ABSTRACT
Background: Outcomes after oesophagogastric cancer surgery remain poor. Cardiopulmonary exercise testing (CPET) used for risk stratification before oesophagogastric cancer surgery is based on conflicting evidence. This study explores the relationship between CPET and postoperative outcomes, specifically for patients undergoing neoadjuvant treatment. Methods: Patients undergoing oesophagogastric cancer resection and CPET (pre- or post-neoadjuvant treatment, or both) were retrospectively enrolled into a multicentre pooled cohort study. Oxygen uptake at peak exercise (VO2 peak) was compared with 1-yr postoperative survival. Secondary analyses explored relationships between patient characteristics, tumour pathology characteristics, CPET variables (absolute, relative to weight, ideal body weight, and body surface area), and postoperative outcomes (morbidity, 1-yr and 3-yr survival) were assessed using logistic regression analyses. Results: Seven UK centres recruited 611 patients completing a 3-yr postoperative follow-up period. Oesophagectomy was undertaken in 475 patients (78%). Major complications occurred in 25%, with 18% 1-yr and 43% 3-yr mortality. No association between VO2 peak or other selected CPET variables and 1-yr survival was observed in the overall cohort. In the overall cohort, the anaerobic threshold relative to ideal body weight was associated with 3-yr survival (P=0.013). Tumour characteristics (ypT/ypN/tumour regression/lymphovascular invasion/resection margin; P<0.001) and Clavien-Dindo ≥3a (P<0.001) were associated with 1-yr and 3-yr survival. On subgroup analyses, pre-neoadjuvant treatment CPET; anaerobic threshold (absolute; P=0.024, relative to ideal body weight; P=0.001, body surface area; P=0.009) and VE/VCO2 at anaerobic threshold (P=0.026) were associated with 3-yr survival. No other CPET variables (pre- or post-neoadjuvant treatment) were associated with survival. Conclusions: VO2 peak was not associated with 1-yr survival after oesophagogastric cancer resection. Tumour characteristics and major complications were associated with survival; however, only some selected pre-neoadjuvant treatment CPET variables were associated with 3-yr survival. CPET in this cohort of patients demonstrates limited outcome predictive precision. Clinical trial registration: NCT03637647.
ABSTRACT
Cryptochromes (Crys) represent a multi-facetted class of proteins closely associated with circadian clocks. They have been shown to function as photoreceptors but also to fulfill light-independent roles as transcriptional repressors within the negative feedback loop of the circadian clock. In addition, there is evidence for Crys being involved in light-dependent magneto-sensing, and regulation of neuronal activity in insects, adding to the functional diversity of this cryptic protein class. In mammals, Crys are essential components of the circadian clock, but their role in other vertebrates is less clear. In invertebrates, Crys can function as circadian photoreceptors, or as components of the circadian clock, while in some species, both light-receptive and clock factor roles coexist. In the current study, we investigate the function of Cry proteins in zebrafish (Danio rerio), a freshwater teleost expressing 6 cry genes. Zebrafish peripheral circadian clocks are intrinsically light-sensitive, suggesting the involvement of Cry in light-resetting. Echinoderms (Strongylocentrotus purpuratus) represent the only class of deuterostomes that possess an orthologue (SpuCry) of the light-sensitive Drosophila melanogaster Cry, which is an important component of the light-resetting pathway, but also works as transcriptional repressor in peripheral clocks of fruit flies. We therefore investigated the potential of different zebrafish cry genes and SpuCry to replace the light-resetting and repressor functions of Drosophila Cry by expressing them in fruit flies lacking endogenous cry function. Using various behavioral and molecular approaches, we show that most Cry proteins analyzed are able to fulfill circadian repressor functions in flies, except for one of the zebrafish Crys, encoded by cry4a. Cry4a also shows a tendency to support light-dependent Cry functions, indicating that it might act in the light-input pathway of zebrafish.
Subject(s)
Circadian Clocks , Circadian Rhythm , Cryptochromes , Drosophila melanogaster , Zebrafish , Animals , Cryptochromes/genetics , Cryptochromes/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/physiology , Zebrafish/genetics , Circadian Clocks/genetics , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Light , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism , Aquatic Organisms/geneticsABSTRACT
The agile wallaby (Notamacropus agilis) is one of the most abundant marsupial species in northern Queensland and a competent host for the zoonotic Ross River virus. Despite their increased proximity and interactions with humans, little is known about the viruses carried by these animals, and whether any are of conservation or zoonotic importance. Metagenomics and molecular techniques were used in a complementary manner to identify and characterize novel viruses in the fecal samples of free-ranging agile wallabies. We detected a variety of novel marsupial-related viral species including agile wallaby atadenovirus 1, agile wallaby chaphamaparvovirus 1-2, agile wallaby polyomavirus 1-2, agile wallaby associated picobirnavirus 1-9, and a known macropod gammaherpesvirus 3. Phylogenetic analyses indicate that most of these novel viruses would have co-evolved with their hosts (agile wallabies). Additionally, non-marsupial viruses that infect bacteria (phages), plants, insects, and other eukaryotes were identified. This study highlighted the utility of non-invasive sampling as well as the integration of broad-based molecular assays (consensus PCR and next generation sequencing) for monitoring the emergence of potential pathogenic viruses in wildlife species. Furthermore, the novel marsupial viruses identified in this study will enrich the diversity of knowledge about marsupial viruses, and may be useful for developing diagnostics and vaccines.
Subject(s)
Macropodidae , Viruses , Animals , Humans , Phylogeny , Animals, Wild , FecesABSTRACT
Most animals on earth have evolved under daily light-dark cycles and consequently possess a circadian clock which regulates much of their biology, from cellular processes to behaviour. There are however some animals that have invaded dark ecosystems and have adapted to an apparently arrhythmic environment. One such example is the Mexican blind cavefish Astyanax mexicanus, a species complex with over 30 different isolated cave types, including the founding surface river fish. These cavefish have evolved numerous fascinating adaptations to the dark, such as loss of eyes, reduced sleep phenotype and alterations in their clock and light biology. While cavefish are an excellent model for studying circadian adaptations to the dark, their rarity and long generational time makes many studies challenging. To overcome these limitations, we established embryonic cell cultures from cavefish strains and assessed their potential as tools for circadian and light experiments. Here, we show that despite originating from animals with no eyes, cavefish cells in culture are directly light responsive and show an endogenous circadian rhythm, albeit that light sensitivity is relatively reduced in cave strain cells. Expression patterns are similar to adult fish, making these cavefish cell lines a useful tool for further circadian and molecular studies.
Subject(s)
Circadian Clocks , Ecosystem , Animals , Fishes , Acclimatization , CavesABSTRACT
Here, we report the draft genome sequence of a novel agile wallaby adenovirus that was detected in the fecal metagenome of agile wallabies. The genome is 31,512 bp long, with a G+C content of 34.4%. Currently, the pathogenic and zoonotic potential of this novel virus is unknown.
ABSTRACT
Consensus PCR assays that can be used to sensitively detect several herpesvirus (HV) species across the different subfamilies were developed in this study. Primers containing degenerate bases were designed to amplify regions of the DNA polymerase (DPOL) gene of alpha- and gamma-HVs, and the glycoprotein B (gB) gene of beta-HVs in a singleplex, non-nested touchdown PCR format. The singleplex touchdown consensus PCR (STC-PCR) was used to amplify the DNA of eight human and 24 animal HVs. The assay was able to detect the lowest DNA dilution of 10-5 for alpha-HVs and 10-3 for beta- and gamma-HVs. In comparison, lowest detection limits of 10-5, 10-3, and 10-2 were obtained for alpha-, beta-, and gamma-HVs respectively when a nested PCR was used. The findings in this study suggest that the STC-PCR assays can be employed for the molecular surveys and clinical detection of novel and known HVs.
Subject(s)
DNA, Viral , Herpesviridae , Animals , Humans , Consensus , DNA, Viral/genetics , DNA, Viral/analysis , Herpesviridae/genetics , DNA Primers/genetics , Polymerase Chain ReactionABSTRACT
BACKGROUND: Cytomegalovirus (CMV) is an important opportunistic pathogen after transplantation. Some virological variation in transplant recipients is explained by donor and recipient CMV serostatus, but not all. Circadian variability of herpesviruses has been described, so we investigated the effect of time of day of transplantation on posttransplant CMV viremia. METHODS: We performed a retrospective analysis of 1517 patients receiving liver or kidney allografts at a single center from 2002 to 2018. All patients were given preemptive therapy with CMV viremia monitoring after transplantation. Circulatory arrest and reperfusion time of donor organ were categorized into 4 periods. Patients were divided into serostatus groups based on previous CMV infection in donor and recipient. CMV viremia parameters were compared between time categories for each group. Factor analysis of mixed data was used to interrogate this complex data set. RESULTS: Live-donor transplant recipients were less likely to develop viremia than recipients of deceased-donor organs (48% vs 61%; Pâ <â .001). After controlling for this, there was no evidence of time of day of transplantation affecting CMV parameters in any serostatus group, by logistic regression or factor analysis of mixed data. DISCUSSION: We found no evidence for a circadian effect of transplantation on CMV viremia, but these novel results warrant confirmation by other centers.
Subject(s)
Cytomegalovirus Infections , Organ Transplantation , Antiviral Agents/therapeutic use , Circadian Rhythm , Cytomegalovirus , Humans , Organ Transplantation/adverse effects , Retrospective Studies , Viral Load , Viremia/etiologyABSTRACT
Since the 1970s, several species of herpesviruses have been identified and associated with significant diseases in reptiles. Earlier discoveries placed these viruses into different taxonomic groups on the basis of morphological and biological characteristics, while advancements in molecular methods have led to more recent descriptions of novel reptilian herpesviruses, as well as providing insight into the phylogenetic relationship of these viruses. Herpesvirus infections in reptiles are often characterised by non-pathognomonic signs including stomatitis, encephalitis, conjunctivitis, hepatitis and proliferative lesions. With the exception of fibropapillomatosis in marine turtles, the absence of specific clinical signs has fostered misdiagnosis and underreporting of the actual disease burden in reptilian populations and hampered potential investigations that could lead to the effective control of these diseases. In addition, complex life histories, sampling bias and poor monitoring systems have limited the assessment of the impact of herpesvirus infections in wild populations and captive collections. Here we review the current published knowledge of the taxonomy, pathogenesis, pathology and epidemiology of reptilian herpesviruses.
ABSTRACT
It is a well-established fact that different tissues within the body contain their own circadian clocks or pacemakers, where it is proposed that the clock controls the local, daily cell biology of that organ.1,2 In mammals, these peripheral clocks work in concert with and are entrained by rhythmic signals arising from the suprachiasmatic nucleus (SCN) in the hypothalamus of the animal, among other systemic cues.2 In the case of zebrafish, the circadian system appears to be highly decentralized with each tissue not only having an internal circadian clock, but also being directly light entrained.1 Several years ago, we showed that the zebrafish heart contains its own circadian pacemaker at the gene expression level.1 This is also the case in mammals, where the circadian clock controls approximately 10% of the genes expressed in the heart.3 However, heart rate itself is generally thought to be regulated by several well-described autonomic cues, neurotransmitters, and hormones. In this study, we report that, for larval zebrafish hearts, the daily change in heartbeat rate is not only clock-controlled in vivo, but that this rhythm also persists in vitro, indicating that the cardiac circadian clock itself can directly drive this major physiological oscillation.
Subject(s)
Circadian Clocks , Circadian Rhythm , Heart Rate , Zebrafish/anatomy & histology , Zebrafish/physiology , AnimalsABSTRACT
Hundreds of human genes are associated with neurological diseases, but translation into tractable biological mechanisms is lagging. Larval zebrafish are an attractive model to investigate genetic contributions to neurological diseases. However, current CRISPR-Cas9 methods are difficult to apply to large genetic screens studying behavioural phenotypes. To facilitate rapid genetic screening, we developed a simple sequencing-free tool to validate gRNAs and a highly effective CRISPR-Cas9 method capable of converting >90% of injected embryos directly into F0 biallelic knockouts. We demonstrate that F0 knockouts reliably recapitulate complex mutant phenotypes, such as altered molecular rhythms of the circadian clock, escape responses to irritants, and multi-parameter day-night locomotor behaviours. The technique is sufficiently robust to knockout multiple genes in the same animal, for example to create the transparent triple knockout crystal fish for imaging. Our F0 knockout method cuts the experimental time from gene to behavioural phenotype in zebrafish from months to one week.
Subject(s)
CRISPR-Cas Systems , Gene Knockout Techniques , Genetic Testing/methods , RNA, Guide, Kinetoplastida/analysis , Zebrafish/genetics , Animals , Behavior, Animal , Embryo, Nonmammalian , Phenotype , Zebrafish/embryologyABSTRACT
One of the key defining features of an endogenous circadian clock is that it can be entrained or set to local time. Though a number of cues can perform this role, light is the predominant environmental signal that acts to entrain circadian pacemakers in most species. For the past 20 years, a great deal of work has been performed on the light input pathway in mammals and the role of intrinsically photosensitive retinal ganglion cells (ipRGCs)/melanopsin in detecting and sending light information to the suprachiasmatic nucleus (SCN). In teleost fishes, reptiles and birds, the biology of light sensitivity is more complicated as cells and tissues can be directly light responsive. Non-visual light signalling was described many years ago in the context of seasonal, photoperiodic responses in birds and lizards. In the case of teleosts, in particular the zebrafish model system, not only do peripheral tissues have a circadian pacemaker, but possess clear, direct light sensitivity. A surprisingly wide number of opsin photopigments have been described within these tissues, which may underpin this fundamental ability to respond to light, though no specific functional link for any given opsin yet exists. In this study, we show that zebrafish cells show wide spectral sensitivities, as well as express a number of opsin photopigments - several of which are under direct clock control. Furthermore, we also show that light outside the visual range, both ultraviolet and infrared light, can induce clock genes in zebrafish cells. These same wavelengths can phase shift the clock, except infrared light, which generates no shift even though genes such as per2 and cry1a are induced.
ABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
The methyl cycle is a universal metabolic pathway providing methyl groups for the methylation of nuclei acids and proteins, regulating all aspects of cellular physiology. We have previously shown that methyl cycle inhibition in mammals strongly affects circadian rhythms. Since the methyl cycle and circadian clocks have evolved early during evolution and operate in organisms across the tree of life, we sought to determine whether the link between the two is also conserved. Here, we show that methyl cycle inhibition affects biological rhythms in species ranging from unicellular algae to humans, separated by more than 1 billion years of evolution. In contrast, the cyanobacterial clock is resistant to methyl cycle inhibition, although we demonstrate that methylations themselves regulate circadian rhythms in this organism. Mammalian cells with a rewired bacteria-like methyl cycle are protected, like cyanobacteria, from methyl cycle inhibition, providing interesting new possibilities for the treatment of methylation deficiencies.
Subject(s)
Circadian Rhythm , Methylation , Animals , Arabidopsis/physiology , Caenorhabditis elegans/physiology , Chlamydomonas reinhardtii/physiology , Chlorophyta/physiology , Drosophila melanogaster/physiology , Humans , Mice/physiology , Synechococcus/physiology , Zebrafish/physiologyABSTRACT
Zebrafish represent the one alternative vertebrate, genetic model system to mice that can be easily manipulated in a laboratory setting. With the teleost Medaka (Oryzias latipes), which now has a significant following, and over 30,000 other fish species worldwide, there is great potential to study the biology of environmental adaptation using teleosts. Zebrafish are primarily used for research on developmental biology, for obvious reasons. However, fish in general have also contributed to our understanding of circadian clock biology in the broadest sense. In this review, we will discuss selected areas where this contribution seems most unique. This will include a discussion of the issue of central versus peripheral clocks, in which zebrafish played an early role; the global nature of light sensitivity; and the critical role played by light in regulating cell biology. In addition, we also discuss the importance of the clock in controlling the timing of fundamental aspects of cell biology, such as the temporal control of the cell cycle. Many of these findings are applicable to the majority of vertebrate species. However, some reflect the unique manner in which "fish" can solve biological problems, in an evolutionary context. Genome duplication events simply mean that many fish species have more gene copies to "throw at a problem", and evolution seems to have taken advantage of this "gene abundance". How this relates to their poor cousins, the mammals, remains to be seen.
ABSTRACT
Most animals and plants live on the planet exposed to periods of rhythmic light and dark. As such, they have evolved endogenous circadian clocks to regulate their physiology rhythmically, and non-visual light detection mechanisms to set the clock to the environmental light-dark cycle. In the case of fish, circadian pacemakers are not only present in the majority of tissues and cells, but these tissues are themselves directly light-sensitive, expressing a wide range of opsin photopigments. This broad non-visual light sensitivity exists to set the clock, but also impacts a wide range of fundamental cell biological processes, such as DNA repair regulation. In this context, Astyanax mexicanus is a very intriguing model system with which to explore non-visual light detection and circadian clock function. Previous work has shown that surface fish possess the same directly light entrainable circadian clocks, described above. The same is true for cave strains of Astyanax in the laboratory, though no daily rhythms have been observed under natural dark conditions in Mexico. There are, however, clear alterations in the cave strain light response and changes to the circadian clock, with a difference in phase of peak gene expression and a reduction in amplitude. In this study, we expand these early observations by exploring the development of non-visual light sensitivity and clock function between surface and cave populations. When does the circadian pacemaker begin to oscillate during development, and are there differences between the various strains? Is the difference in acute light sensitivity, seen in adults, apparent from the earliest stages of development? Our results show that both cave and surface populations must experience daily light exposure to establish a larval gene expression rhythm. These oscillations begin early, around the third day of development in all strains, but gene expression rhythms show a significantly higher amplitude in surface fish larvae. In addition, the light induction of clock genes is developmentally delayed in cave populations. Zebrafish embryonic light sensitivity has been shown to be critical not only for clock entrainment, but also for transcriptional activation of DNA repair processes. Similar downstream transcriptional responses to light also occur in Astyanax. Interestingly, the establishment of the adult timing profile of clock gene expression takes several days to become apparent. This fact may provide mechanistic insight into the key differences between the cave and surface fish clock mechanisms.
Subject(s)
Characiformes/physiology , Circadian Clocks/physiology , Gene Expression Regulation/physiology , Light , Transcription, Genetic/physiology , AnimalsABSTRACT
Studies from a number of model systems have shown that the circadian clock controls expression of key cell cycle checkpoints, thus providing permissive or inhibitory windows in which specific cell cycle events can occur. However, a major question remains: Is the clock actually regulating the cell cycle through such a gating mechanism or, alternatively, is there a coupling process that controls the speed of cell cycle progression? Using our light-responsive zebrafish cell lines, we address this issue directly by synchronizing the cell cycle in culture simply by changing the entraining light-dark (LD) cycle in the incubator without the need for pharmacological intervention. Our results show that the cell cycle rapidly reentrains to a shifted LD cycle within 36 h, with changes in p21 expression and subsequent S phase timing occurring within the first few hours of resetting. Reentrainment of mitosis appears to lag S phase resetting by 1 circadian cycle. The range of entrainment of the zebrafish clock to differing LD cycles is large, from 16 to 32 hour periods. We exploited this feature to explore cell cycle entrainment at both the population and single cell levels. At the population level, cell cycle length is shortened or lengthened under corresponding T-cycles, suggesting that a 1:1 coupling mechanism is capable of either speeding up or slowing down the cell cycle. However, analysis at the single cell level reveals that this, in fact, is not true and that a gating mechanism is the fundamental method of timed cell cycle regulation in zebrafish. Cell cycle length at the single cell level is virtually unaltered with varying T-cycles.
Subject(s)
Cell Cycle/physiology , Circadian Clocks , Zebrafish/physiology , Animals , Cell Cycle/genetics , Cell Line , Circadian Rhythm/physiology , Cyclin-Dependent Kinase Inhibitor p21/genetics , Light , Photoperiod , Single-Cell Analysis , Zebrafish/geneticsABSTRACT
Most of the life with which humans interact is exposed to highly rhythmic and extremely predictable changes in illumination that occur with the daily events of sunrise and sunset. However, while the influence of the sun feels omnipotent to surface dwellers such as ourselves, life on earth is dominated, in terms of biomass, by organisms isolated from the direct effects of the sun. A limited understanding of what life is like away from the sun can be inferred from our knowledge of physiology and ecology in the light biosphere, but a full understanding can only be gained by studying animals from the dark biosphere, both in the laboratory and in their natural habitats. One of the least understood aspects of life in the dark biosphere is the rhythmicity of physiology and what it means to live in an environment of low or no rhythmicity. Here we describe methods that may be used to understand rhythmic physiology in the dark and summarise some of the studies of rhythmic physiology in "arrhythmic" environments, such as the poles, deep sea and caves. We review what can be understood about the adaptive value of rhythmic physiology on the Earth's surface from studies of animals from arrhythmic environments and what role a circadian clock may play in the dark.
Subject(s)
Circadian Clocks/physiology , Circadian Rhythm/physiology , Ecosystem , Zoology/methods , Animals , Aquatic Organisms , Arctic Regions , Behavior, Animal , Biological Evolution , Caves , LightABSTRACT
The circadian clock plays a pivotal role in modulating physiological processes and has been implicated, either directly or indirectly, in a range of pathological states including cancer. Here we investigate how the circadian clock is entrained by external cues such as light. Working with zebrafish cell lines and combining light pulse experiments with simulation efforts focused on the role of synchronization effects, we find that even very modest doses of light exposure are sufficient to trigger some entrainment, whereby a higher light intensity or duration correlates with strength of the circadian signal. Moreover, we observe in the simulations that stochastic effects may be considered an essential feature of the circadian clock in order to explain the circadian signal decay in prolonged darkness, as well as light initiated resynchronization as a strong component of entrainment.
Subject(s)
Circadian Clocks/physiology , Zebrafish/physiology , Animals , Cell Line , Circadian Clocks/radiation effects , Computational Biology , Computer Simulation , Mathematical Concepts , Models, Biological , Photic Stimulation , Stochastic ProcessesABSTRACT
Light affects animal physiology and behavior more than simply through classical visual, image-forming pathways. Nonvisual photoreception regulates numerous biological systems, including circadian entrainment, DNA repair, metabolism, and behavior. However, for the majority of these processes, the photoreceptive molecules involved are unknown. Given the diversity of photophysiological responses, the question arises whether a single photopigment or a greater diversity of proteins within the opsin superfamily detect photic stimuli. Here, a functional genomics approach identified the full complement of photopigments in a highly light-sensitive model vertebrate, the zebrafish (Danio rerio), and characterized their tissue distribution, expression levels, and biochemical properties. The results presented here reveal the presence of 42 distinct genes encoding 10 classical visual photopigments and 32 nonvisual opsins, including 10 novel opsin genes comprising four new pigment classes. Consistent with the presence of light-entrainable circadian oscillators in zebrafish, all adult tissues examined expressed two or more opsins, including several novel opsins. Spectral and electrophysiological analyses of the new opsins demonstrate that they form functional photopigments, each with unique chromophore-binding and wavelength specificities. This study has revealed a remarkable number and diversity of photopigments in zebrafish, the largest number so far discovered for any vertebrate. Found in amphibians, reptiles, birds, and all three mammalian clades, most of these genes are not restricted to teleosts. Therefore, nonvisual light detection is far more complex than initially appreciated, which has significant biological implications in understanding photoreception in vertebrates.
Subject(s)
Gene Expression Regulation , Opsins/genetics , Zebrafish/genetics , Amphibians/genetics , Animals , Birds/genetics , Evolution, Molecular , Gene Expression Profiling , Genome , Genomics , Light , Mammals/genetics , Opsins/metabolism , Phylogeny , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
The circadian clock controls the timing of the cell cycle in healthy tissues and clock disruption is known to increase tumourigenesis. Melanoma is one of the most rapidly increasing forms of cancer and the precise molecular circadian changes that occur in a melanoma tumor are unknown. Using a melanoma zebrafish model, we have explored the molecular changes that occur to the circadian clock within tumors. We have found disruptions in melanoma clock gene expression due to a major impairment to the light input pathway, with a parallel loss of light-dependent activation of DNA repair genes. Furthermore, the timing of mitosis in tumors is perturbed, as well as the regulation of certain key cell cycle regulators, such that cells divide arhythmically. The inability to co-ordinate DNA damage repair and cell division is likely to promote further tumourigenesis and accelerate melanoma development.