Characterizing enteric neurons in dopamine transporter (DAT)-Cre reporter mice reveals dopaminergic subtypes with dual-transmitter content.

The enteric nervous system (ENS) comprises a complex network of neurons whereby a subset appears to be dopaminergic although the characteristics, roles, and implications in disease are less understood. Most investigations relating to enteric dopamine (DA) neurons rely on immunoreactivity to tyrosine hydroxylase (TH)-the rate-limiting enzyme in the production of DA. However, TH immunoreactivity is likely to provide an incomplete picture. This study herein provides a comprehensive characterization of DA neurons in the gut using a reporter mouse line, expressing a fluorescent protein (tdTomato) under control of the DA transporter (DAT) promoter. Our findings confirm a unique localization of DA neurons in the gut and unveil the discrete subtypes of DA neurons in this organ, which we characterized using both immunofluorescence and single-cell transcriptomics, as well as validated using in situ hybridization. We observed distinct subtypes of DAT-tdTomato neurons expressing co-transmitters and modulators across both plexuses; some of them likely co-releasing acetylcholine, while others were positive for a slew of canonical DAergic markers (TH, VMAT2 and GIRK2). Interestingly, we uncovered a seemingly novel population of DA neurons unique to the ENS which was ChAT/DAT-tdTomato-immunoreactive and expressed Grp, Calcb, and Sst. Given the clear heterogeneity of DAergic gut neurons, further investigation is warranted to define their functional signatures and decipher their implication in disease.

miR-301a Regulates Inflammatory Response to Japanese Encephalitis Virus Infection via Suppression of NKRF Activity.

Microglia being the resident macrophage of brain provides neuroprotection following diverse microbial infections. Japanese encephalitis virus (JEV) invades the CNS, resulting in neuroinflammation, which turns the neuroprotective role of microglia detrimental as characterized by increased microglial activation and neuronal death. Several host factors, including microRNAs, play vital roles in regulating virus-induced inflammation. In the current study, we demonstrate that the expression of miR-301a is increased in JEV-infected microglial cells and human brain. Overexpression of miR-301a augments the JEV-induced inflammatory response, whereas inhibition of miR-301a completely reverses the effects. Mechanistically, NF-κB-repressing factor (NKRF) functioning as inhibitor of NF-κB activation is identified as a potential target of miR-301a in JEV infection. Consequently, miR-301a-mediated inhibition of NKRF enhances nuclear translocation of NF-κB, which, in turn, resulted in amplified inflammatory response. Conversely, NKRF overexpression in miR-301a-inhibited condition restores nuclear accumulation of NF-κB to a basal level. We also observed that JEV infection induces classical activation (M1) of microglia that drives the production of proinflammatory cytokines while suppressing alternative activation (M2) that could serve to dampen the inflammatory response. Furthermore, in vivo neutralization of miR-301a in mouse brain restores NKRF expression, thereby reducing inflammatory response, microglial activation, and neuronal apoptosis. Thus, our study suggests that the JEV-induced expression of miR-301a positively regulates inflammatory response by suppressing NKRF production, which might be targeted to manage viral-induced neuroinflammation.

Japanese Encephalitis Virus-induced let-7a/b interacted with the NOTCH-TLR7 pathway in microglia and facilitated neuronal death via caspase activation.

MicroRNAs (miRNAs) released from the activated microglia upon neurotropic virus infection may exacerbate the neuronal damage. Here, we identified let-7a and let-7b (let-7a/b) as one of the essential miRNAs over-expressed upon Japanese Encephalitis virus (JEV) infection and released in the culture supernatant of the JEV-infected microglial cells through extracellular vesicles. The let-7a/b was previously reported to modulate inflammation in microglial cells through Toll-like receptor 7 (TLR7) pathways; although their role in accelerating JEV pathogenesis remain unexplored. Therefore, we studied the role of let-7a/b in modulating microglia-mediated inflammation during JEV infection and investigated the effect of let-7a/b-containing exosomes on primary neurons. To this end, we examined let-7a/b and NOTCH signaling pathway in TLR7 knockdown (KD) mice. We observed that TLR7 KD or inhibition of let-7a/b suppressed the JEV-induced NOTCH activation possibly via NF-κB dependent manner and subsequently, attenuated JEV-induced TNFα production in microglial cells. Furthermore, exosomes secreted from let-7a/b over-expressed microglia when transferred to uninfected mice brain induced caspase activation. Exosomes secreted from virus-infected or let-7a/b over-expressed microglia when co-incubated with mouse neuronal (Neuro2a) cells or primary cortical neurons also facilitated caspase activation leading to neuronal death. Thus, our results provide evidence for the multifaceted role of let-7a/b miRNAs in JEV pathogenesis. Let-7a/b can interact with TLR7 and NOTCH signaling pathway and enhance TNFα release from microglia. On the other hand, the exosomes secreted by JEV-infected microglia can activate caspases in uninfected neuronal cells which possibly contribute to bystander neuronal death. Cover Image for this issue: doi: 10.1111/jnc.14506.

Using Ultrafast X-ray Spectroscopy To Address Questions in Ligand-Field Theory: The Excited State Spin and Structure of [Fe(dcpp)2]2.

We have employed a range of ultrafast X-ray spectroscopies in an effort to characterize the lowest energy excited state of [Fe(dcpp)2]2+ (where dcpp is 2,6-(dicarboxypyridyl)pyridine). This compound exhibits an unusually short excited-state lifetime for a low-spin Fe(II) polypyridyl complex of 270 ps in a room-temperature fluid solution, raising questions as to whether the ligand-field strength of dcpp had pushed this system beyond the 5T2/3T1 crossing point and stabilizing the latter as the lowest energy excited state. Kα and Kß X-ray emission spectroscopies have been used to unambiguously determine the quintet spin multiplicity of the long-lived excited state, thereby establishing the 5T2 state as the lowest energy excited state of this compound. Geometric changes associated with the photoinduced ligand-field state conversion have also been monitored with extended X-ray absorption fine structure. The data show the typical average Fe-ligand bond length elongation of ∼0.18 Å for a 5T2 state and suggest a high anisotropy of the primary coordination sphere around the metal center in the excited 5T2 state, in stark contrast to the nearly perfect octahedral symmetry that characterizes the low-spin 1A1 ground state structure. This study illustrates how the application of time-resolved X-ray techniques can provide insights into the electronic structures of molecules-in particular, transition metal complexes-that are difficult if not impossible to obtain by other means.

GRP78 Is an Important Host Factor for Japanese Encephalitis Virus Entry and Replication in Mammalian Cells.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is the leading cause of viral encephalitis in Southeast Asia with potential to become a global pathogen. Here, we identify glucose-regulated protein 78 (GRP78) as an important host protein for virus entry and replication. Using the plasma membrane fractions from mouse neuronal (Neuro2a) cells, mass spectroscopy analysis identified GRP78 as a protein interacting with recombinant JEV envelope protein domain III. GRP78 was found to be expressed on the plasma membranes of Neuro2a cells, mouse primary neurons, and human epithelial Huh-7 cells. Antibodies against GRP78 significantly inhibited JEV entry in all three cell types, suggesting an important role of the protein in virus entry. Depletion of GRP78 by small interfering RNA (siRNA) significantly blocked JEV entry into Neuro2a cells, further supporting its role in virus uptake. Immunofluorescence studies showed extensive colocalization of GRP78 with JEV envelope protein in virus-infected cells. This interaction was also confirmed by immunoprecipitation studies. Additionally, GRP78 was shown to have an important role in JEV replication, as treatment of cells post-virus entry with subtilase cytotoxin that specifically cleaved GRP78 led to a substantial reduction in viral RNA replication and protein synthesis, resulting in significantly reduced extracellular virus titers. Our results indicate that GRP78, an endoplasmic reticulum chaperon of the HSP70 family, is a novel host factor involved at multiple steps of the JEV life cycle and could be a potential therapeutic target.IMPORTANCE Recent years have seen a rapid spread of mosquito-borne diseases caused by flaviviruses. The flavivirus family includes West Nile, dengue, Japanese encephalitis, and Zika viruses, which are major threats to public health with potential to become global pathogens. JEV is the major cause of viral encephalitis in several parts of Southeast Asia, affecting a predominantly pediatric population with a high mortality rate. This study is focused on identification of crucial host factors that could be targeted to cripple virus infection and ultimately lead to development of effective antivirals. We have identified a cellular protein, GRP78, that plays a dual role in virus entry and virus replication, two crucial steps of the virus life cycle, and thus is a novel host factor that could be a potential therapeutic target.

Comparison of Interfacial Electron Transfer Efficiency in [Fe(ctpy)2]2+-TiO2 and [Fe(cCNC)2]2+-TiO2 Assemblies: Importance of Conformational Sampling.

Fe(II)-polypyridines have limited applications as chromophores in dye-sensitized solar cells due to the short lifetimes (∼100 fs) of their photoactive metal-to-ligand charge transfer (MLCT) states formed upon photoexcitation. Recently, a 100-fold increase in the MLCT lifetime was observed in a [Fe(CNC)2]2+ complex (CNC = 2,6-bis(3-methylimidazole-1-ylidine)pyridine) which has strong σ-donating N-heterocyclic carbene ligand in comparison to the weaker field parent [Fe(tpy)2]2+ complex (tpy = 2,2':6',2″-terpyridine). This study utilizes density functional theory (DFT), time-dependent DFT, and quantum dynamics simulations to investigate the interfacial electron transfer (IET) in [Fe(cCNC)2]2+ (cCNC = 4'-carboxy-2,6-bis(3-methylimidazole-1-ylidine)pyridine) and [Fe(ctpy)2]2+ (ctpy = 4'-carboxy-2,2':6',2″-terpyridine) sensitized TiO2. Our results suggest that the replacement of tpy by CNC ligand does not significantly speed up the IET kinetics in the [Fe(cCNC)2]2+-TiO2 assembly in comparison to the [Fe(ctpy)2]2+-TiO2 analogue. The high IET efficiency in the [Fe(cCNC)2]2+-TiO2 assemblies is therefore due to longer lifetime of [Fe(cCNC)2]2+ photoactive 3MLCT states rather than faster electron injection kinetics. It was also found that the inclusion of conformational sampling in the computational model is important for proper description of the IET processes in these systems, as the models relying on the use of only fully optimized structures may yield misleading results. The simulations presented in this work also illustrate various pitfalls of utilizing properties such as electronic coupling, number of available acceptor states, and driving force, as well as calculations based on Fermi's golden rule framework, to reach conclusions on the IET efficiency in dye-semiconductor systems.

Chandipura virus perturbs cholesterol homeostasis leading to neuronal apoptosis.

Chandipura virus (CHPV; genus Vesiculovirus, family Rhabdoviridae) induces neuronal death through the Fas-mediated extrinsic apoptosis pathway. What propels this apoptosis remains unclear, although oxysterols have been reported to be key players in neurodegeneration. In our study of CHPV-infected brain samples, we observed over-expression of genes such as apolipoprotein E, Cyp46a1, Srebf-1 and Nsdhl. This backs up the hypothesis that CHPV replication demands cholesterol that is supplied by apolipoprotein E through low density lipid receptors, lipid metabolism being pivotal for viral replication. We were able to illustrate this with over-expression of low density lipid receptors in CHPV-infected neurons. An upsurge of cholesterol concentration has been observed in neurons, triggering the expression of Cyp46a1 enzyme and culminating into the conversion of cholesterol to 24(S)-hydroxycholesterol. Increased 24(S)-hydroxycholesterol concentration is toxic to neurons, propelling neuronal apoptosis through the Fas-mediated extrinsic apoptosis pathway. For the first time, perturbation of cholesterol homeostasis in brain is shown to be utilized by the viruses for both maturation and the release of its matured virions outside the cells for continuous neuropathogenesis.

RIG-I knockdown impedes neurogenesis in a murine model of Japanese encephalitis.

Retinoic acid inducible gene I (RIG-I) is a well established pattern recognition receptor (PRR) in neurons infected with Japanese encephalitis virus (JEV) as reported previously from our laboratory. Japanese encephalitis (JE) virus infection in brain has been shown to decrease the proliferation of neural stem/progenitor cells (NSPCs) which has its implications in neurological sequelae in JE survivors. We have found that ablation of RIG-I both in vivo and in vitro models results in significant decrease in NSPC proliferation post JEV infection. We hypothesize that knockdown of RIG-I diminishes the expression of antiviral molecules resulting in an increase in viral replication, which in turn results in enhancement of the expression of cell cycle inhibitors, hence affecting the proliferation of NSPCs.

Tuning the Electronic Structure of Fe(II) Polypyridines via Donor Atom and Ligand Scaffold Modifications: A Computational Study.

Fe(II) polypyridines are an important class of pseudo-octahedral metal complexes known for their potential applications in molecular electronic switches, data storage and display devices, sensors, and dye-sensitized solar cells. Fe(II) polypyridines have a d(6) electronic configuration and pseudo-octahedral geometry and can therefore possess either a high-spin (quintet) or a low-spin (singlet) ground state. In this study, we investigate a series of complexes based on [Fe(tpy)2](2+) (tpy = 2,2';6',2″-terpyridine) and [Fe(dcpp)2](2+) (dcpp = 2,6-bis(2-carboxypyridyl)pyridine). The ligand field strength in these complexes is systematically tuned by replacing the central pyridine with five-membered (N-heterocyclic carbene, pyrrole, furan) or six-membered (aryl, thiazine-1,1-dioxide, 4-pyrone) moieties. To determine the impact of ligand substitutions on the relative energies of metal-centered states, the singlet, triplet, and quintet states of the Fe(II) complexes were optimized in water (PCM) using density functional theory at the B3LYP+D2 level with 6-311G* (nonmetals) and SDD (Fe) basis sets. It was found that the dcpp ligand scaffold allows for a more ideal octahedral coordination environment in comparison to the tpy ligand scaffold. The presence of six-membered central rings also allows for a more ideally octahedral coordination environment relative to five-membered central rings, regardless of the ligand scaffold. We find that the ligand field strength in the Fe(II) polypyridines can be tuned by altering the donor atom identity, with C donor atoms providing the strongest ligand field.

Cyclometalated Fe(II) complexes as sensitizers in dye-sensitized solar cells.

Dye-sensitized solar cells (DSSCs) often utilize transition metal-based chromophores for light absorption and semiconductor sensitization. Ru(II)-based dyes are among the most commonly used sensitizers in DSSCs. As ruthenium is both expensive and rare, complexes based on cheaper and more abundant iron could serve as a good alternative. In this study, we investigate Fe(II)-bis(terpyridine) and its cyclometalated analogues, in which pyridine ligands are systematically replaced by aryl groups, as potential photosensitizers in DSSCs. We employ density functional theory at the B3LYP/6-31G*,SDD level to obtain the ground state electronic structure of these complexes. Quantum dynamics simulations are utilized to study interfacial electron transfer between the Fe(II) photosensitizers and a titanium dioxide semiconductor. We find that cyclometalation stabilizes the singlet ground state of these complexes by 8-19 kcal/mol but reduces the electron density on the carboxylic acid attached to the aryl ring. The results suggest that cyclometalation provides a feasible route to increasing the efficiency of Fe(II) photosensitizers but that care should be taken in choosing the substitution position for the semiconductor anchoring group.

Insights into the Spin-State Transitions in [Fe(tpy)2]2+: Importance of the Terpyridine Rocking Motion.

Iron(II) polypyridine complexes have the potential for numerous applications on a global scale, such as sensitizers, sensors, and molecular memory. The excited-state properties of these systems, particularly the intersystem crossing (ISC) rates, are sensitive to the choice of ligands and can be significantly altered depending on the coordination environment. We employ density functional theory and Smolyak's sparse grid interpolation algorithm to construct potential energy surfaces (PESs) for the photophysically relevant states ((1)A, (3,5)MC, and (1,3)MLCT) of the [Fe(tpy)2](2+) (tpy = 2,2':6',2"-terpyridine) complex, with the goal of obtaining a deeper understanding of the ground- and excited-state electronic structure of this system. The three dimensions that define our adiabatic PESs consist of equatorial and axial metal-ligand bond length distortions and a terpyridine ligand "rocking angle", which has not previously been investigated. The intersection crossing seams and minimum energy crossing points (MECPs) between surfaces are also determined. Overall, we find that the PESs of all electronic excited states investigated are characterized by low-energy valleys along the tpy rocking-angle coordinate. This results in the presence of large low-energy areas around the MECPs on the intersection seams of different electronic states and indicates that inclusion of this third coordinate is crucial for an adequate description of the PESs and surface crossing seams of the [Fe(tpy)2](2+) complex. Finally, we suggest that tuning the energetics of the tpy ligand rocking motion could provide a way to control the ISC process in this complex.

TLR7 is a key regulator of innate immunity against Japanese encephalitis virus infection.

Toll-like receptor 7 (TLR7) known to recognize guanidine-rich ssRNA has been shown to mount vital host defense mechanism against many viruses including flaviviruses. Signal transduction through TLR7 has been shown to produce type-1 interferon and proinflammatory mediators, thereby initiating essential innate immune response against ssRNA viruses in hosts. Systemic and brain specific TLR7 knock-down mice (TLR7(KD)) were generated using vivo-morpholinos. These mice were then subcutaneously challenged with lethal dose of JEV (GP78 strain) and were subsequently analyzed for survival. Significant difference in susceptibility to JEV between wild-type and systemic TLR7(KD) mice was observed whereas, no difference in susceptibility to JEV infection was seen in brain-specific TLR7(KD) mice. Significant decreases in IFN-α and antiviral proteins were also observed in both TLR7(KD) mice along with increased viral loads in their brain. Owing to increased viral load, increases in levels of various proinflammatory cyto/chemokines, increased microglial activation and infiltration of peripheral immune cells in brain of TLR7(KD) mice were also observed. Immunocytochemistry and RNA co-immunoprecipitation performed with JEV-infected N2a or HT22 cells indicated endosomal localization and confirmed interaction between JEV ssRNA with TLR7. Treatment of mice with imiquimod, a TLR7 agonist, prior to JEV infection resulted in their increased survival. Overall, our results suggest that the TLR7 response following JEV infection promotes type-1 interferon production and generation of antiviral state which might contribute to protective effect in systemic infection.

Synaptotagmin-1-dependent phasic axonal dopamine release is dispensable for basic motor behaviors in mice.

In Parkinson's disease (PD), motor dysfunctions only become apparent after extensive loss of DA innervation. This resilience has been hypothesized to be due to the ability of many motor behaviors to be sustained through a diffuse basal tone of DA; but experimental evidence for this is limited. Here we show that conditional deletion of the calcium sensor synaptotagmin-1 (Syt1) in DA neurons (Syt1 cKODA mice) abrogates most activity-dependent axonal DA release in the striatum and mesencephalon, leaving somatodendritic (STD) DA release intact. Strikingly, Syt1 cKODA mice showed intact performance in multiple unconditioned DA-dependent motor tasks and even in a task evaluating conditioned motivation for food. Considering that basal extracellular DA levels in the striatum were unchanged, our findings suggest that activity-dependent DA release is dispensable for such tasks and that they can be sustained by a basal tone of extracellular DA. Taken together, our findings reveal the striking resilience of DA-dependent motor functions in the context of a near-abolition of phasic DA release, shedding new light on why extensive loss of DA innervation is required to reveal motor dysfunctions in PD.

Conditional deletion of neurexins dysregulates neurotransmission from dopamine neurons.

Midbrain dopamine (DA) neurons are key regulators of basal ganglia functions. The axonal domain of these neurons is highly complex, with a large subset of non-synaptic release sites and a smaller subset of synaptic terminals from which in addition to DA, glutamate or GABA are also released. The molecular mechanisms regulating the connectivity of DA neurons and their neurochemical identity are unknown. An emerging literature suggests that neuroligins, trans-synaptic cell adhesion molecules, regulate both DA neuron connectivity and neurotransmission. However, the contribution of their major interaction partners, neurexins (Nrxns), is unexplored. Here, we tested the hypothesis that Nrxns regulate DA neuron neurotransmission. Mice with conditional deletion of all Nrxns in DA neurons (DAT::NrxnsKO) exhibited normal basic motor functions. However, they showed an impaired locomotor response to the psychostimulant amphetamine. In line with an alteration in DA neurotransmission, decreased levels of the membrane DA transporter (DAT) and increased levels of the vesicular monoamine transporter (VMAT2) were detected in the striatum of DAT::NrxnsKO mice, along with reduced activity-dependent DA release. Strikingly, electrophysiological recordings revealed an increase of GABA co-release from DA neuron axons in the striatum of these mice. Together, these findings suggest that Nrxns act as regulators of the functional connectivity of DA neurons.

The human brain contains billions of nerve cells, known as neurons, which receive input from the outside world and process this information in the brain. Neurons communicate with each other by releasing chemical messengers from specialized structures, called axon terminals, some of which form junctions known as synapses. These messengers then generate signals in the target neurons. Based on the type of chemical they release, neurons can be classified into different types. For example, neurons releasing dopamine are considered to act as key regulators of learning, movements and motivation. Such neurons establish very large numbers of axon terminals, but very few of them form synapses. Specific sets of proteins, including neurexins and neuroligins, are thought to help regulate the activity of the connexions between these neurons. Previous research has shown that when neuroligins were removed from the neurons of worms or mice, it affected the ability of the animals to move. So far, the role of neurexins in managing the connectivity of regulatory neurons, such as those releasing dopamine, has received much less attention. To bridge this knowledge gap, Ducrot et al. explored how removing neurexins from dopamine neurons in mice affected their behaviour. The experiments revealed that eliminating neurexins did not affect their motor skills on a rotating rod, but it did reduce their movements in response to the psychostimulant amphetamine, a molecule known to enhance dopamine-associated behaviours. The cellular structure of dopamine neurons lacking neurexins was the same as in neurons containing this protein. But dopamine neurons without neurexins were slower to recycle dopamine, and they released a higher amount of the inhibitory messenger GABA. This suggests that neurexin acts as an important suppressor of GABA secretion to help regulate the signals released by dopamine neurons. These findings set the stage for further research into the role of neurexins in regulating dopamine and other populations of neurons in conditions such as Parkinson's disease, where movement and coordination are affected.

Bone marrow-derived extracellular vesicles modulate the abundance of infiltrating immune cells in the brain and exert an antiviral effect against the Japanese encephalitis virus.

Mesenchymal stem cells (MSCs) have regenerative capacity and have reported a beneficial effect on the Japanese encephalitis virus (JEV) in an encephalitis model. However, the MSCs do not cross the blood-brain barrier and have other disadvantages limiting their therapeutic utility scope. Recently, there has been a shift in concept from a cell-based to a cell-free approach using MSCs-derived extracellular vesicles (MSC-EVs). The MSC-EVs retain regenerative and immunomodulatory capacity as their parental cells. However, the role of MSC-EVs in limiting JEV pathology remains elusive. In this study, we have used Bone marrow (BM)-derived EV (BM-EVs) and assessed their effect on JEV replication and pathogenesis in primary neuronal stem cells and a murine model. The in vitro and in vivo studies suggested that BM-derived EVs delay JEV-induced symptoms and death in mice, improve the length of survival, accelerate neurogenesis in primary neuronal stem cells, reduce JEV-induced neuronal death, and attenuate viral replication. BM-EVs treatment upregulated interferon-stimulated genes. Flow cytometry analysis revealed a reduction in the frequency of macrophages. At the same time, CD4+ T cells and neutrophils were significantly augmented, accompanied by the alteration of cytokine expression with the administration of BM-EVs, reinforcing the immunomodulatory role of EVs during JEV-induced encephalitis. In conclusion, our study describes the beneficial role of BM-EVs in limiting JEV pathology by attenuating virus replication, enhancing antiviral response, and neurogenesis in primary neuronal stem cells. However, BM-EVs do not seem to protect BBB integrity and alter immune cell infiltration into the treated brain.

Involvement of RIG-I Pathway in Neurotropic Virus-Induced Acute Flaccid Paralysis and Subsequent Spinal Motor Neuron Death.

Poliomyelitis-like illness is a common clinical manifestation of neurotropic viral infections. Functional loss and death of motor neurons often lead to reduced muscle tone and paralysis, causing persistent motor sequelae among disease survivors. Despite several reports demonstrating the molecular basis of encephalopathy, the pathogenesis behind virus-induced flaccid paralysis remained largely unknown. The present study for the first time aims to elucidate the mechanism responsible for limb paralysis by studying clinical isolates of Japanese encephalitis virus (JEV) and Chandipura virus (CHPV) responsible for causing acute flaccid paralysis (AFP) in vast regions of Southeast Asia and the Indian subcontinent. An experimental model for studying virus-induced AFP was generated by intraperitoneal injection of 10-day-old BALB/c mice. Progressive decline in motor performance of infected animals was observed, with paralysis being correlated with death of motor neurons (MNs). Furthermore, we demonstrated that upon infection, MNs undergo an extrinsic apoptotic pathway in a RIG-I-dependent fashion via transcription factors pIRF-3 and pIRF-7. Both gene-silencing experiments using specific RIG-I-short interfering RNA and in vivo morpholino abrogated cellular apoptosis, validating the important role of pattern recognition receptor (PRR) RIG-I in MN death. Hence, from our experimental observations, we hypothesize that host innate response plays a significant role in deterioration of motor functioning upon neurotropic virus infections. IMPORTANCE Neurotropic viral infections are an increasingly common cause of immediate or delayed neuropsychiatric sequelae, cognitive impairment, and movement disorders or, in severe cases, death. Given the highest reported disability-adjusted life years and mortality rate worldwide, a better understanding of molecular mechanisms for underlying clinical manifestations like AFP will help in development of more effective tools for therapeutic solutions.

Experimental autoimmune vasculitis: an animal model of anti-neutrophil cytoplasmic autoantibody-associated systemic vasculitis.

The morbidity burden associated with anti-neutrophil cytoplasmic autoantibody-associated vasculitis is increasing, and many novel biological therapies are now entering the drug development pipeline. There is thus an urgent need to develop a representative animal model to facilitate testing of these agents. We previously examined the effect of antineutrophil cytoplasmic autoantibody on leukocyte-endothelial interactions in WKY rats via immunization with human myeloperoxidase. We now seek to extend this model so that all animals reliably develop crescentic glomerulonephritis and lung hemorrhage. We also wish to investigate whether there is a genetic contribution to vasculitis development in this rat strain. Using escalating doses of human myeloperoxidase, we found that a dose of 1600 microg/kg induced pauci-immune crescentic glomerulonephritis and lung hemorrhage in all immunized animals. We also found that the addition of pertussis toxin and killed Mycobacterium tuberculosis to the adjuvant when immunizing with 400 microg/kg of myeloperoxidase resulted in crescentic glomerulonephritis and lung hemorrhage in all animals. However, when Lewis, Wistar Furth, or Brown Norway rats were immunized using a similar protocol, no animals developed hematuria or glomerulonephritis, despite having identical levels of anti-human myeloperoxidase antibodies. We conclude that, by adjusting the immunization regimen, all WKY rats immunized with myeloperoxidase develop experimental autoimmune vasculitis, thus facilitating future therapeutic studies. The resistance of Lewis rats to experimental autoimmune vasculitis provides a genetic basis for future studies of anti-myeloperoxidase antibody-associated vasculitis.

Atorvastatin ameliorates viral burden and neural stem/ progenitor cell (NSPC) death in an experimental model of Japanese encephalitis.

Japanese encephalitis virus, a neurotropic flavivirus, causes sporadic encephalitis with nearly 25% fatal case reports. JEV infects neural stem/progenitor cells (NSPCs) and decreases their proliferation. Statin, a commonly used class of cholesterol lowering drug, has been shown to possess potent anti-inflammatory and neuroprotective effects in acute brain injury and chronic neurodegenerative conditions. Here, we aimed to check the efficacy of atorvastatin in alleviating the symptoms of Japanese encephalitis (JE). Using BALB/c mouse model of JEV infection, we observed that atorvastatin effectively reduces viral load in the subventricular zone (SVZ) of infected pups and decreases the resultant cell death. Furthermore, atorvastatin abrogates microglial activation and production of proinflammatory cyto/chemokine production post JEV infection in vivo. It also reduced interferon-ß response in the neurogenic environs. The neuroprotective role of atorvastatin is again evident from the rescued neurosphere size and decreased cell death in vitro. It has also been observed that upon atorvastatin administration, cell cycle regulatory proteins and cell survival proteins are also restored to their respective expression level as observed in uninfected animals. Thus the antiviral, immunomodulatory and neuroprotective roles of atorvastatin reflect in our experimental observations. Therefore, this drug broadens a path for future therapeutic measures against JEV infection.

Designing air-stable cyclometalated Fe(ii) complexes: stabilization via electrostatic effects.

Designing efficient Fe(ii) chromophores requires optimization of numerous, at times conflicting, properties. It has been suggested that replacement of polypyridine ligands with cyclometalated analogs will be effective at destabilizing the quintet state and therefore extending the lifetime of photoactive metal-to-ligand charge transfer states. However, cyclometalated Fe(ii) complexes are not oxidatively stable due to the strong electron-donating nature of this ligand, which limits their applicability. Here we use density functional theory calculations to show how simple addition of nitro and carboxylic acid groups to these cyclometalated complexes can engender a less oxidizable Fe(ii) center while maintaining, or even improving, the favorable ligand field strength.

Identification and Classification of Hubs in microRNA Target Gene Networks in Human Neural Stem/Progenitor Cells following Japanese Encephalitis Virus Infection.

RNA viruses are known to modulate host microRNA (miRNA) machinery for their own benefit. Japanese encephalitis virus (JEV), a neurotropic RNA virus, has been reported to manipulate several miRNAs in neurons or microglia. However, no report indicates a complete sketch of the miRNA profile of neural stem/progenitor cells (NSPCs), hence the focus of our current study. We used an miRNA array of 84 miRNAs in uninfected and JEV-infected human neuronal progenitor cells and primary neural precursor cells isolated from aborted fetuses. Severalfold downregulation of hsa-miR-9-5p, hsa-miR-22-3p, hsa-miR-124-3p, and hsa-miR-132-3p was found postinfection in both of the cell types compared to the uninfected cells. Subsequently, we screened for the target genes of these miRNAs and looked for the biological pathways that were significantly regulated by the genes. The target genes involved in two or more pathways were sorted out. Protein-protein interaction (PPI) networks of the miRNA target genes were formed based on their interaction patterns. A binary adjacency matrix for each gene network was prepared. Different modules or communities were identified in those networks by community detection algorithms. Mathematically, we identified the hub genes by analyzing their degree centrality and participation coefficient in the network. The hub genes were classified as either provincial (P < 0.4) or connector (P > 0.4) hubs. We validated the expression of hub genes in both cell line and primary cells through qRT-PCR after JEV infection and respective miR mimic transfection. Taken together, our findings highlight the importance of specific target gene networks of miRNAs affected by JEV infection in NSPCs.IMPORTANCE JEV damages the neural stem/progenitor cell population of the mammalian brain. However, JEV-induced alteration in the miRNA expression pattern of the cell population remains an open question, hence warranting our present study. In this study, we specifically address the downregulation of four miRNAs, and we prepared a protein-protein interaction network of miRNA target genes. We identified two types of hub genes in the PPI network, namely, connector hubs and provincial hubs. These two types of miRNA target hub genes critically influence the participation strength in the networks and thereby significantly impact up- and downregulation in several key biological pathways. Computational analysis of the PPI networks identifies key protein interactions and hubs in those modules, which opens up the possibility of precise identification and classification of host factors for viral infection in NSPCs.