Immunogold Electron Microscopy Research Articles

An Inactivated Recombinant Rabies Virus Displaying the Zika Virus prM-E Induces Protective Immunity Against Both Pathogens

Background: The global spread of Zika virus (ZIKV), which caused a pandemic associated with microcephaly and neuropathology in newborns and adults, prompted pursuit of a safe and effective vaccine. Methods: Rabies virus (RABV)-based ZIKV vaccines encoding prM-E protein of ZIKV were constructed with signal peptide or transmembrane (TM) domain replaced by corresponding region of RABV G gene. The recombinant viruses were characterized by confocal microscopy analysis, western blotting, immunogold electron microscopy and one-step growth curves. The immunogenicity of recombinant viruses mixed with a complex adjuvant of ISA 201 VG and poly(I:C) was tested in Balb/c mice. Results: Three recombinant viruses expressing ZIKV prM-E were constructed (named ZI-D, ZI-E and ZI-F). The change of signal peptide was not conducive to protein expression. TM replacement promoted the increase of protein expression. After immunization, the anti-ZIKV IgG antibody lasted for at least 10 weeks. The titer of neutralizing antibodies (NAbs) at week 6 against ZIKV and RABV were all above protective titer. Moreover, ZI-E could stimulate the proliferation of spleen lymphocytes and promoted the secretion of cytokines. It also promoted the production of central memory T cell (TCM) among CD4+/CD8+ T cells and stimulates B cell activation and maturation. Interpretation: These results indicate that ZI-E all could induce ZIKV-specific humoral and cellular immune responses, which have the potential to be developed into promising vaccines for protection against both ZIKV and RABV infections. Funding Statement: This study was funded in part by the National Key Research and Development Program of China (grant numbers 2016YFD0500400) and the Fundamental Research Funds for the Central Universities, JLU. Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: The study was approved by the Animal Welfare and Ethics Committee of the Institute of Veterinary Medicine of the Military Academy of Sciences (Laboratory Animal Care and Use Committee Authorization permit number JSY-DW-2018-02).

Isolation and characterization of a γ-carbonic anhydrase localized in the mitochondria of Saccharina japonica

Saccharina japonica is an ecologically and economically important seaweed that is dominant in the rocky shores of cold-temperate regions, forms the major component of productive beds, and affects marine environments. S. japonica exhibits a high photosynthetic efficiency in natural seawater with low dissolved CO2 concentration, thus suggesting the presence of its carbon-concentrating mechanism (CCM). However, the genes, proteins, and pathways involved in the CCM of S. japonica have not been fully identified and characterized. Carbonic anhydrase (CA) is a crucial component of CCM in macroalgae. In this study, the cloning, characterization, and subcellular localization of a specific CA were described. Multisequence alignment and phylogenetic analysis indicated that this CA belonged to the gamma (Sjγ-CA) class. This enzyme has a full-length cDAN of 1370 bp, encodes a protein with 246 amino acids (aa; ca. 25.7 kDa), and contains the mitochondrial transit peptide of 16 aa and LbH_gama_CA_like domain of 159 aa that defined the γ-CA region. The Sjγ-CA was successfully expressed in E. coli BL21 and purified as an active recombinant CA. Immunogold electron microscopy and fluorescence localization illustrated that this enzyme is localized in the mitochondria, and its transcription level is up-regulated by low CO2 concentration. These findings showed that Sjγ-CA is a possible component of the CCM in S. japonica. This work is the first to report about the mtCA of macroalgae and provides a basis for further analysis on seaweed CCM.

A Novel Major Pilin Subunit Protein FimM Is Involved in Adhesion of Bifidobacterium longum BBMN68 to Intestinal Epithelial Cells.

Adhesion to the gastrointestinal tract is considered to be important for bifidobacteria to colonize the human gut and exert their probiotic effects. Some cell surface proteins of bifidobacteria, known as adhesins, play critical roles in the binding to host cells or the extracellular matrix (ECM). To elucidate the mechanisms associated with the adhesion of Bifidobacterium longum BBMN68, a centenarian originated potential probiotic, PSORTdb was employed to identify putative extracellular localized proteins in the B. longum BBMN68. Of the 560 predicted extracellular proteins, 21 were further identified as putative adhesion proteins using the conserved domain database of NCBI, and four were successfully overexpressed in the heterologous host, Lactococcus lactis NZ9000. Notably, a recombinant strain expressing FimM showed a significantly increased adhesive affinity for both HT-29 and mucus-secreting LS174T goblet cells (2.2- and 5.4-fold higher than that of the control strain, respectively). Amino acid sequence alignment showed that FimM is a major pilin subunit protein containing a Cna-B type domain and a C-terminal LPKTG sequence. However, in silico analysis of the fimM-coding cluster revealed that BBMN68_RS10200, encoding a pilus-specific class C sortase, was a pseudogene, indicating that FimM may function as a surface adhesin that cannot polymerize into a pili-like structure. Immunogold electron microscopy results further confirmed that FimM localized to the surface of L. lactis NZfimM and B. longum BBMN68 but did not assemble into pilus filaments. Moreover, the adhesive affinity of L. lactis NZfimM to fibronectin, fibrinogen, and mucin were 3.8-, 2.1-, and 3.1-fold higher than that of the control. The affinity of FimM for its attachment receptors was further verified through an inhibition assay using anti-FimM antibodies. In addition, homologs of FimM were found in Bifidobacterium bifidum 85B, Bifidobacterium gallinarum CACC 514, and 23 other B. longum strains by sequence similarity analysis using BLASTP. Our results suggested that FimM is a novel surface adhesin that is mainly present in B. longum strains.

Inhibitory Action of Antidiabetic Drugs on the Free Radical Production by the Rod Outer Segment Ectopic Aerobic Metabolism.

Rod outer segments (OS) express the FoF1-ATP synthase and the respiratory chain, conducting an ectopic aerobic metabolism that produces free radicals in vitro. Diabetic retinopathy, a leading cause of vision loss, is associated with oxidative stress in the outer retina. Since metformin and glibenclamide, two anti-type 2 diabetes drugs, target the respiratory complexes, we studied the effect of these two drugs, individually or in association, on the free radical production in purified bovine rod OS. ATP synthesis, oxygen consumption, and oxidative stress production were assayed by luminometry, oximetry and flow cytometry, respectively. The expression of FoF1-ATP synthase was studied by immunogold electron microscopy. Metformin had a hormetic effect on the OS complex I and ATP synthetic activities, being stimulatory at concentrations below 1 mM, and inhibitory above. Glibenclamide inhibited complexes I and III, as well as ATP production in a concentration-dependent manner. Maximal concentrations of both drugs inhibited the ROI production by the light-exposed OS. Data, consistent with the delaying effect of these drugs on the onset of diabetic retinopathy, suggest that a combination of the two drugs at the beginning of the treatment might reduce the oxidative stress production helping the endogenous antioxidant defences in avoiding retinal damage.

The Whey Acidic Protein WFDC12 Is Specifically Expressed in Terminally Differentiated Keratinocytes and Regulates Epidermal Serine Protease Activity

WFDC proteins such as peptidase inhibitor 3 and SLPI inhibit proteases in the epidermis and other tissues. In this study, we tested the hypothesis that further WFDC protein family members might contribute to epidermal homeostasis. We found that in addition to peptidase inhibitor 3 and SLPI, WFDC5 and WFDC12 were expressed in human epidermis. In contrast to WFDC5, the expression of WFDC12 was induced during the late differentiation of keratinocytes and was restricted to the outermost layer of live cells. Single-cell RNA sequencing demonstrated that WFDC12-positive keratinocytes were characterized by the upregulation of LCE mRNA expression and downregulated the expression of keratins and claudins. Immunogold-electron microscopy revealed the colocalization of WFDC12 with corneodesmosomes in the lower stratum corneum. WFDC12 was elevated in the affected skin of patients with psoriasis, atopic dermatitis, and Darier disease. By contrast, WFDC12 expression was strongly upregulated not only in the affected but even more so in clinically normal-appearing skin of patients with Netherton syndrome. Finally, functional analysis showed distinct inhibitory activity of WFDC12 on neutrophil elastase and epidermal kallikrein‒related peptidase. Altogether, our study identified WFDC12 as a marker of the last stage of epidermal keratinocyte differentiation and suggests that WFDC12 contributes to the control of protease activity in the stratum corneum.

Metabolic acidosis and hyperkalemia differentially regulate cation HCN3 channel in the rat nephron.

The kidney controls body fluids, electrolyte and acid-base balance. Previously, we demonstrated that hyperpolarization-activated and cyclic nucleotide-gated (HCN) cation channels participate in ammonium excretion in the rat kidney. Since acid-base balance is closely linked to potassium metabolism, in the present work we aim to determine the effect of chronic metabolic acidosis (CMA) and hyperkalemia (HK) on protein abundance and localization of HCN3 in the rat kidney. CMA increased HCN3 protein level only in the outer medulla (2.74±0.31) according to immunoblot analysis. However, immunofluorescence assays showed that HCN3 augmented in cortical proximal tubules (1.45±0.11) and medullary thick ascending limb of Henle's loop (4.48±0.45) from the inner stripe of outer medulla. HCN3 was detected in brush border membranes (BBM) and mitochondria of the proximal tubule by immunogold electron and confocal microscopy in control conditions. Acidosis did not alter HCN3 levels in BBM and mitochondria but augmented them in lysosomes. HCN3 was also immuno-detected in mitoautophagosomes. In the distal nephron, HCN3 was expressed in principal and intercalated cells from cortical to medullary collecting ducts. CMA did not change HCN3 abundance in these nephron segments. In contrast, HK doubled HCN3 level in cortical collecting ducts and favored its basolateral localization in principal cells from the inner medullary collecting ducts. These findings further support HCN channels contribution to renal acid-base and potassium balance.

Stimulation-induced differential redistributions of clathrin and clathrin-coated vesicles in axons compared to soma/dendrites

Clathrin-mediated endocytosis plays an important role in the recycling of synaptic vesicle in presynaptic terminals, and in the recycling of transmitter receptors in neuronal soma/dendrites. The present study uses electron microscopy (EM) and immunogold EM to document the different categories of clathrin-coated vesicles (CCV) and pits (CCP) in axons compared to soma/dendrites, and the depolarization-induced redistribution of clathrin in these two polarized compartments of the neuron. The size of CCVs in presynaptic terminals (~ 40 nm; similar to the size of synaptic vesicles) is considerably smaller than the size of CCVs in soma/dendrites (~ 90 nm). Furthermore, neuronal stimulation induces an increase in the number of CCV/CCP in presynaptic terminals, but a decrease in soma/dendrites. Immunogold labeling of clathrin revealed that in presynaptic terminals under resting conditions, the majority of clathrin molecules are unassembled and concentrated outside of synaptic vesicle clusters. Upon depolarization with high K+, label for clathrin became scattered among de-clustered synaptic vesicles and moved closer to the presynaptic active zone. In contrast to axons, clathrin-labeled CCVs and CCPs were prominent in soma/dendrites under resting conditions, and became inconspicuous upon depolarization with high K+. Thus, EM examination suggests that the regulation and mechanism of clathrin-mediated endocytosis differ between axon and dendrite, and that clathrin redistributes differently in these two neuronal compartments upon depolarization.

Adhesion-GPCR Gpr116 (ADGRF5) expression inhibits renal acid secretion

The diversity and near universal expression of G protein-coupled receptors (GPCR) reflects their involvement in most physiological processes. The GPCR superfamily is the largest in the human genome, and GPCRs are common pharmaceutical targets. Therefore, uncovering the function of understudied GPCRs provides a wealth of untapped therapeutic potential. We previously identified an adhesion-class GPCR, Gpr116, as one of the most abundant GPCRs in the kidney. Here, we show that Gpr116 is highly expressed in specialized acid-secreting A-intercalated cells (A-ICs) in the kidney using both imaging and functional studies, and we demonstrate in situ receptor activation using a synthetic agonist peptide unique to Gpr116. Kidney-specific knockout (KO) of Gpr116 caused a significant reduction in urine pH (i.e., acidification) accompanied by an increase in blood pH and a decrease in pCO2 compared to WT littermates. Additionally, immunogold electron microscopy shows a greater accumulation of V-ATPase proton pumps at the apical surface of A-ICs in KO mice compared to controls. Furthermore, pretreatment of split-open collecting ducts with the synthetic agonist peptide significantly inhibits proton flux in ICs. These data suggest a tonic inhibitory role for Gpr116 in the regulation of V-ATPase trafficking and urinary acidification. Thus, the absence of Gpr116 results in a primary excretion of acid in KO mouse urine, leading to mild metabolic alkalosis ("renal tubular alkalosis"). In conclusion, we have uncovered a significant role for Gpr116 in kidney physiology, which may further inform studies in other organ systems that express this GPCR, such as the lung, testes, and small intestine.

Deep Learning-Assisted High-Throughput Analysis of Freeze-Fracture Replica Images Applied to Glutamate Receptors and Calcium Channels at Hippocampal Synapses.

The molecular anatomy of synapses defines their characteristics in transmission and plasticity. Precise measurements of the number and distribution of synaptic proteins are important for our understanding of synapse heterogeneity within and between brain regions. Freeze–fracture replica immunogold electron microscopy enables us to analyze them quantitatively on a two-dimensional membrane surface. Here, we introduce Darea software, which utilizes deep learning for analysis of replica images and demonstrate its usefulness for quick measurements of the pre- and postsynaptic areas, density and distribution of gold particles at synapses in a reproducible manner. We used Darea for comparing glutamate receptor and calcium channel distributions between hippocampal CA3-CA1 spine synapses on apical and basal dendrites, which differ in signaling pathways involved in synaptic plasticity. We found that apical synapses express a higher density of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and a stronger increase of AMPA receptors with synaptic size, while basal synapses show a larger increase in N-methyl-D-aspartate (NMDA) receptors with size. Interestingly, AMPA and NMDA receptors are segregated within postsynaptic sites and negatively correlated in density among both apical and basal synapses. In the presynaptic sites, Cav2.1 voltage-gated calcium channels show similar densities in apical and basal synapses with distributions consistent with an exclusion zone model of calcium channel-release site topography.

A Novel Interaction of Translocator Protein 18\xa0kDa (TSPO) with NADPH Oxidase in Microglia

In the brain neuropil, translocator protein 18 kDa (TSPO) is a stress response protein that is upregulated in microglia and astrocytes in diverse central nervous system pathologies. TSPO is widely used as a biomarker of neuroinflammation in preclinical and clinical neuroimaging studies. However, there is a paucity of knowledge on the function(s) of TSPO in glial cells. In this study, we explored a putative interaction between TSPO and NADPH oxidase 2 (NOX2) in microglia. We found that TSPO associates with gp91phox and p22phox, the principal subunits of NOX2 in primary murine microglia. The association of TSPO with gp91phox and p22phox was observed using co-immunoprecipitation, confocal immunofluorescence imaging, and proximity ligation assay. We found that besides gp91phox and p22phox, voltage-dependent anion channel (VDAC) also co-immunoprecipitated with TSPO consistent with previous reports. When we compared lipopolysaccharide (LPS) stimulated microglia to vehicle control, we found that a lower amount of gp91phox and p22phox protein co-immunoprecipitated with TSPO suggesting a disruption of the TSPO-NOX2 subunits association. TSPO immuno-gold electron microscopy confirmed that TSPO is present in the outer mitochondrial membrane but it is also found in the endoplasmic reticulum (ER), mitochondria-associated ER membrane (MAM), and in the plasma membrane. TSPO localization at the MAM may represent a subcellular site where TSPO interacts with gp91phox and p22phox since the MAM is a point of communication between outer mitochondria membrane proteins (TSPO) and ER proteins (gp91phox and p22phox) where they mature and form the cytochrome b558 (Cytb558) heterodimer. We also found that an acute burst of reactive oxygen species (ROS) increased TSPO levels on the surface of microglia and this effect was abrogated by a ROS scavenger. These results suggest that ROS production may alter the subcellular distribution of TSPO. Collectively, our findings suggest that in microglia, TSPO is associated with the major NOX2 subunits gp91phox and p22phox. We hypothesize that this interaction may regulate Cytb558 formation and modulate NOX2 levels, ROS production, and redox homeostasis in microglia.

Delta opioid receptor activation modulates affective pain and modality-specific pain hypersensitivity associated with chronic neuropathic pain.

Delta opioid receptor (DOR) agonists alleviate nociceptive behaviors in various chronic pain models, including neuropathic pain, while having minimal effect on sensory thresholds in the absence of injury. The mechanisms underlying nerve injury-induced enhancement of DOR function are unclear. We used a peripheral nerve injury (PNI) model of neuropathic pain to assess changes in the function and localization of DORs in mice and rats. Intrathecal administration of DOR agonists reversed mechanical allodynia and thermal hyperalgesia. The dose-dependent thermal antinociceptive effects of DOR agonists were shifted to the left in PNI rats. Administration of DOR agonists produced a conditioned place preference in PNI, but not in sham, animals, whereas the DOR antagonist naltrindole produced a place aversion in PNI, but not in sham, mice, suggesting the engagement of endogenous DOR activity in suppressing pain associated with the injury. GTPγS autoradiography revealed an increase in DOR function in the dorsal spinal cord, ipsilateral to PNI. Immunogold electron microscopy and in vivo fluorescent agonist assays were used to assess changes in the ultrastructural localization of DORs in the spinal dorsal horn. In shams, DORs were primarily localized within intracellular compartments. PNI significantly increased the cell surface expression of DORs within lamina IV-V dendritic profiles. Using neonatal capsaicin treatment, we identified that DOR agonist-induced thermal antinociception was mediated via receptors expressed on primary afferent sensory neurons but did not alter mechanical thresholds. These data reveal that the regulation of DORs following PNI and suggest the importance of endogenous activation of DORs in regulating chronic pain states.

The Rice BZ1 Locus Is Required for Glycosylation of Arabinogalactan Proteins and Galactolipid and Plays a Role in both Mechanical Strength and Leaf Color

BackgroundThe cell wall and chloroplast are two fundamental structures determining plant mechanical strength and grain yield. Therefore, understanding mechanisms that improve plants’ ability to develop a robust cell wall and well-developed chloroplast is of utmost importance for agricultural activities.ResultsIn this study, we report the functional characterization of a novel rice mutant, brittle stem and zebra leaf (bz1), which displays altered cell wall composition and collapsed chloroplast membrane. Molecular and biochemical analysis revealed that BZ1 encodes a functional UDP-galactose/glucose epimerase (UGE) and is ubiquitously expressed with higher expression in stem and leaf tissues. Multiple techniques analyses, including immunoblots, immuno-gold, and cryogenic scanning electron microscopy, demonstrated a significantly impaired glycosylation of arabinogalactan proteins (AGPs) and disordered cellulose microfibril deposition in bz1. Lipid profiling assay showed that the amount of monogalactosyldiacylglycerols (MGDG), a major chloroplast membrane glycolipid, was significantly decreased in bz1. Taken together, these results strongly demonstrate that BZ1 participates in UDP-galactose supply for the sugar chains biosynthesis of AGPs and MGDG, which thereby, respectively, results in altered cell wall and abnormal chloroplast development. Due to inferior mechanical strength and reduced photosynthesis, bz1 plants displayed detrimental agronomic traits, whereas BZ1 overexpressing lines showed enhanced plant growth. Transcriptome analysis of stems and leaves further showed that numerous key genes involved in AGPs biosynthesis and photosynthesis metabolism were substantially suppressed in bz1.ConclusionsOur finding identifies BZ1 as a dual-targeting UGE protein for glycosylation of AGPs and MGDG and suggests a strategy for breeding robust elite crops.

Rlip Depletion Suppresses Growth of Breast Cancer

RLIP76 (RAL-binding protein-1, Rlip) is a stress-protective mercapturic-acid-pathway transporter protein that also plays a key role in regulating clathrin-dependent endocytosis as a Ral effector. Targeted inhibition or depletion of Rlip causes regression of xenografts of many cancers and is capable of abrogating tumor formation in p53-null mice. This is associated with the reversion of the abnormal methylomic profile of p53-null mice to wild-type. In a query of The Cancer Genome Atlas (TCGA) databases, we found that Rlip expression was associated with poor survival and with significant differences in the frequencies of PIK3CA mutation, MYC amplification, and CDKN2A/B deletion, which were the most commonly mutated, amplified, and deleted genes, respectively, among TCGA breast cancer patients. We conducted the present study to further examine the effects of Rlip inhibition and to evaluate the in vitro and in vivo efficacy in breast cancer. Using immunogold electron microscopy, we found that plasma-membrane Rlip was accessible to cell-surface antibodies in the MCF7 (ER+) breast cancer cell line. Rlip depletion resulted in decreased survival of MCF7 and MDA-MB-231 cells and increased terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positivity and DNA laddering, indicating apoptotic cell death. Additionally, in vitro knockdown of Rlip inhibited EGF endocytosis and WNT/MAPK signaling. Xenograft studies in nude mice showed regression of breast cancer via antisense-mediated depletion of Rlip mRNA as well as by anti-Rlip antibody. Finally, knockdown of Rlip by antisense locked nucleic acid oligonucleotides increased markers for apoptotic signaling and decreased markers for proliferation, angiogenesis, and cell cycling in MCF7 and MDA-MB-231luc xenografts. Our findings validate Rlip as an attractive target in breast cancer.

Dynamin regulates the dynamics and mechanical strength of the actin cytoskeleton as a multifilament actin-bundling protein.

The dynamin GTPase is known to bundle actin filaments, but the underlying molecular mechanism and physiological relevance remain unclear. Our genetic analyses revealed a function of dynamin in propelling invasive membrane protrusions during myoblast fusion in vivo. Using biochemistry, total internal reflection fluorescence microscopy, electron microscopy and cryo-electron tomography, we show that dynamin bundles actin while forming a helical structure. At its full capacity, each dynamin helix captures 12-16 actin filaments on the outer rim of the helix. GTP hydrolysis by dynamin triggers disassembly of fully assembled dynamin helices, releasing free dynamin dimers/tetramers and facilitating Arp2/3-mediated branched actin polymerization. The assembly/disassembly cycles of dynamin promote continuous actin bundling to generate mechanically stiff actin super-bundles. Super-resolution and immunogold platinum replica electron microscopy revealed dynamin along actin bundles at the fusogenic synapse. These findings implicate dynamin as a unique multifilament actin-bundling protein that regulates the dynamics and mechanical strength of the actin cytoskeletal network.

Interaction with the Receptor SLAM and Baculovirus Surface Display of Peste des petits ruminants Virus Hemagglutinin.

Peste des petits ruminants (PPR) is an acute, highly infectious, and highly pathogenic disease, which mainly damages small ruminants such as goats and sheep. Hemagglutinin protein (H), the main antigenic protein of peste des petits ruminants virus (PPRV), has been a hot spot in the research of genetic engineering vaccine for PPRV. In this study, the silkworm baculovirus surface display technology is combined with the transmembrane structure of the silkworm baculovirus envelope protein GP64 and different characteristics of the promoters to display four kinds of fusion proteins, which contain Pph-H, Pph-HJ, Pie1-H, and Pie1-HJ. The fusion proteins displayed on baculovirus surface have been detected by western blotting, cell surface immunofluorescence, and immunogold electron microscopy. In addition, the dominant form of PPR H displayed on baculovirus surface has been determined which is fusion protein mediated by Pph containing the hemagglutinin protein and full-length GP64, Pph-H. Furthermore, by comparing the fluorescence intensity of binding of hemagglutinin protein and signaling lymphocyte activation molecules (SLAM) in Vero-SLAM cells by immunocytochemistry, Pph-H can be combined with the receptor protein of PPRV, SLAM. It provides technical support for displaying the different structure of hemagglutinin and exploring the key sites of hemagglutinin and SLAM binding. Meanwhile, it is important for exploring the pathogenesis and immune mechanism of PPRV.

Myoendothelial Junctions of Mature Coronary Vessels Express Notch Signaling Proteins

The Notch signaling pathway, via cell‐cell communication, is critical during normal vascular development and homeostasis, and it is known to regulate vascular smooth muscle (VSMC) phenotype, including proliferation; however, few studies have addressed the role of Notch signaling in the mature vasculature. The current study tested the hypothesis that Notch signaling is present and active at mature myoendothelial junctions (MEJs), signaling hubs that occur at breaks in the internal elastic lamina (IEL). In mature, intact normal adult mouse coronary resistance microvessels (CRMs) embedded within heart sections, we observed Notch1, Notch2, Notch3, and Jagged1 expression within the fenestrae of the CRM IEL where MEJs are known to occur. Immunofluorescence clearly shows bridging from the VSMCs to endothelial cells (ECs). Immunofluorescence in larger mouse left anterior descending (LAD) coronary arteries demonstrate the expression Notch2 and Notch3 protein immunofluorescence within fenestrae of the IEL. We found that, on average, 39% and 33% of IEL fenestrae were positive for Notch2 and Notch3 expression, respectively. We also confirmed the presence of Notch3 expression in the MEJ by immunogold electron microscopy. In normal mouse femoral arteries, we found that, on average, 46%, 51%, and 58% of IEL fenestrae were positive for Notch1, Notch3, and Jagged1 expression, respectively. To determine whether Notch signaling components were localized to the MEJ, we co‐cultured primary human coronary ECs (hcECs) and primary human coronary VSMCs (hcVSMCs) across transwell inserts, and we utilized fluorescent microscopy to immunostain for Pai‐1, Jagged1, Notch1, and Notch2. PAI‐1 immunofluorescence was observed on the EC side of the transwells and within the transwell pores, indicating the formation of in vitro MEJs. We also identified distinct staining of Notch1, Notch2, and Jagged1 in the pores of the co‐culture transwells, which co‐localized with PAI‐1 within the pores. To assess whether Notch signaling was active at the MEJ, we co‐cultured hcECs and hcVSMCs on transwells to create in vitro MEJs. Smooth muscle cells were first transfected with a Notch‐sensor luciferase plasmid followed by transwell co‐culture of the transfected cells with endothelial cells. Notch activity was stimulated by 3‐fold across the MEJ in hcVSMC (1.00 ± 0.15 vs. 3.02 ± 0.43, p<0.001), which was completely abrogated by the non‐selective Notch γ‐secretase inhibitor, DAPT (1.15 ± 0.16, p<0.01). In this study, we demonstrate for the first time that Notch receptors and ligands are expressed within and are active at mature coronary MEJs, demonstrating a previously unrecognized mode of Notch signaling regulation between the endothelium and smooth muscle.Support or Funding InformationFunding: EW was supported by NIH R25HD086885. NIH R00HL116769, R21EB026518, and S10OD023438 to AJT.

Role of MnSOD in AIFm2‐Mediated Apoptosis During Cardiac Ischemia/Reperfusion Injury

Role of MnSOD in AIFm2‐Mediated Apoptosis During Cardiac Ischemia/Reperfusion Injury

Microtubule‐associated protein 2 mediates induction of long‐term potentiation in hippocampal neurons

Microtubule-associated protein (MAP) 2 has been perceived as a static cytoskeletal protein enriched in neuronal dendritic shafts. Emerging evidence indicates dynamic functions for various MAPs in activity-dependent synaptic plasticity. However, it is unclear how MAP2 is associated with synaptic plasticity mechanisms. Here, we demonstrate that specific silencing of high-molecular-weight MAP2 in vivo abolished induction of long-term potentiation (LTP) in the Schaffer collateral pathway of CA1 pyramidal neurons and in vitro blocked LTP-induced surface delivery of AMPA receptors and spine enlargement. In mature hippocampal neurons, we observed rapid translocation of a subpopulation of MAP2, present in dendritic shafts, to spines following LTP stimulation. Time-lapse confocal imaging showed that spine translocation of MAP2 was coupled with LTP-induced spine enlargement. Consistently, immunogold electron microscopy revealed that LTP stimulation of the Schaffer collateral pathway promoted MAP2 labeling in spine heads of CA1 neurons. This translocation depended on NMDA receptor activation and Ras-MAPK signaling. Furthermore, LTP stimulation led to an increase in surface-expressed AMPA receptors specifically in the neurons with MAP2 spine translocation. Altogether, this study indicates a novel role for MAP2 in LTP mechanisms and suggests that MAP2 participates in activity-dependent synaptic plasticity in mature hippocampal networks.

Differential expression of the five redox complexes in the retinal mitochondria or rod outer segment disks is consistent with their different functionality.

PurposeThe retinal rod outer segment (OS) disk membranes, devoid of mitochondria, conducts oxidative phosphorylation (OxPhos). This study aimed at identifying which proteins expressed in the retinal rod OS disks determined the considerable adenosine‐5'‐triphosphate production and oxygen consumption observed in comparison with retinal mitochondria.ProceduresCharacterization was conducted by immunogold transmission electron microscopy on retinal sections. OxPhos was studied by oximetry and luminometry. The proteomes of OS disks and mitochondria purified from bovine retinas were studied by mass spectrometry. Statistical and bioinformatic analyses were conducted by univariate, multivariate, and machine learning methods.ResultsWeighted gene coexpression network analysis identified two protein expression profile modules functionally associated with either retinal mitochondria or disk samples, in function of a strikingly different ability of each sample to utilized diverse substrate for F1Fo‐ATP synthase. The OS disk proteins correlated better than mitochondria with the tricarboxylic acids cycle and OxPhos proteins.ConclusionsThe differential enrichment of the expression profile of the OxPhos proteins in the disks versus mitochondria suggests that these proteins may represent a true proteome component of the former, with different functionality. These findings may shed new light on the pathogenesis of rod‐driven retinal degenerative diseases.

Clinical histopathologic characteristics of lacrimal glands in lacrimal gland prolapse with blepharochalasis

Objective: To analyze the clinical histopathologic characteristics of lacrimal glands and possible mechanisms of lacrimal gland prolapse in blepharochalasis (BC). Methods: A case-controlled study of 23 consecutive patients with prolapse of lacrimal glands in BC was performed. All samples were obtained during surgery from the Department of Ophthalmology, Beijing Tongren Hospital, Capital Medical University between January 2009 and December 2016. The lacrimal tissue included prolapsed lacrimal glands (30 samples) and controls from the donors in the eye bank of Beijing Tongren Hospital, Capital Medical University (8 samples). Hematoxylin-eosin staining, special staining, immunohistochemistry and colloidal gold-labeled pre-embedded indirect immunogold electron microscopy (Gold-IIEM) were performed to analyze the histopathologic characteristics of the samples. The nonparametric Wilcoxon signed-ranks test was carried out for statistical analysis. Results: Among the 23 patients with lacrimal gland prolapse in BC, there were 3 males and 20 females. The mean age of morbidity was 11 years old (7-16 years). In the 8 normal control cases, 3 males and 5 females were included. The mean age was 15 years (10-20 years). In the 30 prolapsed lacrimal gland samples, hematoxylin-eosin staining showed enlargement of glandular lumina accompanied by inflammatory infiltrates of interstitial tissue in 2 samples. Marked loosening of collagen fibers of the obtainable lacrimal fascia was observed. The results on immunohistochemical staining demonstrated an increased level of immunocytes in the 30 prolapsed lacrimal gland samples, including IgA (+++, ++, +, -; 12, 11, 4, 3 vs. 0, 0, 1, 7; Z=-3.892), CD3(+)T cells (+++, ++, +, -; 2, 19, 7, 2 vs. 0, 0, 1, 7; Z=-4.168), matrix metalloproteinase (MMP)-3 (+++, ++, +, -; 0, 0, 11, 19 vs. 0, 0, 0, 8; Z=-2.005) and MMP-9 (+++, ++, +, -; 14, 14, 0, 2 vs. 0, 0, 0, 8; Z=-4.552) (all P<0.05). IgG, IgM, CD20 and C1-inhibitor were either absent or expressed at background level in the 30 prolapsed lacrimal gland samples (all P>0.05). Gold-IIEM showed zymogon granules in lacrimal glands were out of shape. MMP-3 and MMP-9 colloidal gold particles existed on the zymogon granules, and MMP-3 colloidal gold particles also existed on the surface of lacrimal gland epithelial cells. Conclusions: The histopathological changes in the lacrimal glands of lacrimal gland prolapse with BC include inflammatory infiltration, elastic fiber degeneration, marked loosening of the supporting fascia tissue, and an increased level of immunocytes, including IgA, CD3(+)T cells, MMP-3 and MMP-9. The results suggest that lacrimal gland prolapse with BC may result in the immuno-pathogenetic mechanisms with the involvement of cell-mediated immune responses. (Chin J Ophthalmol, 2020, 56: 205-210).