AVP-eGFP was significantly upregulated by hypovolemia in the parvocellular division of the paraventricular nucleus in the transgenic rats

Arginine vasopressin (AVP) is produced in the paraventricular (PVN) and supraoptic nuclei (SON). Peripheral AVP, which is secreted from the posterior pituitary, is produced in the magnocellular division of the PVN (mPVN) and SON. In addition, AVP is produced in the parvocellular division of the PVN (pPVN), where corticotrophin releasing factor (CRF) is synthesized. These peptides synergistically modulate the hypothalamic-pituitary-adrenal (HPA) axis. Previous studies have revealed that the HPA axis was activated by the hypovolemia. However, the detailed dynamics of AVP in the pPVN under hypovolemic state has not been elucidated. Here, we evaluated the effects of hypovolemia and hyperosmolality on the hypothalamus, using AVP-enhanced green fluorescent protein (eGFP) transgenic rats. Polyethylene glycol (PEG) or 3% hypertonic saline (HTN) was intraperitoneally administered in order to develop hypovolemia or hyperosmolality. AVP-eGFP intensity was robustly upregulated at 3 and 6 h after intraperitoneal (i.p.) administration of PEG or HTN in the mPVN. While in the pPVN, eGFP intensity was significantly increased at 6 h after i.p. administration of PEG with significant induction of Fos-immunoreactive (-ir) neurons. Consistently, eGFP mRNA, AVP hnRNA, and CRF mRNA in the pPVN and plasma AVP and corticosterone were significantly increased at 6 h after i.p. administration of PEG. The results suggest that AVP and CRF syntheses in the pPVN were activated by hypovolemia, resulting in the activation of the HPA axis.

Simultaneous Monitoring of pH and Chloride (Cl−) in Brain Slices of Transgenic Mice

Optosensorics is the direction of research possessing the possibility of non-invasive monitoring of the concentration of intracellular ions or activity of intracellular components using specific biosensors. In recent years, genetically encoded proteins have been used as effective optosensory means. These probes possess fluorophore groups capable of changing fluorescence when interacting with certain ions or molecules. For monitoring of intracellular concentrations of chloride ([Cl−]i) and hydrogen ([H+] i) the construct, called ClopHensor, which consists of a H+- and Cl−-sensitive variant of the enhanced green fluorescent protein (E2GFP) fused with a monomeric red fluorescent protein (mDsRed) has been proposed [1]. We recently developed a line of transgenic mice expressing ClopHensor in neurons and obtained the map of its expression in different areas of the brain [2]. The purpose of this study was to examine the effectiveness of transgenic mice expressing ClopHensor for estimation of [H+]i and [Cl−]i concentrations in neurons of brain slices. We performed simultaneous monitoring of [H+]i and [Cl−]i under different experimental conditions including changing of external concentrations of ions (Ca2+, Cl−, K+, Na+) and synaptic stimulation of Shaffer’s collaterals of hippocampal slices. The results obtained illuminate different pathways of regulation of Cl− and pH equilibrium in neurons and demonstrate that transgenic mice expressing ClopHensor represent a reliable tool for non-invasive simultaneous monitoring of intracellular Cl− and pH.

Preexposure and Postexposure Prophylaxis of Rabies With Adeno-Associated Virus Expressing Virus-Neutralizing Antibody in Rodent Models

Rabies, a fatal disease in humans and other mammals, is caused by the rabies virus (RABV), and it poses a public health threat in many parts of the world. Once symptoms of rabies appear, the mortality is near 100%. There is currently no effective treatment for rabies. In our study, two human-derived RABV-neutralizing antibodies (RVNA), CR57 and CR4098, were cloned into adeno-associated virus (AAV) vectors, and recombinant AAVs expressing RVNA were evaluated for postexposure prophylaxis after intrathecal injection into RABV-infected rats. At 4days post-infection with a lethal dose of RABV, 60% of the rats that received an intrathecal injection of AAV-CR57 survived, while 100% of the rats inoculated with AAV-enhanced green fluorescent protein (EGFP) succumbed to rabies. Overall, these results demonstrate that AAV-encoding RVNA can be utilized as a potential human rabies postexposure prophylaxis.

Astrocyte-to-neuron transportation of enhanced green fluorescent protein in cerebral cortex requires F-actin dependent tunneling nanotubes

Tunneling nanotube (TNT), a dynamic cell–cell contact, is dependent on actin polymerization. TNTs are efficient in transporting ions, proteins and organelles intercellularly, which are important mechanisms in physiological and pathological processes. Reported studies on the existence and function of TNTs among neural cells focus on cultured cell for the convenience in detecting TNTs’ ultrastructure. In this study, the adeno-associated virus (AAV-GFAP-EGFP-p2A-cre) was injected into the cerebral cortex of knock-in mice ROSA26 GNZ. GFAP promoter initiated the expression of enhanced green fluorescent protein (EGFP) in infected astrocytes. At 10 days post injection (10 DPI), EGFP transferred from astrocytes in layer I–III to neurons in layer V. The dissemination of EGFP was not through endocytosis or exosome. Applying microscopes, we found that the intercellular transportation of EGFP through contact connection was F-actin dependent. Therefore, we concluded that EGFP transported from astrocytes to neurons in cortex via F-actin dependent TNTs. This study first proved that proteins transported intercellularly via TNTs in brain.

Chromophore of an Enhanced Green Fluorescent Protein Can Play a Photoprotective Role Due to Photobleaching

Under stress conditions, elevated levels of cellular reactive oxygen species (ROS) may impair crucial cellular structures. To counteract the resulting oxidative damage, living cells are equipped with several defense mechanisms, including photoprotective functions of specific proteins. Here, we discuss the plausible ROS scavenging mechanisms by the enhanced green fluorescent protein, EGFP. To check if this protein could fulfill a photoprotective function, we employed electron spin resonance (ESR) in combination with spin-trapping. Two organic photosensitizers, rose bengal and methylene blue, as well as an inorganic photocatalyst, nano-TiO2, were used to photogenerate ROS. Spin-traps, TMP-OH and DMPO, and a nitroxide radical, TEMPOL, served as molecular targets for ROS. Our results show that EGFP quenches various forms of ROS, including superoxide radicals and singlet oxygen. Compared to the three proteins PNP, papain, and BSA, EGFP revealed high ROS quenching ability, which suggests its photoprotective role in living systems. Damage to the EGFP chromophore was also observed under strong photo-oxidative conditions. This study contributes to the discussion on the protective function of fluorescent proteins homologous to the green fluorescent protein (GFP). It also draws attention to the possible interactions of GFP-like proteins with ROS in systems where such proteins are used as biological markers.

Expression of Secreted Neutrophil Gelatinase-Associated Lipocalin in 293T Cell Using the Inducible Dual-Function System

Neutrophil gelatinase-associated lipocalin (NGAL) has emerged as a promising biomarker for the early prediction of acute kidney injury (AKI). The production of recombinant NGAL is considered to be necessary for the development of a detection method. This study intended to express the recombinant NGAL protein in 293T cell under the Tet-On inducible system and human serum albumin signal sequence (HSA-SS). The transfection efficiency and protein modulation were assessed by detecting the expression of the enhanced green fluorescent protein (EGFP) and secreted NGAL protein. Both proteins were detected only in the presence of a doxycycline (Dox) inducer. Cell toxicity was not found under any conditions. Moreover, a higher level of soluble NGAL protein in the supernatant secreted by HSA-SS compared with a native signal peptide (Nat-SS) was observed. In summary, this work successfully optimized the conditions for induction of NGAL expression. This system will provide as an efficient strategy to produce other recombinant proteins secreted from a mammalian cell.

Development of a stable transgenic Theileria equi parasite expressing an enhanced green fluorescent protein/blasticidin S deaminase

Theileria equi, an intraerythrocytic protozoan parasite, causes equine piroplasmosis, a disease which negatively impacts the global horse industry. Genetic manipulation is one of the research tools under development as a control method for protozoan parasites, but this technique needs to be established for T. equi. Herein, we report on the first development of a stable transgenic T. equi line expressing enhanced green fluorescent protein/blasticidin S deaminase (eGFP/BSD). To express the exogenous fusion gene in T. equi, regulatory regions of the elongation factor-1 alpha (ef-1α) gene were identified in T. equi. An eGFP/BSD-expression cassette containing the ef-1α gene promoter and terminator regions was constructed and integrated into the T. equi genome. On day 9 post-transfection, blasticidin-resistant T. equi emerged. In the clonal line of T. equi obtained by limiting dilution, integration of the eGFP/BSD-expression cassette was confirmed in the designated B-locus of the ef-1α gene via PCR and Southern blot analyses. Parasitaemia dynamics between the transgenic and parental T. equi lines were comparable in vitro. The eGFP/BSD-expressing transgenic T. equi and the methodology used to generate it offer new opportunities for better understanding of T. equi biology, with the add-on possibility of discovering effective control methods against equine piroplasmosis.

Development and Characterization of a cDNA-Launch Recombinant Simian Hemorrhagic Fever Virus Expressing Enhanced Green Fluorescent Protein: ORF 2b’ Is Not Required for In Vitro Virus Replication

Simian hemorrhagic fever virus (SHFV) causes acute, lethal disease in macaques. We developed a single-plasmid cDNA-launch infectious clone of SHFV (rSHFV) and modified the clone to rescue an enhanced green fluorescent protein-expressing rSHFV-eGFP that can be used for rapid and quantitative detection of infection. SHFV has a narrow cell tropism in vitro, with only the grivet MA-104 cell line and a few other grivet cell lines being susceptible to virion entry and permissive to infection. Using rSHFV-eGFP, we demonstrate that one cricetid rodent cell line and three ape cell lines also fully support SHFV replication, whereas 55 human cell lines, 11 bat cell lines, and three rodent cells do not. Interestingly, some human and other mammalian cell lines apparently resistant to SHFV infection are permissive after transfection with the rSHFV-eGFP cDNA-launch plasmid. To further demonstrate the investigative potential of the infectious clone system, we introduced stop codons into eight viral open reading frames (ORFs). This approach suggested that at least one ORF, ORF 2b’, is dispensable for SHFV in vitro replication. Our proof-of-principle experiments indicated that rSHFV-eGFP is a useful tool for illuminating the understudied molecular biology of SHFV.