Natural Rabies Infection in a Domestic Fowl (Gallus domesticus): A Report from India

Background

Rabies is a fatal encephalitis caused by viruses belonging to the genus Lyssavirus of the family Rhabdoviridae. It is a viral disease primarily affecting mammals, though all warm blooded animals are susceptible. Experimental rabies virus infection in birds has been reported, but naturally occurring infection of birds has been documented very rarely.

Principal Findings

The carcass of a domestic fowl (Gallus domesticus), which had been bitten by a stray dog one month back, was brought to the rabies diagnostic laboratory. A necropsy was performed and the brain tissue obtained was subjected to laboratory tests for rabies. The brain tissue was positive for rabies viral antigens by fluorescent antibody test (FAT) confirming a diagnosis of rabies. Phylogenetic analysis based on nucleoprotein gene sequencing revealed that the rabies virus strain from the domestic fowl belonged to a distinct and relatively rare Indian subcontinent lineage.

Significance

This case of naturally acquired rabies infection in a bird species, Gallus domesticus, being reported for the first time in India, was identified from an area which has a significant stray dog population and is highly endemic for canine rabies. It indicates that spill over of infection even to an unusual host is possible in highly endemic areas. Lack of any clinical signs, and fewer opportunities for diagnostic laboratory testing of suspected rabies in birds, may be the reason for disease in these species being undiagnosed and probably under-reported. Butchering and handling of rabies virus- infected poultry may pose a potential exposure risk.     

Identification of the synaptic vesicle glycoprotein 2 receptor binding site in botulinum neurotoxin A

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release by hydrolysing SNARE proteins. The most important serotype BoNT/A employs the synaptic vesicle glycoprotein 2 (SV2) isoforms A-C as neuronal receptors. Here, we identified their binding site by blocking SV2 interaction using monoclonal antibodies with characterised epitopes within the cell binding domain (H_C). The site is located on the backside of the conserved ganglioside binding pocket at the interface of the H_CC and H_CN subdomains. The dimension of the binding pocket was characterised in detail by site directed mutagenesis allowing the development of potent inhibitors as well as modifying receptor binding properties.     

Running as fast as it can: How spiking dynamics form object groupings in the laminar circuits of visual cortex

How spiking neurons cooperate to control behavioral processes is a fundamental problem in computational neuroscience. Such cooperative dynamics are required during visual perception when spatially distributed image fragments are grouped into emergent boundary contours. Perceptual grouping is a challenge for spiking cells because its properties of collinear facilitation and analog sensitivity occur in response to binary spikes with irregular timing across many interacting cells. Some models have demonstrated spiking dynamics in recurrent laminar neocortical circuits, but not how perceptual grouping occurs. Other models have analyzed the fast speed of certain percepts in terms of a single feedforward sweep of activity, but cannot explain other percepts, such as illusory contours, wherein perceptual ambiguity can take hundreds of milliseconds to resolve by integrating multiple spikes over time. The current model reconciles fast feedforward with slower feedback processing, and binary spikes with analog network-level properties, in a laminar cortical network of spiking cells whose emergent properties quantitatively simulate parametric data from neurophysiological experiments, including the formation of illusory contours; the structure of non-classical visual receptive fields; and self-synchronizing gamma oscillations. These laminar dynamics shed new light on how the brain resolves local informational ambiguities through the use of properly designed nonlinear feedback spiking networks which run as fast as they can, given the amount of uncertainty in the data that they process.     

Nucleotides affect neurogenesis and dopaminergic differentiation of mouse fetal midbrain-derived neural precursor cells

The fetal midbrain is a preferred source for isolating and producing dopaminergic neurons for subsequent grafting and replacement of damaged or lost dopaminergic midbrain neurons. We analysed the potential of a variety of nucleotides and of adenosine to support dopaminergic neuron formation from primary mouse fetal midbrain-derived cells, harvested at E10.5 and at E13.5 and subjected to adherent cell culture. In contrast to cells derived at E13.5, cells derived at E10.5 have the potential to produce dopaminergic neurons in culture. These neurons express tyrosine hydroxylase and the dopamine transporter. The fetal ventral midbrain contained mRNA encoding almost all P2X and P2Y receptors, all adenosine receptors as well as the ectonucleotidases nucleoside triphosphate diphosphohydrolase 2 and tissue nonspecific alkaline phosphatase. Essentially, all components of the purinergic signalling pathway were also expressed by the cultured cells. ATP, ADPβS, 2MeSATP, 2ClATP and adenosine increased neuron formation. There was, however, no preference for the formation of dopaminergic neurons—with the exception of 2ClATP that increased the relative contribution of tyrosine hydroxylase-positive neurons. In cells isolated at E13.5 UTP promoted neuron survival but ADPβS and ATPγS essentially eliminated neurons. These data showed that the outcome of nucleotide application was different even though cells isolated at E10.5 and E13.5 expressed very similar receptor mRNA profiles. They suggest that purinergic agonists carry potential for stimulating neurogenesis and enriching the contribution of dopaminergic neurons in vitro. Nucleotide receptor agonists may be of value for contributing to the formation and survival of dopaminergic neurons in vivo.     

Chemoattraction of macrophages, T lymphocytes, and mast cells is evolutionarily conserved within the human alpha-defensin family

Human defensins are natural peptide antibiotics. On the basis of the position and bonding of six conserved cysteine residues, they are divided into two families, designated alpha- and beta-defensins. Human alpha-defensins are expressed predominantly in neutrophils (human neutrophil peptides (HNP) 1-4) or intestinal Paneth cells (human defensins (HD) 5 and 6). Although alpha-defensins have been implicated in the pathogenesis of inflammatory bowel disease, their immunomodulatory functions are poorly understood. In the present study, HNP-1, HNP-3, and HD5 were found to be potent chemotaxins for macrophages but not dendritic cells using Galphai proteins and MAPK as signal transducers. Alpha-defensins were also chemoattractive for the human mast cell line HMC-1 but lacked, in contrast to beta-defensins, the ability to induce intracellular calcium fluxes. Furthermore, HNP-1, HNP-3, and HD5 comparably mobilized naive as well as memory T lymphocytes. Using the protein kinase C (PKC) inhibitors GF109 and G?6976, we observed a PKC-independent functional desensitization to occur between human alpha-defensins, which suggests a common receptor for HNP-1, HNP-3, and HD5 on immune cells. This alpha-defensin receptor was subject to heterologous desensitization by the PKC activator PMA and to PKC-dependent cross-desensitization by human beta-defensins. Conversely, alpha-defensins desensitized beta-defensin-mediated migration of immune cells in a PKC-dependent manner, suggesting unique receptors for both defensin families. Taken together, our observations indicate that chemoattraction of macrophages, T lymphocytes, and mast cells represents an immunomodulatory function which is evolutionarily conserved within the human alpha-defensin family and tightly regulated by beta-defensins.     

Evolution of a single niche specialist in variable environments

The pattern (space versus time) and scale (relative to the lifetime of individuals) of environmental variation is thought to play a central role in governing the evolution of the ecological niche and the maintenance of genetic variance in fitness. To evaluate this idea, we serially propagated an initially genetically uniform population of the bacterium Pseudomonas fluorescens for a few hundred generations in environments that differed in both the pattern and scale at which two highly contrasted carbon substrates were experienced. We found that, contrary to expectations, populations often evolved into a single niche specialist adapted to the less-productive substrate in variable environments and that the genetic variance in fitness across different components of the environment was not generally higher in variable environments when compared with constant environments. We provide evidence to suggest that our results reflect a novel constraint on niche evolution imposed by the supply of beneficial mutations available to selection in variable environments.     

Differential response of etiolated pea seedlings to inoculation with rhizobacteria capable of utilizing 1-aminocyclopropane-1-carboxylate or L-methionine

The majority of soil microorganisms can derive ethylene from L-methionine (L-MET), while some rhizobacteria can hydrolyze 1-aminocyclopropane-1-carboxylate (ACC) due to their ACC-deaminase activity. In this study, three strains having either ACC-deaminase activity (Pseudomonas putida biotype A, A7), or the ability to produce ethylene from L-MET (Acinetobacter calcoaceticus, M9) or both (Pseudomonas fluorescens, AM3) were used for inoculation. The highly ethylene specific bioassay of a classical "triple" response in pea seedlings was used to investigate the effect of the inoculation with the rhizobacteria in the presence of 10 mM ACC or L-MET. The exogenous application of ACC had a concentration-dependent effect on the etiolated pea seedlings in creating the classical "triple" response. The inoculation with P. putida diluted the effect of ACC, which was most likely due to its ACC-deaminase activity. Similarly, the application of Co2+ reduced the ACC-imposed effect on etiolated pea seedlings. In contrast, the inoculation of A. calcoaceticus or P. fluorescens in the presence of L-MET caused a stronger classical "triple" response in etiolated pea seedlings; most likely by producing ethylene from L-MET. This is the first study, to our knowledge, reporting on the comparative effect of rhizobacteria capable of utilizing ACC vs L-MET on etiolated pea seedlings.     

2-Cysteine Peroxiredoxins and Thylakoid Ascorbate Peroxidase Create a Water-Water Cycle That Is Essential to Protect the Photosynthetic Apparatus under High Light Stress Conditions

Different peroxidases, including 2-cysteine (2-Cys) peroxiredoxins (PRXs) and thylakoid ascorbate peroxidase (tAPX), have been proposed to be involved in the water-water cycle (WWC) and hydrogen peroxide (H₂O₂)-mediated signaling in plastids. We generated an Arabidopsis (Arabidopsis thaliana) double-mutant line deficient in the two plastid 2-Cys PRXs (2-Cys PRX A and B, 2cpa 2cpb) and a triple mutant deficient in 2-Cys PRXs and tAPX (2cpa 2cpb tapx). In contrast to wild-type and tapx single-knockout plants, 2cpa 2cpb double-knockout plants showed an impairment of photosynthetic efficiency and became photobleached under high light (HL) growth conditions. In addition, double-mutant plants also generated elevated levels of superoxide anion radicals, H₂O₂, and carbonylated proteins but lacked anthocyanin accumulation under HL stress conditions. Under HL conditions, 2-Cys PRXs seem to be essential in maintaining the WWC, whereas tAPX is dispensable. By comparison, this HL-sensitive phenotype was more severe in 2cpa 2cpb tapx triple-mutant plants, indicating that tAPX partially compensates for the loss of functional 2-Cys PRXs by mutation or inactivation by overoxidation. In response to HL, H₂O₂- and photooxidative stress-responsive marker genes were found to be dramatically up-regulated in 2cpa 2cpb tapx but not 2cpa 2cpb mutant plants, suggesting that HL-induced plastid to nucleus retrograde photooxidative stress signaling takes place after loss or inactivation of the WWC enzymes 2-Cys PRX A, 2-Cys PRX B, and tAPX.Plants are frequently exposed to different abiotic stresses, including high light (HL), UV irradiation, heat, cold, and drought. A component common to these stresses is the rapid formation of reactive oxygen species (ROS) as the result of metabolic dysbalances. A major ROS produced under moderate light (ML) and, in particular, HL photooxidative stress conditions was shown to be singlet oxygen, ¹O₂, that is produced in illuminated chloroplasts predominantly at the PSII (Triantaphylidès et al., 2008). Most of the singlet oxygen is quenched by carotenoids and tocopherols or reacts with galactolipids in thylakoid membranes, yielding galactolipid hydroperoxides (Zoeller et al., 2012; Farmer and Mueller, 2013). In addition, superoxide radicals, O₂·⁻, are produced predominantly at the PSI and rapidly dismutate to hydrogen peroxide (H₂O₂) either spontaneously or because of being catalyzed by superoxide dismutase. Hence, lipid peroxides and H₂O₂ are produced close to the photosystems and may damage thylakoid proteins. In this context, 2-Cys peroxiredoxin (PRX) enzymes have been implicated in the reductive detoxification of lipid peroxides and H₂O₂ (König et al., 2002).During photosynthesis, light energy absorbed by PSII is used to split water molecules, and the electrons are channeled from PSII through PSI to ferredoxin (Fd). As a result, electrons flow from water to Fd. The main electron sink reaction is the Fd NADP oxidoreductase-catalyzed production of NADPH that functions as an electron donor to reduce carbon dioxide to sugars. Under HL conditions, excessive excitation energy is dissipated into heat, which was indicated by nonphotochemical quenching of chlorophyll fluorescence. In addition, excessive photosynthetic electrons can be donated from PSI to O₂, yielding O₂·⁻ (Miyake, 2010). This process, the Mehler reaction, creates an alternative electron sink and electron flow. Superoxide anion radicals, O₂·⁻, can be dismutated to O₂ and H₂O₂ by a thylakoid-attached copper/zinc superoxide dismutase (Cu/ZnSOD; Rizhsky et al., 2003). H₂O₂ can then be reduced to water by peroxidases. As a result, O₂ molecules originating from the water-splitting process at PSII are reduced to water by electrons originating from PSI. This process is termed the water-water cycle (WWC) that is thought to protect the photosynthetic apparatus from excessive light and alleviate photoinhibition.In the classical WWC, the Mehler-ascorbate peroxidase (MAP) pathway, ascorbate peroxidases (APXs) have been considered as key enzymes in the reductive detoxification of H₂O₂ in chloroplasts (Kangasjärvi et al., 2008). APXs reduce H₂O₂ to water and oxidize ascorbate to monodehydroascorbate radicals. NADPH functions as an electron donor to regenerate ascorbate by monodehydroascorbate radical reductase. There are two functional APX homologs in plastids: a 33-kD stromal ascorbate peroxidase (sAPX) and a 38-kD thylakoid ascorbate peroxidase (tAPX). The latter tAPX is thought to reside close to the site of H₂O₂ generation at PSI. Surprisingly, knockout-tAPX mutants as well as double mutants lacking both the tAPX and the sAPX exhibited no visible symptoms of stress after long-term (1–14 d) HL (1.000 µmol photons m⁻² s⁻¹) exposure (Giacomelli et al., 2007; Kangasjärvi et al., 2008; Maruta et al., 2010). Moreover, the photosynthetic efficiency of PSII (as judged by the maximum photochemical efficiency of PSII in the dark-adapted state [F_v/F_m]), H₂O₂ production, antioxidant levels (ascorbate, glutathione, and tocopherols), protein oxidation, and anthocyanin accumulation were similar between light-stressed mutant and wild-type plants. Hence, other H₂O₂ detoxification mechanisms can efficiently compensate for the lack of the sAPX and tAPX detoxification system.In addition to APX, glutathione peroxidases and PRXs may reduce H₂O₂ to water. It has been postulated that, in the chloroplast, two highly homologous thylakoid-associated 2-Cys peroxiredoxins (2CPs), 2CPA and 2CPB, can create an alternative ascorbate-independent WWC (Dietz et al., 2006). In support of this concept, HL stress-acclimated tapx sapx double-mutant plants showed increased levels of 2-Cys PRX compared with wild-type plants (Kangasjärvi et al., 2008). Because the two plastidial 2CPA and 2CPB dynamically interact with the stromal side of thylakoid membranes and are capable of reducing peroxides, 2-Cys PRX enzymes may be involved in both H₂O₂ detoxification and reduction of lipid peroxides in thylakoids (König et al., 2002).The reaction mechanism of 2-Cys PRX is highly conserved and involves a Cys residue, which becomes transiently oxidized to sulphenic acid (termed the peroxidatic Cys residue), thereby reducing H₂O₂ to water. The sulphenic acid is subsequently attacked by a second Cys residue, termed resolving Cys residue, yielding an intermolecular disulfide bridge and water (Dietz, 2011).At high peroxide concentrations, the peroxidase function of 2-Cys PRX becomes inactivated through overoxidation, and excess H₂O₂ may function as a redox signal (Puerto-Galán et al., 2013). It has been postulated that 2-Cys PRXs function as a floodgate that allows H₂O₂ signaling only under oxidative stress conditions (Wood et al., 2003; Dietz, 2011; Puerto-Galán et al., 2013). In addition to its function as peroxidase, 2-Cys PRX may also serve as proximity-based thiol oxidases and chaperones (König et al., 2013).The genome of Arabidopsis (Arabidopsis thaliana) contains two 2CP genes. To study 2-Cys PRX function, transgenic plants with reduced 2-Cys PRX levels were generated by antisense suppression (Baier et al., 2000) as well as crossing of transfer DNA (T-DNA) insertion mutants (Pulido et al., 2010). The T-DNA insertion double mutant was shown to contain less than 5% of the wild-type content of 2CPA and no 2CPB. Hence, full knockout lines lacking both 2-Cys PRXs have not yet been established. Under standard growth conditions, 2-Cys PRX double mutants (similar to plastid APX-deficient plants) also did not show a photooxidative stress phenotype that might be because of compensation by alternative H₂O₂ reduction systems (Pulido et al., 2010). Because of the lack of a clear phenotype of the 2-Cys PRX double-knockdown mutant under ML conditions, the physiological functions of 2CPA and 2CPB remain to be elucidated.The main aim of this study was to identify the physiological function of 2CPA and 2CPB under HL stress conditions, when the WWC is of particular importance in protecting the photosynthetic apparatus from photooxidative damage. We investigated mutants completely deficient in 2-Cys PRX (2cpa 2cpb) or tAPX (tapx) and in addition, 2cpa 2cpb tapx triple knockout plants to study the extent of the functional overlap between these enzymes. Results suggest that 2-Cys PRXs are involved in a 2-Cys PRX-dependent WWC that seems to be more important in protecting the photosynthetic apparatus than the tAPX-dependent WWC, the MAP cycle.     

Spectroscopic investigation on glutamic acid by Coulomb-attenuating and double hybrid density functional theory methods

This study deals with the identification of glutamic acid by means of quantum chemical approach. FT-IR, FT-Raman and UV–vis spectra were recorded in the region 4000–400, 4000–50 cm^? 1 and 200–600 nm, respectively. CAM-B3LYP/6-31G(d,p) and B2PLYP/6-31G(d,p) calculations were performed to obtain the optimised molecular structures, vibrational frequencies and corresponding vibrational assignment, thermodynamic properties and natural bonding orbital (NBO) analysis. The results show that the obtained optimised geometric parameters (bond lengths, bond angles and bond dihedrals) and vibrational frequencies were found to be in good agreement with the experimental results. The calculations of the electronic spectra were compared with the experimental ones. Furthermore, highest occupied molecular orbital and lowest unoccupied molecular orbital analyses and UV–vis spectral analysis were also performed to determine the energy band gaps and transition states. NBO analysis, calculated using density functional theory methods (CAM-B3LYP/6-31G(d,p) and B2PLYP/6-31G(d,p)), was induced to find inter-molecular atoms. ¹³C and ¹H NMR isotropic chemical shifts were calculated and the assignments made were compared with the ChemDraw Ultra values.