Colisepticaemia caused by avian pathogenic Escherichia coli (APEC) is a challenging disease due to its high economic importance in poultry, dubious pathogenesis and potential link with zoonosis and food safety. The existing in vitro studies can\'t define hallmark traits of APEC isolates, suggesting a paradigm shift towards host response to understand pathogenesis. This study investigated the comprehensive pathological and microbial progression of colisepticaemia, and transmission of E. coli into eggs using novel tools. In total 48 hens were allocated into three groups and were inoculated intratracheally with ilux2-E. coli PA14/17480/5-/ovary (bioluminescent strain), E. coli PA14/17480/5-/ovary or phosphate buffered saline. Infection with both strains led to typical clinical signs and lesions of colibacillosis as in field outbreaks. Based on lung histopathology, colisepticaemia progression was divided into four disease stages as: stage I (1-3 days post infection (dpi)), stage II (6 dpi), stage III (9 dpi) and stage IV (16 dpi) that were histologically characterized by predominance of heterophils, mixed cells, pyogranuloma, and convalescence, respectively. As disease progressed, bacterial colonization in host organs also decreased, revealed by the quantification of bacterial bioluminescence, bacteriology, and quantitative immunohistochemistry. Furthermore, immunofluorescence, immunohistochemistry, and bacteria re-isolation showed that E. coli colonized the reproductive tract of infected hens and reached to egg yolk and albumen. In conclusion, the study provides novel insights into the pathogenesis of colisepticemia by characterizing microbial and pathological changes at different disease stages, and of the bacteria transmission to table eggs, which have serious consequences on poultry health and food safety.

Approximately 80% of luminous organisms live in the oceans, and considerable diversity of life dependence on bioluminescence has been observed in marine organisms. Among vertebrates, luminous fish species are the only group of vertebrates that have the ability to emit bioluminescent light. Meanwhile, the lantern fish family (Myctophidae), with 33 genera all of which have the ability to emit light, is considered the most prominent family among the luminous fish of the deep oceans and seas. Lantern fish Benthosema pterotum has bioluminescence properties due to the presence of photophores scattered in its ventral-lateral region. However, no research has been performed on its bioluminescence system and light emission mechanism. The present research aimed to assess the type of bioluminescence, pigment, photoprotein, or luciferin-luciferase system in B. pterotum. In order to determine the type of light-emitting system in B. pterotum species, several specific experiments were designed and performed. It was shown that the light emission system in B. pterotum species is categorized into the luciferin-luciferase type. Conducting this research was not only innovative, but it also could be the beginning of further research in the field of marine biochemistry and production of the recombinant active forms of enzymes for industrial, commercial, medical, and pharmaceutical purposes.

A complex heterogeneous intracellular environment seems to affect enzymatic catalysis by changing the mobility of biomolecules, their stability, and their conformational states, as well as by facilitating or hindering continuously occurring interactions. The evaluation and description of the influence of the cytoplasmic matrix components on enzymatic activity are problems that remain unsolved. In this work, we aimed to determine the mechanisms of action of two-component media with cosolvents of various molecular sizes on the complex multi-stage bioluminescent reaction catalyzed by bacterial luciferase. Kinetic and structural effects of ethylene glycol, glycerol, sorbitol, glucose, sucrose, dextran, and polyethylene glycol on bacterial luciferase were studied using stopped-flow and fluorescence spectroscopy techniques and molecular dynamics simulations. We have found that diffusion limitations in the presence of cosolvents promote the stabilization of flavin substrate and peroxyflavin intermediate of the reaction, but do not provide any advantages in bioluminescence quantum yield, because substrate binding is slowed down as well. The catalytic constant of bacterial luciferase has been found to be viscosity-independent and correlated with parameters of water-cosolvent interactions (Norrish constant, van der Waals interaction energies). Crowding agents, in contrast to low-molecular-weight cosolvents, had little effect on peroxyflavin intermediate decay and enzyme catalytic constant. We attributed specific kinetic effects to the preferential interaction of the cosolvents with enzyme surface and their penetration into the active site.

The two forward-looking eyes and their ultrastructural organization of an 18 mm long adult bioluminescent female millipede (Paraspirobolus lucifugus) were investigated by transmission electron microscopy and energy-dispersive X-ray spectroscopy. Each eye contained approximately 23 ommatidia with 50-60 μm wide and 80 um thick corneal lenses that contained calcium and silicon and proximally ended in truncated flat surfaces of around 20 μm in diameter. A maximally 28 μm thick and 25 μm long rhabdom, made up of at least 12-14 retinula cells and a 4 μm thick sleeve of screening pigment granules in a light-adapted position was present. Compared with the eyes of non-luminescent julid millipede species, those of P. lucifugus share their basic anatomy, but also exhibit features like the wide possible binocular frontal visual overlap, somewhat narrower interommatidial angles combined with relatively larger rhabdoms, which suggests that P. lucifugus has more efficient eyes and makes greater use of its photoreceptors. P. lucifugus is negatively phototactic and strictly nocturnal and its activity rhythm is apparently governed by a circadian clock.

Thorium is one of the most widespread radioactive elements in natural ecosystems, along with uranium, it is the most important source of nuclear energy. However, the effects of thorium on living organisms have not been thoroughly studied. Marine luminescent bacteria and their enzymes are optimal bioassays for studying low-dose thorium exposures. Luminescent bioassays provide a quantitative measure of toxicity and are characterized by high rates, sensitivity, and simplicity. It is known that the metabolic activity of bacteria is associated with the production of reactive oxygen species (ROS). We studied the effects of thorium-232 (10-11-10-3 M) on Photobacterium phosphoreum and bacterial enzymatic reactions; kinetics of bacterial bioluminescence and ROS content were investigated in both systems. Bioluminescence activation was revealed under low-dose exposures (<0.1 Gy) and discussed in terms of \"radiation hormesis\". The activation was accompanied by an intensification of the oxidation of a low-molecular reducer, NADH, during the enzymatic processes. Negative correlations were found between the intensity of bioluminescence and the content of ROS in bacteria and enzyme systems; an active role of ROS in the low-dose activation by thorium was discussed. The results contribute to radioecological potential of bioluminescence techniques adapted to study low-intensity radioactive exposures.

Agroinfiltration in Nicotiana benthamiana is widely used to transiently express heterologous proteins in plants. However, the state of Agrobacterium itself is not well studied in agroinfiltrated tissues, despite frequent studies of immunity genes conducted through agroinfiltration. Here, we generated a bioluminescent strain of Agrobacterium tumefaciens GV3101 to monitor the luminescence of Agrobacterium during agroinfiltration. By integrating a single copy of the lux operon into the genome, we generated a stable \'AgroLux\' strain, which is bioluminescent without affecting Agrobacterium growth in vitro and in planta. To illustrate its versatility, we used AgroLux to demonstrate that high light intensity post infiltration suppresses both Agrobacterium luminescence and protein expression. We also discovered that AgroLux can detect Avr/Cf-induced immune responses before tissue collapse, establishing a robust and rapid quantitative assay for the hypersensitive response (HR). Thus, AgroLux provides a non-destructive, versatile and easy-to-use imaging tool to monitor both Agrobacterium and plant responses.

Annelids constitute a diverse phylum with more than 19,000 species, which exhibit greatly varying morphologies and lifestyles ranging from sessile detritivores to fast swimming active predators. The lifestyle of an animal is closely linked to its sensory systems, not least the visual equipment. Interestingly, many errantian annelid species from different families, such as the scale worms (Polynoidae), have two pairs of eyes on their prostomium. These eyes are typically 100-200 µm in diameter and structurally similar judged from their gross morphology. The polynoids Harmothoe imbricata and Lepidonotus squamatus from the North Atlantic are both benthic predators preying on small invertebrates but only H. imbricata can produce bioluminescence in its scales. Here, we examined the eye morphology, photoreceptor physiology and light-guided behaviour in these two scale worms to assess their visual capacity and visual ecology. The structure and physiology of the two pairs of eyes are remarkably similar within each species, with the only difference being the gaze direction. The photoreceptor physiology, however, differs between species. Both species express a single opsin in their eyes, but in H. imbricata the peak sensitivity is green shifted and the temporal resolution is lower, suggesting that the eyes of H. imbricata are adapted to detect their own bioluminescence. The behavioural experiments showed that both species are strictly night active but yielded no support for the hypothesis that H. imbricata is repelled by its own bioluminescence.

Hormesis is a biphasic dose-response relationship featured by low-dose stimulation and high-dose inhibition. Although the hormetic phenomenon has been extensively studied over the past decades, there is little information regarding the influence of energy source on the occurrence of hormesis, especially the time-dependent one. In this study, to explore the role of cultivation system\'s energy source in time-dependent hormesis, the toxic dose-responses of Aliivibrio fischeri (A. fischeri) bioluminescence to Sulfadoxine (SDX) during 24 h were determined in four cultivation systems with different energy source conditions. The results indicated that the time-dependent hormetic effects were induced by SDX in all cultivation systems: SDX triggered hormetic phenomenon on the bioluminescence at each growth stage over 24 h in the cultivation systems with sufficient and insufficient energy source; due to the diauxic growth of A. fischeri under multiple energy source conditions, the hormetic effects of SDX gradually disappeared after the preferred energy source was used up. It was speculated that the inhibitory action of SDX was derived from its interaction with DHPS to impede the synthesis of proteins, and SDX bound with AC to upregulate the quorum sensing (QS) system to exhibit the stimulatory action. Comparing the time-dependent hormesis in each cultivation system, it was obtained that the energy source could impact the hourly maximum stimulatory rate, the EC50 of SDX, and the time point that hormesis occurred, which might result from the influence of energy source on the stimulatory and inhibitory actions of SDX through regulating the metabolic system (individual level) and QS system (group level) of bacteria. This study clarifies the importance of energy source for hormesis occurrence, which may further promote the development of hormesis.

A fundamental feature of circadian clock neurons across species is that they express circadian rhythms in spontaneous spike frequency. Spike frequency rhythms serve as both output timing signals of clock neurons as well as resonant elements of rhythms generation. Importantly, optogenetics, as applied to clock neurons, can enable investigation of the roles of clock neuron electrical activity in circadian timing. Here we describe protocols for using both in vitro and in vivo optogenetics directed to mammalian clock neurons in the suprachiasmatic nucleus to study circadian physiology and behavior. Optogenetic stimulation via channelrhodopsin, or inhibition via halorhodopsin, allows for the precise manipulation of neuronal firing rates across the SCN, and within specific neuronal subpopulations thereof, and can be combined with actigraphy and gene expression analysis.

Little is known on the phenotypic characteristics of stem cells (SCs) after they are transplanted to the myocardium, in part due to lack of noninvasive platforms to study SCs directly in the living subject. Reporter gene imaging has played a valuable role in the noninvasive assessment of cell fate in vivo. In this study, we validated a pathway-specific reporter gene that can be used to noninvasively image the phenotype of SCs transplanted to the myocardium. Rat mesenchymal SCs (MSCs) were studied for phenotypic evidence of myogenic characteristics under in vitro conditions. After markers of myogenic characteristics were identified, we constructed a reporter gene sensor, comprising the firefly luciferase (Fluc) reporter gene driven by the troponin T (TnT) promoter (cardio MSCs had threefold expression in polymerase chain reaction compared to control MSCs) using a two-step signal amplification strategy. MSCs transfected with TnT-Fluc were studied and validated under in vitro conditions, showing a strong signal after MSCs acquired myogenic characteristics. Lastly, we observed that cardio MSCs had higher expression of the reporter sensor compared to control cells (0.005 ± 0.0005 vs 0.0025 ± 0.0008 Tnt-Fluc/ubiquitin-Fluc, P < .05), and that this novel sensor can detect the change in the phenotype of MSCs directly in the living subject. Pathway-specific reporter gene imaging allows assessment of changes in the phenotype of MSCs after delivery to the ischemic myocardium, providing important information on the phenotype of these cells. Imaging sensors like the one described here are critical to better understanding of the changes that SCs undergo after transplantation.