Myosin VIIA, important for human auditory function, is necessary for Drosophila auditory organ development.

BACKGROUND: Myosin VIIA (MyoVIIA) is an unconventional myosin necessary for vertebrate audition [1]-[5]. Human auditory transduction occurs in sensory hair cells with a staircase-like arrangement of apical protrusions called stereocilia. In these hair cells, MyoVIIA maintains stereocilia organization [6]. Severe mutations in the Drosophila MyoVIIA orthologue, crinkled (ck), are semi-lethal [7] and lead to deafness by disrupting antennal auditory organ (Johnston's Organ, JO) organization [8]. ck/MyoVIIA mutations result in apical detachment of auditory transduction units (scolopidia) from the cuticle that transmits antennal vibrations as mechanical stimuli to JO. PRINCIPAL FINDINGS: Using flies expressing GFP-tagged NompA, a protein required for auditory organ organization in Drosophila, we examined the role of ck/MyoVIIA in JO development and maintenance through confocal microscopy and extracellular electrophysiology. Here we show that ck/MyoVIIA is necessary early in the developing antenna for initial apical attachment of the scolopidia to the articulating joint. ck/MyoVIIA is also necessary to maintain scolopidial attachment throughout adulthood. Moreover, in the adult JO, ck/MyoVIIA genetically interacts with the non-muscle myosin II (through its regulatory light chain protein and the myosin binding subunit of myosin II phosphatase). Such genetic interactions have not previously been observed in scolopidia. These factors are therefore candidates for modulating MyoVIIA activity in vertebrates. CONCLUSIONS: Our findings indicate that MyoVIIA plays evolutionarily conserved roles in auditory organ development and maintenance in invertebrates and vertebrates, enhancing our understanding of auditory organ development and function, as well as providing significant clues for future research.

Activation and targeting of extracellular signal-regulated kinases by beta-arrestin scaffolds.

Using both confocal immunofluorescence microscopy and biochemical approaches, we have examined the role of beta-arrestins in the activation and targeting of extracellular signal-regulated kinase 2 (ERK2) following stimulation of angiotensin II type 1a receptors (AT1aR). In HEK-293 cells expressing hemagglutinin-tagged AT1aR, angiotensin stimulation triggered beta-arrestin-2 binding to the receptor and internalization of AT1aR-beta-arrestin complexes. Using red fluorescent protein-tagged ERK2 to track the subcellular distribution of ERK2, we found that angiotensin treatment caused the redistribution of activated ERK2 into endosomal vesicles that also contained AT1aR-beta-arrestin complexes. This targeting of ERK2 reflects the formation of multiprotein complexes containing AT1aR, beta-arrestin-2, and the component kinases of the ERK cascade, cRaf-1, MEK1, and ERK2. Myc-tagged cRaf-1, MEK1, and green fluorescent protein-tagged ERK2 coprecipitated with Flag-tagged beta-arrestin-2 from transfected COS-7 cells. Coprecipitation of cRaf-1 with beta-arrestin-2 was independent of MEK1 and ERK2, whereas the coprecipitation of MEK1 and ERK2 with beta-arrestin-2 was significantly enhanced in the presence of overexpressed cRaf-1, suggesting that binding of cRaf-1 to beta-arrestin facilitates the assembly of a cRaf-1, MEK1, ERK2 complex. The phosphorylation of ERK2 in beta-arrestin complexes was markedly enhanced by coexpression of cRaf-1, and this effect is blocked by expression of a catalytically inactive dominant inhibitory mutant of MEK1. Stimulation with angiotensin increased the binding of both cRaf-1 and ERK2 to beta-arrestin-2, and the association of beta-arrestin-2, cRaf-1, and ERK2 with AT1aR. These data suggest that beta-arrestins function both as scaffolds to enhance cRaf-1 and MEK-dependent activation of ERK2, and as targeting proteins that direct activated ERK to specific subcellular locations.

Astrocytes refine cortical connectivity at dendritic spines.

During cortical synaptic development, thalamic axons must establish synaptic connections despite the presence of the more abundant intracortical projections. How thalamocortical synapses are formed and maintained in this competitive environment is unknown. Here, we show that astrocyte-secreted protein hevin is required for normal thalamocortical synaptic connectivity in the mouse cortex. Absence of hevin results in a profound, long-lasting reduction in thalamocortical synapses accompanied by a transient increase in intracortical excitatory connections. Three-dimensional reconstructions of cortical neurons from serial section electron microscopy (ssEM) revealed that, during early postnatal development, dendritic spines often receive multiple excitatory inputs. Immuno-EM and confocal analyses revealed that majority of the spines with multiple excitatory contacts (SMECs) receive simultaneous thalamic and cortical inputs. Proportion of SMECs diminishes as the brain develops, but SMECs remain abundant in Hevin-null mice. These findings reveal that, through secretion of hevin, astrocytes control an important developmental synaptic refinement process at dendritic spines.

Autophagy enhances NFκB activity in specific tissue macrophages by sequestering A20 to boost antifungal immunity.

Immune responses must be well restrained in a steady state to avoid excessive inflammation. However, such restraints are quickly removed to exert antimicrobial responses. Here we report a role of autophagy in an early host antifungal response by enhancing NFκB activity through A20 sequestration. Enhancement of NFκB activation is achieved by autophagic depletion of A20, an NFκB inhibitor, in F4/80(hi) macrophages in the spleen, peritoneum and kidney. We show that p62, an autophagic adaptor protein, captures A20 to sequester it in the autophagosome. This allows the macrophages to release chemokines to recruit neutrophils. Indeed, mice lacking autophagy in myeloid cells show higher susceptibility to Candida albicans infection due to impairment in neutrophil recruitment. Thus, at least in the specific aforementioned tissues, autophagy appears to break A20-dependent suppression in F4/80(hi) macrophages, which express abundant A20 and contribute to the initiation of efficient innate immune responses.

An enteroendocrine cell-enteric glia connection revealed by 3D electron microscopy.

The enteroendocrine cell is the cornerstone of gastrointestinal chemosensation. In the intestine and colon, this cell is stimulated by nutrients, tastants that elicit the perception of flavor, and bacterial by-products; and in response, the cell secretes hormones like cholecystokinin and peptide YY--both potent regulators of appetite. The development of transgenic mice with enteroendocrine cells expressing green fluorescent protein has allowed for the elucidation of the apical nutrient sensing mechanisms of the cell. However, the basal secretory aspects of the enteroendocrine cell remain largely unexplored, particularly because a complete account of the enteroendocrine cell ultrastructure does not exist. Today, the fine ultrastructure of a specific cell can be revealed in the third dimension thanks to the invention of serial block face scanning electron microscopy (SBEM). Here, we bridged confocal microscopy with SBEM to identify the enteroendocrine cell of the mouse and study its ultrastructure in the third dimension. The results demonstrated that 73.5% of the peptide-secreting vesicles in the enteroendocrine cell are contained within an axon-like basal process. We called this process a neuropod. This neuropod contains neurofilaments, which are typical structural proteins of axons. Surprisingly, the SBEM data also demonstrated that the enteroendocrine cell neuropod is escorted by enteric glia--the cells that nurture enteric neurons. We extended these structural findings into an in vitro intestinal organoid system, in which the addition of glial derived neurotrophic factors enhanced the development of neuropods in enteroendocrine cells. These findings open a new avenue of exploration in gastrointestinal chemosensation by unveiling an unforeseen physical relationship between enteric glia and enteroendocrine cells.

Characterization of basal pseudopod-like processes in ileal and colonic PYY cells.

The peptide tyrosine tyrosine (PYY) is produced and secreted from L cells of the gastrointestinal mucosa. To study the anatomy and function of PYY-secreting L cells, we developed a transgenic PYY-green fluorescent protein mouse model. PYY-containing cells exhibited green fluorescence under UV light and were immunoreactive to antibodies against PYY and GLP-1 (glucagon-like peptide-1, an incretin hormone also secreted by L cells). PYY-GFP cells from 15 μm thick sections were imaged using confocal laser scanning microscopy and three-dimensionally (3D) reconstructed. Results revealed unique details of the anatomical differences between ileal and colonic PYY-GFP cells. In ileal villi, the apical portion of PYY cells makes minimal contact with the lumen of the gut. Long pseudopod-like basal processes extend from these cells and form an interface between the mucosal epithelium and the lamina propria. Some basal processes are up to 50 μm in length. Multiple processes can be seen protruding from one cell and these often have a terminus resembling a synapse that appears to interact with neighboring cells. In colonic crypts, PYY-GFP cells adopt a spindle-like shape and weave in between epithelial cells, while maintaining contact with the lumen and lamina propria. In both tissues, cytoplasmic granules containing the hormones PYY and GLP-1 are confined to the base of the cell, often filling the basal process. The anatomical arrangement of these structures suggests a dual function as a dock for receptors to survey absorbed nutrients and as a launching platform for hormone secretion in a paracrine fashion.

Anatomical identification of extracellularly recorded cells in large-scale multielectrode recordings.

This study combines for the first time two major approaches to understanding the function and structure of neural circuits: large-scale multielectrode recordings, and confocal imaging of labeled neurons. To achieve this end, we develop a novel approach to the central problem of anatomically identifying recorded cells, based on the electrical image: the spatiotemporal pattern of voltage deflections induced by spikes on a large-scale, high-density multielectrode array. Recordings were performed from identified ganglion cell types in the macaque retina. Anatomical images of cells in the same preparation were obtained using virally transfected fluorescent labeling or by immunolabeling after fixation. The electrical image was then used to locate recorded cell somas, axon initial segments, and axon trajectories, and these signatures were used to identify recorded cells. Comparison of anatomical and physiological measurements permitted visualization and physiological characterization of numerically dominant ganglion cell types with high efficiency in a single preparation.

Atherosclerosis: Viewing the problem from a different perspective including possible treatment options

This paper proposes that atherosclerosis is initiated by a signaling event that deposits calcium hydroxyapatite (Ca-HAP). This event is preceded by a loss of mechanical structure in the arterial wall. After Ca-HAP has been deposited, it is unlikely that it will be reabsorbed because the solubility product constant (K sp) is very small, and the large stores of Ca +2 and PO 4-3 in the bones oppose any attempts to dissolve Ca-HAP by decreasing the common ions. The hydroxide ion (OH -) of Ca-HAP can be displaced in nature by fluoride (F -) and carbonate (CO 3-2) ions, and it is proposed that anions associated with cholesterol ester hydrolysis and, in very small quantities, the enolate of 7-ketocholesterol could also displace the OH -of Ca-HAP, forming an ionic bond. The free energy of hydration of Ca-HAP at 310 K is most likely negative, and the ionic radii of the anions associated with the hydrolysis of cholesterol ester are compatible with the substitution. Furthermore, examination of the pathology of atherosclerotic lesions by Raman and NMR spectroscopy and confocal microscopy supports deposition of Ca-HAP associated with cholesterol. Investigating the affinity of intermediates of cholesterol hydrolysis for Ca-HAP compared to lipoproteins such as HDL, LDL, and VLDL using isothermic titration calorimetry could add proof of this concept and may lead to the development of a new class of medications targeted at the deposition of cholesterol within Ca-HAP. Treatment of acute ischemic events as a consequence of atherosclerosis with denitrogenation and oxygenation is discussed. © the author(s), publisher and licensee Libertas Academica Ltd.

Micro-Anatomical Quantitative Imaging Towards Enabling Automated Diagnosis of Thick Tissues at the Point of Care

Histopathology is the clinical standard for tissue diagnosis. However, histopathology has several limitations including that it requires tissue processing, which can take 30 minutes or more, and requires a highly trained pathologist to diagnose the tissue. Additionally, the diagnosis is qualitative, and the lack of quantitation leads to possible observer-specific diagnosis. Taken together, it is difficult to diagnose tissue at the point of care using histopathology.

Several clinical situations could benefit from more rapid and automated histological processing, which could reduce the time and the number of steps required between obtaining a fresh tissue specimen and rendering a diagnosis. For example, there is need for rapid detection of residual cancer on the surface of tumor resection specimens during excisional surgeries, which is known as intraoperative tumor margin assessment. Additionally, rapid assessment of biopsy specimens at the point-of-care could enable clinicians to confirm that a suspicious lesion is successfully sampled, thus preventing an unnecessary repeat biopsy procedure. Rapid and low cost histological processing could also be potentially useful in settings lacking the human resources and equipment necessary to perform standard histologic assessment. Lastly, automated interpretation of tissue samples could potentially reduce inter-observer error, particularly in the diagnosis of borderline lesions.

To address these needs, high quality microscopic images of the tissue must be obtained in rapid timeframes, in order for a pathologic assessment to be useful for guiding the intervention. Optical microscopy is a powerful technique to obtain high-resolution images of tissue morphology in real-time at the point of care, without the need for tissue processing. In particular, a number of groups have combined fluorescence microscopy with vital fluorescent stains to visualize micro-anatomical features of thick (i.e. unsectioned or unprocessed) tissue. However, robust methods for segmentation and quantitative analysis of heterogeneous images are essential to enable automated diagnosis. Thus, the goal of this work was to obtain high resolution imaging of tissue morphology through employing fluorescence microscopy and vital fluorescent stains and to develop a quantitative strategy to segment and quantify tissue features in heterogeneous images, such as nuclei and the surrounding stroma, which will enable automated diagnosis of thick tissues.

To achieve these goals, three specific aims were proposed. The first aim was to develop an image processing method that can differentiate nuclei from background tissue heterogeneity and enable automated diagnosis of thick tissue at the point of care. A computational technique called sparse component analysis (SCA) was adapted to isolate features of interest, such as nuclei, from the background. SCA has been used previously in the image processing community for image compression, enhancement, and restoration, but has never been applied to separate distinct tissue types in a heterogeneous image. In combination with a high resolution fluorescence microendoscope (HRME) and a contrast agent acriflavine, the utility of this technique was demonstrated through imaging preclinical sarcoma tumor margins. Acriflavine localizes to the nuclei of cells where it reversibly associates with RNA and DNA. Additionally, acriflavine shows some affinity for collagen and muscle. SCA was adapted to isolate acriflavine positive features or APFs (which correspond to RNA and DNA) from background tissue heterogeneity. The circle transform (CT) was applied to the SCA output to quantify the size and density of overlapping APFs. The sensitivity of the SCA+CT approach to variations in APF size, density and background heterogeneity was demonstrated through simulations. Specifically, SCA+CT achieved the lowest errors for higher contrast ratios and larger APF sizes. When applied to tissue images of excised sarcoma margins, SCA+CT correctly isolated APFs and showed consistently increased density in tumor and tumor + muscle images compared to images containing muscle. Next, variables were quantified from images of resected primary sarcomas and used to optimize a multivariate model. The sensitivity and specificity for differentiating positive from negative ex vivo resected tumor margins was 82% and 75%. The utility of this approach was further tested by imaging the in vivo tumor cavities from 34 mice after resection of a sarcoma with local recurrence as a bench mark. When applied prospectively to images from the tumor cavity, the sensitivity and specificity for differentiating local recurrence was 78% and 82%. The results indicate that SCA+CT can accurately delineate APFs in heterogeneous tissue, which is essential to enable automated and rapid surveillance of tissue pathology.

Two primary challenges were identified in the work in aim 1. First, while SCA can be used to isolate features, such as APFs, from heterogeneous images, its performance is limited by the contrast between APFs and the background. Second, while it is feasible to create mosaics by scanning a sarcoma tumor bed in a mouse, which is on the order of 3-7 mm in any one dimension, it is not feasible to evaluate an entire human surgical margin. Thus, improvements to the microscopic imaging system were made to (1) improve image contrast through rejecting out-of-focus background fluorescence and to (2) increase the field of view (FOV) while maintaining the sub-cellular resolution needed for delineation of nuclei. To address these challenges, a technique called structured illumination microscopy (SIM) was employed in which the entire FOV is illuminated with a defined spatial pattern rather than scanning a focal spot, such as in confocal microscopy.

Thus, the second aim was to improve image contrast and increase the FOV through employing wide-field, non-contact structured illumination microscopy and optimize the segmentation algorithm for new imaging modality. Both image contrast and FOV were increased through the development of a wide-field fluorescence SIM system. Clear improvement in image contrast was seen in structured illumination images compared to uniform illumination images. Additionally, the FOV is over 13X larger than the fluorescence microendoscope used in aim 1. Initial segmentation results of SIM images revealed that SCA is unable to segment large numbers of APFs in the tumor images. Because the FOV of the SIM system is over 13X larger than the FOV of the fluorescence microendoscope, dense collections of APFs commonly seen in tumor images could no longer be sparsely represented, and the fundamental sparsity assumption associated with SCA was no longer met. Thus, an algorithm called maximally stable extremal regions (MSER) was investigated as an alternative approach for APF segmentation in SIM images. MSER was able to accurately segment large numbers of APFs in SIM images of tumor tissue. In addition to optimizing MSER for SIM image segmentation, an optimal frequency of the illumination pattern used in SIM was carefully selected because the image signal to noise ratio (SNR) is dependent on the grid frequency. A grid frequency of 31.7 mm-1 led to the highest SNR and lowest percent error associated with MSER segmentation.

Once MSER was optimized for SIM image segmentation and the optimal grid frequency was selected, a quantitative model was developed to diagnose mouse sarcoma tumor margins that were imaged ex vivo with SIM. Tumor margins were stained with acridine orange (AO) in aim 2 because AO was found to stain the sarcoma tissue more brightly than acriflavine. Both acriflavine and AO are intravital dyes, which have been shown to stain nuclei, skeletal muscle, and collagenous stroma. A tissue-type classification model was developed to differentiate localized regions (75x75 µm) of tumor from skeletal muscle and adipose tissue based on the MSER segmentation output. Specifically, a logistic regression model was used to classify each localized region. The logistic regression model yielded an output in terms of probability (0-100%) that tumor was located within each 75x75 µm region. The model performance was tested using a receiver operator characteristic (ROC) curve analysis that revealed 77% sensitivity and 81% specificity. For margin classification, the whole margin image was divided into localized regions and this tissue-type classification model was applied. In a subset of 6 margins (3 negative, 3 positive), it was shown that with a tumor probability threshold of 50%, 8% of all regions from negative margins exceeded this threshold, while over 17% of all regions exceeded the threshold in the positive margins. Thus, 8% of regions in negative margins were considered false positives. These false positive regions are likely due to the high density of APFs present in normal tissues, which clearly demonstrates a challenge in implementing this automatic algorithm based on AO staining alone.

Thus, the third aim was to improve the specificity of the diagnostic model through leveraging other sources of contrast. Modifications were made to the SIM system to enable fluorescence imaging at a variety of wavelengths. Specifically, the SIM system was modified to enabling imaging of red fluorescent protein (RFP) expressing sarcomas, which were used to delineate the location of tumor cells within each image. Initial analysis of AO stained panels confirmed that there was room for improvement in tumor detection, particularly in regards to false positive regions that were negative for RFP. One approach for improving the specificity of the diagnostic model was to investigate using a fluorophore that was more specific to staining tumor. Specifically, tetracycline was selected because it appeared to specifically stain freshly excised tumor tissue in a matter of minutes, and was non-toxic and stable in solution. Results indicated that tetracycline staining has promise for increasing the specificity of tumor detection in SIM images of a preclinical sarcoma model and further investigation is warranted.

In conclusion, this work presents the development of a combination of tools that is capable of automated segmentation and quantification of micro-anatomical images of thick tissue. When compared to the fluorescence microendoscope, wide-field multispectral fluorescence SIM imaging provided improved image contrast, a larger FOV with comparable resolution, and the ability to image a variety of fluorophores. MSER was an appropriate and rapid approach to segment dense collections of APFs from wide-field SIM images. Variables that reflect the morphology of the tissue, such as the density, size, and shape of nuclei and nucleoli, can be used to automatically diagnose SIM images. The clinical utility of SIM imaging and MSER segmentation to detect microscopic residual disease has been demonstrated by imaging excised preclinical sarcoma margins. Ultimately, this work demonstrates that fluorescence imaging of tissue micro-anatomy combined with a specialized algorithm for delineation and quantification of features is a means for rapid, non-destructive and automated detection of microscopic disease, which could improve cancer management in a variety of clinical scenarios.