution immuno-correlative light and electron microscopy (iCLEM) methodologies. Taking advantage of the antigenicity preservation of the Tokuyasu technique, we have established robust protocols and workflows and provide a side-by-side comparison of iCLEM used in combination with scanning EM (SEM), scanning TEM (STEM), or transmission EM (TEM). Evaluation of the applications and advantages of each method highlights their practicality for the identification, quantification, and characterization of heterogeneous cell populations in small organisms, organs, or tissues in healthy and diseased states. The iCLEM techniques are broadly applicable and can use either genetically encoded or cytochemical markers on plant, animal and human tissues. We demonstrate how these protocols are particularly suited for investigating neural stem and progenitor cell populations of the vertebrate nervous system.

Development of novel 2D and 3D correlative microscopy to characterise the composition and multiscale structure of suspended sediment aggregates

Suspended cohesive sediments form aggregates or ‘flocs’ and are often closely associated with carbon, nutrients, pathogens and pollutants, which makes understanding their composition, transport and fate highly desirable. Accurate prediction of floc behaviour requires the quantification of 3-dimensional (3D) properties (size, shape and internal structure) that span several scales (i.e. nanometre [nm] to millimetre [mm]-scale). Traditional techniques (optical cameras and electron microscopy [EM]), however, can only provide 2-dimensional (2D) simplifications of 3D floc geometries. Additionally, the existence of a resolution gap between conventional optical microscopy (COM) and transmission EM (TEM) prevents an understanding of how floc nm-scale constituents and internal structure influence mm-scale floc properties. Here, we develop a novel correlative imaging workflow combining 3D X-ray micro-computed tomography (μCT), 3D focused ion beam nanotomography (FIB-nt) and 2D scanning EM (SEM) and TEM (STEM) which allows us to stabilise, visualise and quantify the composition and multi-scale structure of sediment flocs for the first time. This new technique allowed the quantification of 3D floc geometries, the identification of individual floc components (e.g., clays, non-clay minerals and bacteria), and characterisation of particle-particle and structural associations across scales. This novel dataset demonstrates the truly complex structure of natural flocs at multiple scales. The integration of multi scale, state-of-the-art instrumentation/techniques offers the potential to generate fundamental new understanding of floc composition, structure and behaviour.

The phosphodiesterase-4 inhibitor roflumilast impacts Schistosoma mansoni ovipositing in vitro but displays only modest antischistosomal activity in vivo

Praziquantel (PZQ) is the sole drug used to treat schistosomiasis, and the probability of developing resistance is growing the longer it is relied upon, justifying the search for alternatives. Phosphodiesterases (PDEs), particularly the PDE4 family, have attracted considerable attention as drug targets, including in Schistosoma mansoni, and especially SmPDE4A. This study investigates the potential antischistosomal activity of human PDE4 and potent SmPDE4A inhibitor roflumilast, either alone or combined with PZQ. In vitro, roflumilast resulted in a significant, concentration-dependent reduction in egg production but not of worm viability. In vitro exposure to roflumilast in combination with a low concentration of PZQ was less effective than PZQ alone, pointing to antagonism. S. mansoni-infected mice treated with roflumilast showed significant reductions in worm burden (27%) as well as hepatic and intestinal egg burdens (~28%) two weeks post treatment. Scanning EM of worms isolated from roflumilast-treated and untreated mice did not reveal noticeable changes to their tegument. S. mansoni-infected mice treated with a fixed dosage of roflumilast and a variable dosage of PZQ resulted in a higher reduction in worm burden, reduced hepatic egg counts, absence of immature eggs and a marked increase in dead eggs, compared to PZQ alone. However, the combination resulted in increased animal mortality, probably attributable to pharmacodynamic interactions between the two drugs. Although this study marks the first report of in vivo antischistosomal potential by a PDE inhibitor, an important proof of concept, we conclude that the antischistosomal effects of roflumilast are insufficient to warrant further development.

Cytomatrix proteins CAST and ELKS regulate retinal photoreceptor development and maintenance.

At the presynaptic active zone (AZ), the related cytomatrix proteins CAST and ELKS organize the presynaptic release machinery. While CAST is known to regulate AZ size and neurotransmitter release, the role of ELKS and the integral system of CAST/ELKS together is poorly understood. Here, we show that CAST and ELKS have both redundant and unique roles in coordinating synaptic development, function, and maintenance of retinal photoreceptor ribbon synapses. A CAST/ELKS double knockout (dKO) mouse showed high levels of ectopic synapses and reduced responses to visual stimulation. Ectopic formation was not observed in ELKS conditional KO but progressively increased with age in CAST KO mice with higher rates in the dKO. Presynaptic calcium influx was strongly reduced in rod photoreceptors of CAST KO and dKO mice. Three-dimensional scanning EM reconstructions showed structural abnormalities in rod triads of CAST KO and dKO. Remarkably, AAV-mediated acute ELKS deletion after synapse maturation induced neurodegeneration and loss of ribbon synapses. These results suggest that CAST and ELKS work in concert to promote retinal synapse formation, transmission, and maintenance.

Characterizing the adult Hematopoietic Stem cell (HSC) niche in a zebrafish model for fetal bone marrow

Hematopoietic stem and progenitor cells (HSPCs) originate from the dorsal aorta and migrate to the intermediate niche, the fetal liver, where they undergo rapid expansion before migrating to the prospective adult niche, the bone marrow. This early stage of bone marrow colonization is poorly understood. Current imaging technology does not allow direct visualization of the fetal bone marrow niche. Zebrafish embryos can be used to directly visualize HSPC‐niche cell interactions unperturbed because they develop externally and are transparent. Importantly, their HSC ontogeny is highly similar to mammals. The zebrafish kidney is equivalent to the adult bone marrow niche and gets colonized from 4 to 5 days post fertilization (dpf). To track HSPCs during this dynamic process, we used our previously characterized HSPC‐specific transgenic reporter lines (Runx:GFP and Runx:mCherry). We demonstrated by transplantation that these cells mark a highly pure population of long‐term engrafting hematopoietic stem cells. Using a tissue clearing technique that allows visualization of deeper tissues, we detected ~100 HSPCs in the kidney marrow in 5 dpf embryos. These cell numbers could be manipulated through drug treatments and gene knockdowns. For live‐imaging of the kidney marrow, we applied lightsheet microscopy that allows rapid image acquisition with high spatio‐temporal resolution, providing a highly dynamic view of the entire developing niche. By imaging Runx+ HSPC together with flk+ endothelial cells in a double transgenic embryo, we could track interactions between HSPC and the perivascular niche. Previously, we showed that HSPC arrival in the caudal hematopoietic tissue, the zebrafish equivalent of the fetal liver, triggers remodeling of endothelial cells into a surrounding pocket. In this study, we found that a similar cellular structure forms within the kidney marrow niche. To resolve the ultrastructure of the HSPC and its surrounding cells in context of the entire niche, we performed Correlative Light and Electron Microscopy. For this, the embryos imaged on lightsheet were fixed and stained for EM. MicroCT was used to orient and trim the embedded sample to isolate the kidney niche and serial blockface scanning EM to capture high resolution serial sections. By matching anatomical markers such as blood vessels observed in light and EM 3D data sets, a single round HSPC with a large nucleus, little cytoplasm, and a ruffled membrane, was identified lodged just under the vessel wall. This single HSPC was in a distinct anatomical location compared to larger clusters of HSPC in an abluminal space. This is suggestive of distinct anatomical niche sites within the marrow that may support different functions, such as proliferation versus quiescence. Our goal is to gain novel insights into intercellular interactions between HSPC and the surrounding supportive cells in the perivascular niche.Support or Funding InformationASH, NIDDKThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Actinoplanes missouriensiss is a rare actinomycete that forms terminal sporangia containing a few hundred flagellated, motile spores (shown here in a scanning EM image). The TcrA response regulator controls sporangium formation and flagellar biogenesis through the activation of dozens of genes, including three encoding FliA‐family sigma factors. For details, see the article by Mouri et al. on pp. 718–733 of

<i>Actinoplanes missouriensiss</i> is a rare actinomycete that forms terminal sporangia containing a few hundred flagellated, motile spores (shown here in a scanning EM image). The TcrA response regulator controls sporangium formation and flagellar biogenesis through the activation of dozens of genes, including three encoding FliA‐family sigma factors. For details, see the article by Mouri <i>et al</i>. on pp. 718–733 of

Fluorescence Approaches to Image and Quantify the Demarcation Membrane System in Living Megakaryocytes.

The demarcation membrane system (DMS) develops to provide additional surface membrane for the process of platelet production. The DMS is an invagination of the plasma membrane that can extend throughout the extranuclear volume of mature megakaryocytes and its lumen is continuous with the extracellular solution. DMS ultrastructure in fixed samples has been extensively studied using transmission electron microscopy (TEM) and more recently with focused ion beam scanning EM. In addition, whole cell patch clamp membrane capacitance provides a direct measurement of DMS content in living megakaryocytes. However, fluorescence methods to image and quantify the DMS in living megakaryocytes provide several advantages. For example, confocal fluorescence microscopy is easier to use compared to EM or electrophysiological methods and the required equipment is more readily available. In addition, use of living cells avoids artifacts known to occur during the fixation, dehydration, or embedding steps used to prepare EM samples. Here we describe the use of styryl dyes such as FM 1-43 or di-8-ANEPPS and impermeant fluorescent indicators of the extracellular space as simple approaches for imaging and quantification of the DMS.

REEP6 deficiency leads to retinal degeneration through disruption of ER homeostasis and protein trafficking.

Retinitis pigmentosa (RP) is the most common form of inherited retinal dystrophy. We recently identified mutations in REEP6, which encodes the receptor expression enhancing protein 6, in several families with autosomal recessive RP. REEP6 is related to the REEP and Yop1p family of ER shaping proteins and potential receptor accessory proteins, but the role of REEP6 in the retina is unknown. Here we characterize the disease mechanisms associated with loss of REEP6 function using a Reep6 knockout mouse generated by CRISPR/Cas9 gene editing. In control mice REEP6 was localized to the inner segment and outer plexiform layer of rod photoreceptors. The Reep6-/- mice exhibited progressive photoreceptor degeneration from P20 onwards. Ultrastructural analyses at P20 by transmission electron microscopy and 3View serial block face scanning EM revealed an expansion of the distal ER in the Reep6-/- rods and an increase in their number of mitochondria. Electroretinograms revealed photoreceptor dysfunction preceded degeneration, suggesting potential defects in phototransduction. There was no effect on the traffic of rhodopsin, Rom1 or peripherin/rds; however, the retinal guanylate cyclases GC1 and GC2 were severely affected in the Reep6 knockout animals, with almost undetectable expression. These changes correlated with an increase in C/EBP homologous protein (CHOP) expression and the activation of caspase 12, suggesting that ER stress contributes to cell death. Collectively, these data suggest that REEP6 plays an essential role in maintaining cGMP homeostasis though facilitating the stability and/or trafficking of guanylate cyclases and maintaining ER and mitochondrial homeostasis.

The effects of hydrogen sulfide on platelet–leukocyte aggregation and microvascular thrombolysis

The volatile transmitter hydrogen sulfide (H2S) is known for its various functions in vascular biology. This study evaluates the effect of the H2S-donor GYY4137 (GYY) on thrombus stability and microvascular thrombolysis. Human whole blood served for all in vitro studies and was analyzed in a resting state, after stimulation with thrombin-receptor activating peptide (TRAP) and after incubation with 10 or 30 mM GYY or its vehicle DMSO following TRAP-activation, respectively. As a marker for thrombus stability, platelet–leukocyte aggregation was assessed using flow cytometry after staining of human whole blood against CD62P and CD45, respectively. Furthermore, morphology and quantity of platelet–leukocyte aggregation were studied by means of scanning electron microscopy (scanning EM). Therefore, platelets were stained for CD62P followed by immuno gold labeling. In vivo, the dorsal skinfold chamber preparation was performed for light/dye induction of thrombi in arterioles and venules using intravital fluorescence microscopy. Thrombolysis was assessed 10 and 22 h after thrombus induction and treatment with the vehicle, GYY, or recombinant tissue plasminogen activator (rtPA).Flow cytometry revealed an increase of CD62P/CD45 positive aggregates after TRAP stimulation of human whole blood, which was significantly reduced by preincubation with 30 mM GYY. Scanning EM additionally showed a reduced platelet–leukocyte aggregation and a decreased leukocyte count within the aggregates after preincubation with GYY compared to TRAP stimulation alone. Further on, morphological signs of platelet activation were found markedly reduced upon treatment with GYY. In mice, both GYY and rtPA significantly accelerated arteriolar and venular thrombolysis compared to the vehicle control. In conclusion, GYY impairs thrombus stability by reducing platelet–leukocyte aggregation and thereby facilitates endogenous thrombolysis.

The Spectrum of Mitochondrial Ultrastructural Defects in Mitochondrial Myopathy.

Mitochondrial functions are intrinsically linked to their morphology and membrane ultrastructure. Characterizing abnormal mitochondrial structural features may thus provide insight into the underlying pathogenesis of inherited and acquired mitochondrial diseases. Following a systematic literature review on ultrastructural defects in mitochondrial myopathy, we investigated skeletal muscle biopsies from seven subjects with genetically defined mtDNA mutations. Mitochondrial ultrastructure and morphology were characterized using two complimentary approaches: transmission electron microscopy (TEM) and serial block face scanning EM (SBF-SEM) with 3D reconstruction. Six ultrastructural abnormalities were identified including i) paracrystalline inclusions, ii) linearization of cristae and abnormal angular features, iii) concentric layering of cristae membranes, iv) matrix compartmentalization, v) nanotunelling, and vi) donut-shaped mitochondria. In light of recent molecular advances in mitochondrial biology, these findings reveal novel aspects of mitochondrial ultrastructure and morphology in human tissues with implications for understanding the mechanisms linking mitochondrial dysfunction to disease.