A Complete Electron Microscopy Volume of the Brain of Adult Drosophila melanogaster.

Drosophila melanogaster has a rich repertoire of innate and learned behaviors. Its 100,000-neuron brain is a large but tractable target for comprehensive neural circuit mapping. Only electron microscopy (EM) enables complete, unbiased mapping of synaptic connectivity; however, the fly brain is too large for conventional EM. We developed a custom high-throughput EM platform and imaged the entire brain of an adult female fly at synaptic resolution. To validate the dataset, we traced brain-spanning circuitry involving the mushroom body (MB), which has been extensively studied for its role in learning. All inputs to Kenyon cells (KCs), the intrinsic neurons of the MB, were mapped, revealing a previously unknown cell type, postsynaptic partners of KC dendrites, and unexpected clustering of olfactory projection neurons. These reconstructions show that this freely available EM volume supports mapping of brain-spanning circuits, which will significantly accelerate Drosophila neuroscience. VIDEO ABSTRACT

Susceptibility of Marmosets (Callithrix jacchus) to Monkeypox Virus: A Low Dose Prospective Model for Monkeypox and Smallpox Disease.

Although current nonhuman primate models of monkeypox and smallpox diseases provide some insight into disease pathogenesis, they require a high titer inoculum, use an unnatural route of infection, and/or do not accurately represent the entire disease course. This is a concern when developing smallpox and/or monkeypox countermeasures or trying to understand host pathogen relationships. In our studies, we altered half of the test system by using a New World nonhuman primate host, the common marmoset. Based on dose finding studies, we found that marmosets are susceptible to monkeypox virus infection, produce a high viremia, and have pathological features consistent with smallpox and monkeypox in humans. The low dose (48 plaque forming units) required to elicit a uniformly lethal disease and the extended incubation (preclinical signs) are unique features among nonhuman primate models utilizing monkeypox virus. The uniform lethality, hemorrhagic rash, high viremia, decrease in platelets, pathology, and abbreviated acute phase are reflective of early-type hemorrhagic smallpox

A PPIX-binding probe facilitates discovery of PPIX-induced cell death modulation by peroxiredoxin

Abstract While heme synthesis requires the formation of a potentially lethal intermediate, protoporphyrin IX (PPIX), surprisingly little is known about the mechanism of its toxicity, aside from its phototoxicity. The cellular protein interactions of PPIX might provide insight into modulators of PPIX-induced cell death. Here we report the development of PPB, a biotin-conjugated, PPIX-probe that captures proteins capable of interacting with PPIX. Quantitative proteomics in a diverse panel of mammalian cell lines reveal a high degree of concordance for PPB-interacting proteins identified for each cell line. Most differences are quantitative, despite marked differences in PPIX formation and sensitivity. Pathway and quantitative difference analysis indicate that iron and heme metabolism proteins are prominent among PPB-bound proteins in fibroblasts, which undergo PPIX-mediated death determined to occur through ferroptosis. PPB proteomic data (available at PRIDE ProteomeXchange # PXD042631) reveal that redox proteins from PRDX family of glutathione peroxidases interact with PPIX. Targeted gene knockdown of the mitochondrial PRDX3, but not PRDX1 or 2, enhance PPIX-induced death in fibroblasts, an effect blocked by the radical-trapping antioxidant, ferrostatin-1. Increased PPIX formation and death was also observed in a T-lymphoblastoid ferrochelatase-deficient leukemia cell line, suggesting that PPIX elevation might serve as a potential strategy for killing certain leukemias

Altered GM1 catabolism affects NMDAR-mediated Ca2+ signaling at ER-PM junctions and increases synaptic spine formation in a GM1-gangliosidosis model

Summary: Endoplasmic reticulum-plasma membrane (ER-PM) junctions mediate Ca2+ flux across neuronal membranes. The properties of these membrane contact sites are defined by their lipid content, but little attention has been given to glycosphingolipids (GSLs). Here, we show that GM1-ganglioside, an abundant GSL in neuronal membranes, is integral to ER-PM junctions; it interacts with synaptic proteins/receptors and regulates Ca2+ signaling. In a model of the neurodegenerative lysosomal storage disease, GM1-gangliosidosis, pathogenic accumulation of GM1 at ER-PM junctions due to β-galactosidase deficiency drastically alters neuronal Ca2+ homeostasis. Mechanistically, we show that GM1 interacts with the phosphorylated N-methyl D-aspartate receptor (NMDAR) Ca2+ channel, thereby increasing Ca2+ flux, activating extracellular signal-regulated kinase (ERK) signaling, and increasing the number of synaptic spines without increasing synaptic connectivity. Thus, GM1 clustering at ER-PM junctions alters synaptic plasticity and worsens the generalized neuronal cell death characteristic of GM1-gangliosidosis

Histopathological and immunohistochemical findings in the adrenal gland.

<p>Animal #9 (7.8 x 10⁴ PFU group). A) Multifocal degeneration and necrosis of adrenal cortical cells. HE. 40X. B) Replicate section of A. Antigen is present in affected cells. Immunoperoxidase method with hematoxylin counterstain. 40X.</p

Qualitative reduction of secondary plaque formation (comets) in serum samples derived from marmosets exposed to monkeypox virus.

<p>Virus control, A; Day 14 post exposure plaque titration assay (serum), B. Neutralization assay of animal #18, 48 PFU group (heat inactivated serum), C. Notice the focal nature of the plaques from the day 14 post infection serum, B, compared to the virus control, A, indicating a potential anti-EEV effect. The cause of this effect is heat stable and exhibits a dose/dilution response; notice the increase in comets as the heat inactivated serum concentration decreases (dilution annotated by each well), C.</p

Histopathological and immunohistochemical findings in the haired skin from a lesion on the chin.

<p>Animal #9 (7.8 x 10⁴ PFU group). A) Epithelium is hyperplastic with degeneration and necrosis of epithelial cells. Syncytial cells are also present. Note hemorrhage in the underlying dermis. HE. 10X. B) Replicate section of A. Antigen is present in epithelial cells, as well as fibroblasts and inflammatory cells in the dermis. Immunoperoxidase method with hematoxylin counterstain. 10X.</p

Gross pathological findings for monkeypox virus exposed marmosets.

<p>A. Liver. Animal #14 (510 PFU group). The liver is enlarged and pale with diffuse, variably sized flat, tan lesions. B. Esophagus. Animal 12 (7.8 x 10⁴ PFU group). Focal mucosal ulcer (arrow). C) Urinary bladder. Animal 4 (9.5 x 10⁵ PFU group). Multifocal mucosal hemorrhages. D. Testis. Animal #1 (2.4 x 10⁷ PFU group). Multifocal hemorrhage and necrosis.</p