Experimental Lagos bat virus infection in straw-colored fruit bats: A suitable model for bat rabies in a natural reservoir species

Rabies is a fatal neurologic disease caused by lyssavirus infection. Bats are important natural reservoir hosts of various lyssaviruses that can be transmitted to people. The epidemiology and pathogenesis of rabies in bats are poorly understood, making it difficult to prevent zoonotic transmission. To further our understanding of lyssavirus pathogenesis in a natural bat host, an experimental model using straw-colored fruit bats (Eidolon helvum) and Lagos bat virus, an endemic lyssavirus in this species, was developed. To determine the lowest viral dose resulting in 100% productive infection, bats in five groups (four bats per group) were inoculated intramuscularly with one of five doses, ranging from 10^0.1 to 10^4.1 median tissue culture infectious dose (TCID₅₀). More bats died due to the development of rabies after the middle dose (10^2.1 TCID₅₀, 4/4 bats) than after lower (10^1.1, 2/4; 10^1.1, 2/4) or higher (10^3.1, 2/4; 10^4.1, 2/4) doses of virus. In the two highest dose groups, 4/8 bats developed rabies. Of those bats that remained healthy 3/4 bats seroconverted, suggesting that high antigen loads can trigger a strong immune response that abrogates a productive infection. In contrast, in the two lowest dose groups, 3/8 bats developed rabies, 1/8 remained healthy and seroconverted and 4/8 bats remained healthy and did not seroconvert, suggesting these doses are too low to reliably induce infection. The main lesion in all clinically affected bats was meningoencephalitis associated with lyssavirus-positive neurons. Lyssavirus antigen was detected in tongue epithelium (5/11 infected bats) rather than in salivary gland epithelium (0/11), suggesting viral excretion via the tongue. Thus, intramuscular inoculation of 10^2.1 TCID₅₀ of Lagos bat virus into straw-colored fruit bats is a suitable model for lyssavirus associated bat rabies in a natural reservoir host, and can help with the investigation of lyssavirus infection dynamics in bats.     

Breast Cancer‐Derived Exosomes Reflect the Cell‐of‐Origin Phenotype

A manner in which cells can communicate with each other is via secreted nanoparticles termed exosomes. These vesicles contain lipids, nucleic acids, and proteins, and are said to reflect the cell‐of‐origin. However, for the exosomal protein content, there is limited evidence in the literature to verify this statement. Here, proteomic assessment combined with pathway‐enrichment analysis is used to demonstrate that the protein cargo of exosomes reflects the epithelial/mesenchymal phenotype of secreting breast cancer cells. Given that epithelial‐mesenchymal plasticity is known to implicate various stages of cancer progression, the results suggest that breast cancer subtypes with distinct epithelial and mesenchymal phenotypes may be distinguished by directly assessing the protein content of exosomes. Additionally, the work is a substantial step toward verifying the statement that cell‐derived exosomes reflect the phenotype of the cells‐of‐origin.     

Corticospinal and Reticulospinal Contacts on Cervical Commissural and Long Descending Propriospinal Neurons in the Adult Rat Spinal Cord; Evidence for Powerful Reticulospinal Connections

Descending systems have a crucial role in the selection of motor output patterns by influencing the activity of interneuronal networks in the spinal cord. Commissural interneurons that project to the contralateral grey matter are key components of such networks as they coordinate left-right motor activity of fore and hind-limbs. The aim of this study was to determine if corticospinal (CST) and reticulospinal (RST) neurons make significant numbers of axonal contacts with cervical commissural interneurons. Two classes of commissural neurons were analysed: 1) local commissural interneurons (LCINs) in segments C4-5; 2) long descending propriospinal neurons (LDPNs) projecting from C4 to the rostral lumbar cord. Commissural interneurons were labelled with Fluorogold and CST and RST axons were labelled by injecting the b subunit of cholera toxin in the forelimb area of the primary somatosensory cortex or the medial longitudinal fasciculus respectively. The results show that LCINs and LDPNs receive few contacts from CST terminals but large numbers of contacts are formed by RST terminals. Use of vesicular glutamate and vesicular GABA transporters revealed that both types of cell received about 80% excitatory and 20% inhibitory RST contacts. Therefore the CST appears to have a minimal influence on LCINs and LDPNs but the RST has a powerful influence. This suggests that left-right activity in the rat spinal cord is not influenced directly via CST systems but is strongly controlled by the RST pathway. Many RST neurons have monosynaptic input from corticobulbar pathways therefore this pathway may provide an indirect route from the cortex to commissural systems. The cortico-reticulospinal-commissural system may also contribute to functional recovery following damage to the CST as it has the capacity to deliver information from the cortex to the spinal cord in the absence of direct CST input.     

Stimulation of Ca efflux from fura-2-loaded platelets activated by thrombin or phorbol myristate acetate

We investigated the restoration of [Ca²⁺]_i in fura-2-loaded human platelets following discharge of internal Ca²⁺ stores in the absence of external Ca²⁺. After stimulation by thrombin [Ca²⁺]_i returned from a peak level of 0.6 μM to resting levels within 4 min. When ionomycin discharged the internal stores the recovery was slower with [Ca²⁺]_i still elevated at around 0.5 μM after 5 min. Thrombin added shortly after ionomycin could accelerate the recovery of [Ca²⁺]_i and restore resting levels within 5 min, an effect that was mimicked by phorbol-12-myristate-13-acetate (PMA). Since the continued presence of ionomycin precluded reuptake into the internal stores we conclude that thrombin and PMA stimulate Ca²⁺ efflux, perhaps via protein kinase C actions on a plasma membrane Ca²⁺ pump.     

Insulin decreases the secretion of apoB-100 from hepatic HepG2 cells but does not decrease the secretion of apoB-48 from intestinal CaCo-2 cells

We compared the acute effect of insulin on the human colonic intestinal epithelial cell line CaCo-2 and the transformed human hepatic cell line HepG2. Over 24 h, 100 nM and 10 µM insulin significantly inhibited the secretion of apolipoprotein (apo) B-100 from HepG2 cells to 63 and 49% of control, respectively. Insulin had no effect on the secretion of apoB-48 from CaCo-2 cells. There was no effect of insulin on the cholesterol ester or free cholesterol concentrations in HepG2 or CaCo-2 cells. HepG2 and CaCo-2 cells bound insulin with high affinity, leading to similar stimulation of insulin receptor protein tyrosine kinase activation. Protein kinase C or mitogen-activated protein kinase activity in the presence or absence of insulin was not correlated with apoB-48 production in CaCo-2 cells. Therefore, insulin acutely decreases the secretion of apoB-100 in hepatic HepG2 cells, but does not acutely modulate the production or secretion of apoB-48 from CaCo-2 intestinal cells.     

A single fixation protocol for proteome-wide immunofluorescence localization studies

Immunofluorescence microscopy is a valuable tool for analyzing protein expression and localization at a subcellular level thus providing information regarding protein function, interaction partners and its role in cellular processes. When performing sample fixation, parameters such as difference in accessibility of proteins present in various cellular compartments as well as the chemical composition of the protein to be studied, needs to be taken into account. However, in systematic and proteome-wide efforts, a need exists for standard fixation protocol(s) that works well for the majority of all proteins independent of subcellular localization. Here, we report on a study with the goal to find a standardized protocol based on the analysis of 18 human proteins localized in 11 different organelles and subcellular structures. Six fixation protocols were tested based on either dehydration by alcohols (methanol, ethanol or iso-propanol) or cross-linking by paraformaldehyde followed by detergent permeabilization (Triton X-100 or saponin) in three human cell lines. Our results show that cross-linking is essential for proteome-wide localization studies and that cross-linking using paraformaldehyde followed by Triton X-100 permeabilization successfully can be used as a single fixation protocol for systematic studies.     

Caspase-1-driven neutrophil pyroptosis and its role in host susceptibility to Pseudomonas aeruginosa

Multiple regulated neutrophil cell death programs contribute to host defense against infections. However, despite expressing all necessary inflammasome components, neutrophils are thought to be generally defective in Caspase-1-dependent pyroptosis. By screening different bacterial species, we found that several Pseudomonas aeruginosa (P. aeruginosa) strains trigger Caspase-1-dependent pyroptosis in human and murine neutrophils. Notably, deletion of Exotoxins U or S in P. aeruginosa enhanced neutrophil death to Caspase-1-dependent pyroptosis, suggesting that these exotoxins interfere with this pathway. Mechanistically, P. aeruginosa Flagellin activates the NLRC4 inflammasome, which supports Caspase-1-driven interleukin (IL)-1β secretion and Gasdermin D (GSDMD)-dependent neutrophil pyroptosis. Furthermore, P. aeruginosa-induced GSDMD activation triggers Calcium-dependent and Peptidyl Arginine Deaminase-4-driven histone citrullination and translocation of neutrophil DNA into the cell cytosol without inducing extracellular Neutrophil Extracellular Traps. Finally, we show that neutrophil Caspase-1 contributes to IL-1β production and susceptibility to pyroptosis-inducing P. aeruginosa strains in vivo. Overall, we demonstrate that neutrophils are not universally resistant for Caspase-1-dependent pyroptosis.