Single-cell genomics (SCG) methods provide a unique opportunity to analyse whole genome information at the resolution of an individual cell. While SCG has been extensively used to investigate bacterial and archaeal genomes, the technique has been rarely used to access the genetic makeup of uncultivated microbial eukaryotes. In this regard, the use of SCG can provide a wealth of information; not only do the methods allow exploration of the genome, they can also help elucidate the relationship between the cell and intracellular entities extant in nearly all eukaryotes. SCG enables the study of total eukaryotic cellular DNA, which in turn allows us to better understand the evolutionary history and diversity of life, and the physiological interactions that define complex organisms.This article is part of a discussion meeting issue ‘Single cell ecology’. An alphaproteobacterial symbiont that once settled within a pre-eukaryotic cell gave rise to mitochondria . LikewisPerkinsela in Neoparamoeba, [While the evolutionary impact of semiautonomous organelles becomes easily apparent, they are not the only DNA containing entities that contribute to the genomic complexity of eukaryotic cells. Eukaryotes frequently host archaeal, bacterial and even eukaryotic endocytobionts (endosymbionts living intracellularly). Note that we use symbiosis here in its broadest sense—that is, any intimate and constant interaction along the continuum between mutualism and pathogenicity and ranging from harmful to beneficial and from facultative to obligate . Whereasramoeba, ), other ramoeba, . Giant vramoeba,  and can ramoeba, , and theramoeba, .All the above-mentioned intracellular entities contain nucleic acids, and thus, the pool of total cellular DNA in a single eukaryotic cell reflects this sum. Aside from general biological importance, this has practical implications when using single-cell genomics (SCG). Here we do not reflect on these cellular conglomerates as something that weakens the SCG data but as an exciting opportunity to study underexplored multipartite associations within eukaryotic cells. Here, we focus on unicellular eukaryotes and discuss compositions of various possible genomic pools within their single cells. We illustrate how the cultivation skew is limiting our current understanding of eukaryote intracellular associations and how emerging SCG provides a promising tool to enhance our knowledge of the genetic make-up of these associations.2.b), it is clear that Obazoa , Amoebozoa (namely Acanthamoeba strains), and Archaeplastida are overrepresented, suggesting that most of the diversity is still hidden and remains to be discovered. The biases and limitations of laboratory model systems can be partly overcome by employing cultivation-independent methods (e.g. metagenomics) on environmental samples; however, these methods lack single-cell resolution and therefore obscure host–symbiont relationships. Thus, only by focusing on one eukaryotic cell at a time can we obtain a detailed understanding of these fascinating multipartite associations and SCG has already begun to fill this gap (lineages labelled on figure 1a).Over the last decades, the use of easily-maintainable host laboratory cultures has been a standard procedure for studying eukaryote intracellular associations ,14 and ofigure 1et al. [SCG has become a well-established approach for studying the coding potential and evolutionary histories of bacteria and archaea ,23, specet al. . Thus, s3.et al. [Paramecium aurelia, however, it was not possible to unambiguously identify their role inside their hosts. Aside from the ingested prey, infections caused by nanovirus [in silico is of considerable importance or else long-term interactions may remain obscure because of this uncertainty. Even if viruses (only double stranded DNA and single stranded DNA viruses are detectable by SCG) or strictly intracellularly living organisms  are detected, their origin as prey items cannot be renounced. Clustered regularly interspaced short palindromic repeat (CRISPR) systems can link viruses to their host but this approach has two limitations: it is only suited for bacterial/archaeal genomes, and complete or nearly complete genome assemblies from host and virus are necessary [The SCG approach has sufficient sensitivity to recover genomic data of other DNA containing entities present within a single cell. As referenced earlier, Yoon and colleagues  focused et al.  found rianovirus , and putanovirus  were alsanovirus . Anotheranovirus . The mosanovirus . Distingecessary ,42. Thusecessary .Table 1A complementary approach to differentiate free-living and endosymbiotic organisms is to look for typical genomic signatures of an intracellular lifestyle. Generally speaking, bacteria obligately associated with and living intracellularly in other organisms tend to have genomes that are (i) small, (ii) AT-rich, and (iii) rapidly evolving ,45. Thes4.et al. [Escherichia coli tolC knockout strain that not only substituted diverse original bacterial communities in several environmental isolates of Acanthamoeba but also could be later controlled by a sublethal concentration of ampicillin owing to its hypersensitivity.While the aforementioned possible strategies for the distinction between prey and other intracellular entities heavily rely on the genomic sequence data, other approaches can be used to further validate the nature of intracellular associations revealed by SCG. Recurrent observation of the same ‘contaminating’ DNA may suggest the symbiotic lifestyle and rule out the prey origin, especially in combination with starvation experiments. However, it is nearly impossible to eliminate bacteria in environmental samples without depletion of heterotrophic unicellular eukaryotes. A plausible strategy could be the replacement of a diverse bacterial community by a homogeneous and well-defined population of a single bacterium. Lagkouvardos et al.  succeedein situ hybridization via specific oligonucleotide probes. The sample has to be chemically fixed [Coxiella burnetii [Distinction between prey and endosymbionts can also be achieved by localization of the organism(s) detected in the single-cell data using fluorescence ly fixed  for lately fixed . Even thburnetii ).Lastly, single-cell RNA sequencing (scRNA-seq) provides an opportunity to assess the nature of intracellular associations. Though still hampered by technical challenges when applied to single celled eukaryotic microorganisms , progres5.Intracellular associations underlie the emergence and subsequent flourishing and evolutionary trajectory of eukaryotic cells. However, as pointed out by Woyke & Schulz , we stilIn principle, there are two primary directions for exploring diverse eukaryotic lineages and their associated intracellular entities. First, microbial culture collections around the world store strains of unicellular eukaryotes, some of which represent neglected eukaryotic lineages . Most liTo accelerate progress in this field, we encourage the research community to establish a genomic encyclopaedia of eukaryote intracellular associations. A robust genomic database connecting eukaryote host cells and their intracellular entities will enable (i) extension of the tree of life with new microbes  and viruses, (ii) investigation of the intracellular associations in ecological context , and (iii) large-scale comparative genome analyses . By sequencing a single eukaryotic cell at a time, we can bypass cultivation requirements, overcome existing biases and gain insight into naturally occurring associations within so far neglected eukaryotic hosts.


Exposing fish to a stressor can result in altered rhythms of most stress indicators, such as cortisol, glucose, and lactate among others, as well as daily rhythms of most behavioral and physiological functions. In addition, crh and pomca expression profiles can be affected by other factors such as light spectrum, which strongly influence the expression profile of growth-related  genes. Additionally, the daily cycle of water temperature (warmer at day and cooler at night) is another factor that has to be considered. The response to any acute stressor is not only species dependent, but also depends on the time of the day when the stress occurs: nocturnal species show higher responses when stressed during day time, whereas diurnal fish respond stronger at night. Melatonin administration in fish has sedative effects with a reduction in locomotor activity and cortisol levels, as well as reduced liver glycogen and dopaminergic and serotonergic activities within the hypothalamus. In this paper, we are reviewing the role of environmental cycles and biological clocks on the entrainment of daily rhythms in the HPI axis and stress responses in fish.Fish have evolved a biological clock to cope with environmental cycles, so they display circadian rhythms in most physiological functions including stress response. Photoperiodic information is transduced by the pineal organ into a rhythmic secretion of melatonin, which is released into the blood circulation with high concentrations at night and low during the day. The melatonin rhythmic profile is under the control of circadian clocks in most fish , and it is considered as an important output of the circadian system, thus modulating most daily behavioral and physiological rhythms. Lighting conditions (intensity and spectrum) change in the underwater environment and affect fish embryo and larvae development: constant light/darkness or red lights can lead to increased malformations and mortality, whereas blue light usually results in best hatching rates and growth performance in marine fish. Many factors display daily rhythms along the hypothalamus-pituitary-interrenal (HPI) axis that controls stress response in fish, including corticotropin-releasing hormone (Crh) and its binding protein (Crhbp), proopiomelanocortin A and B (Pomca and Pomcb), and plasma cortisol, glucose, and lactate. Many of these circadian rhythms are under the control of endogenous molecular clocks, which consist of self-sustained transcriptional-translational feedback loops involving the cyclic expression of circadian clock genes ( These environmental cycles are governed by geophysical cycles originating from the rotation of the Earth and the Moon around the Sun. Time-keeping systems  have evolved since the most primitive forms of life to cope with natural cycles and anticipate periodic events . In fish reverb) , 3.clock, per, and bmal gene expression was affected by lighting conditions during early larval development. Furthermore, larvae reared under constant darkness became arrhythmic, while under light/dark cycles of different wavelengths their daily activity rhythms appeared earlier under blue than under white or red lights  to entrain biological clocks. Light information is transduced into a nocturnal rhythm of melatonin that acts as an internal zeitgeber setting up the phase of individual pacemakers. Daylength, the basis for photoperiodism and seasonality, is coded by the duration (longer/shorter) of the nocturnal melatonin rhythm . In addid lights .Danio rerio) (er2 and cry1a) do not show rhythmic expression under TC  and DD, which peaked during the hight temperature  synchronizes the circadian clock, which periodicity (tau) is temperature-compensated and remains constant in a wide range of temperatures, with a Q10 value for tau around 1 . Actuallo rerio) , althougunder TC  in lizarperature , 14.Melatonin is a key hormone acting in the circadian system of vertebrates, and it is mainly produced by the pineal gland. In fish, the pineal is a complex structure located in an evagination of the roof of the diencephalon, which exhibits photoreceptive characteristics , 16. Thein situ  transforms N-acetylserotonin in melatonin  and Atlantic salmon , breaks this rule since it lacks an intra-pineal oscillatory mechanism (in vivo and in vitro (Esox lucius), sea bream (Sparus aurata), and zebrafish, with optimal physiological temperatures  axis , 79, whicrh and pituitary pomc gene expression  was recently studied. Decreased amplitude and mean expression levels for most of these genes appeared in stressed trout, except for rev-erbβ-like whose expression increased , presence of pollutants, and oxygen deficits. Routine husbandry in aquaculture also involves further factors, such as stocking conditions, handling, feeding and social interactions, among others –104, sevncreased . Furtherncreased  and fishncreased . Sirt1 dhe liver  and actihe liver . Moreoveed trout . Therefoed trout , 110. Ited trout , suggestin vitro (in vivo (Streptopelia capicola)  cortisol implants reduced melatonin synthesis at night in a similar way than exposure to stressors, and incubation of cultured pineal organs with cortisol reduced melatonin synthesis during the dark phase of the 24-h cycle, with this effect prevented when a GR antagonist was added  . A proloapicola) . Regardiapicola) . A stimuer entry . Other ser entry . A drop as added .Oreochromis mossambicus) (Clarias garieinus), treatment with corticosteroid hormones in a μM to mM range inhibited pineal AANAT activity in a dose-dependent way during different phases of the breeding cycle  mimicking stressed conditions were shown to reduce melatonin secretion from cultured pineal organs of tilapia (ambicus) , althougng cycle . Meanwhing cycle . Since aal organ  it is plal organ . Alternaal organ , therefoal organ .Aequidens pulcher to a mirror presented 20 min later, whereas Larson et al.   administered melatonin at doses as low as 10 μg/g body weight for 7 days resulted in reduced plasma cortisol levels and locomotor activity of goldfish (Carassius auratus) , and the HPI tissues, whose activation by stressors lead to increased catecholamines and cortisol blood levels, respectively . Severaln et al.  reportedn et al. , and Senalensis) , such asalensis) . Accordialensis)  showed tauratus) , thus surh mRNA in sole which was upregulated by environmental stressors   . This suAdditionally, the possibility that melatonin acts directly on adrenal tissue to modulate GC secretion exists, as reported in mammals , and alsigf1a and igf2a) and stress axis in fish (crh and pomca)  . On one ue light .Scophthalmus maximus), larvae reared under shorter wavelengths (blue and green lights) showed significantly enhanced growth in comparison to larvae reared under longer wavelengths (red light)  . Reprodud light) .Rhamdia quelen), a south american aquacultured fish, the combination of tank color and shelter availability reduced stress responses as cortisol levels decreased in fish kept in talks with blue walls and shelter (Rutilus frisii), the color of the tanks  appeared also to influence food intake and lipid content without changing growth or feed conversion rates (Perca fluviatilis) larvae also showed better growth and prey intake when raised in black tanks compared to gray tanks (Huso huso), since red light had a negative impact in growth, while blue light reduced plasma cortisol and glucose , which converts androgens into estrogens and thus led to female differentiation.The role of temperature regulating fish metabolism, reproduction, development and other adaptive responses has been widely reported . Temperahsp70) when reared under a daily thermocycle as compared to a constant rearing temperature .Acclimation to a cyclic thermal environment can increase thermal tolerance, particularly during early development since the thermal history of larvae induces irreversible changes. As reported by Schaefer and Ryan , fish zeThe endocrine system of fish responds differently depending on the time of the day. For instance, daily differences have been reported in the response to exogenous treatments that affect endocrine pathways controlled by the hypothalamus-pituitary (HP) system such as the administration of exogenous Gh or Gnrh agonists (Gnrha) –163.Acipenser medirostris), Senegalese sole, gilthead sea bream and African sharptooth catfish (Clarias gariepinus)  , 164–166iepinus)   . Likewisiepinus)   . The daiThe effectiveness of drug absorption, administration, metabolism and elimination are also subjected to rhythmicity, which affects the final concentration of xenobiotics in the animals' blood and their bioavailability . In mammAnesthetics are administered to fish to immobilize them and minimize their stress response during research and routine procedures in fish farms . HoweverLepeophtheirus salmonis) and amoebic gill disease (AGD) caused by Neoparamoeba perurans, but these treatments can have side effects on fish and trigger a stress response following exposure leading to increased mortalities in some cases  was up-regulated when fish were treated during the first half of the day, and in particular around 6 h after the lights onset  and nuclear receptors (ahr2) showed daily and circadian rhythmicity, in tune with clock genes expression. These transcription factors and nuclear receptors regulate the expression of many detoxifying enzymes and ABC transporters, some of them also displaying rhythmicity in this species   . AltogetThe application of this field of research is evident when designing health strategies in the aquaculture industry. However, it is also important to highlight that zebrafish has become an animal model widely used in biomedical research, to assess the psychoactive and toxic effects of many drugs , 181, inIn conclusion, fish chronotoxicity is a novel area of research that is showing promising prospects for the application of chronobiology concepts to optimize the administration of medicines in fish farms, which can lead to improve welfare of animals in commercial settings. Furthermore, increasing our knowledge about toxicity rhythms of drugs used in biomedical research will also have an impact on the application of therapies in humans.Although light is essential for vision, the trade-off is the production of reactive oxygen species (ROS) that can cause damage within the eye . In vertThe effect of artificial light regimes during early development can be particularly detrimental to fish and have negative effects later during their life cycle. In zebrafish larvae, exposure to abnormal light-rearing conditions (LL or DD) affects their visual behavior and adversely influence the physiological development of the retina, as measured with electroretinogram (ERG) . However2), high green cathode lights  or low green cathode lights , no differences in the outer nuclear layer (ONL) thickness or ONL nuclei number were found between groups or in comparison to the control fish under simulated natural photoperiod  , Atlantic cod and European sea bass  . Howeversea bass . This dasea bass . In albisea bass . In normsea bass .Melatonin is also synthesized in the retina of teleost fish, showing marked daily rhythmicity. However, an inverse melatonin profile has been observed in plasma and eye in some fish species, which could be explained by the existence of two different AANAT isoforms and suggests a local function for ocular melatonin . One of In summary, there is ample scientific evidence that the use of artificial lights and protocols can induce retinal damage in fish, although important differences between light sources and species have been reported. For this reason, it is crucial to develop and test novel illumination technologies before their implementation in aquaculture systems, to ensure that animal welfare is not compromised. In addition, further studies on melatonin effects in the fish retina will be important to enable us to better understand the cellular mechanisms of retinal photodamage and elucidate whether this hormone play a role as a neuroprotector against light-induced oxidative stress in fish.Fish physiology is mainly rhythmic, governed by biological clocks which synchronizes to the (cyclic) environment in order to improve fitness and ultimately survival. Thus, stress responses in fish are not always straight forward, as they may respond differently on a time-dependent basis. Fish in captivity are challenged by many stressors and the chronobiological approach depicted here should be considered to improve their welfare. For instance, in farming conditions fish should be manipulated at the times when stress is better tolerated, whereas anesthetics and medicines should be used at the optimal times to enhance their efficacy while minimizing toxicity and side effects. Finally, keeping conditions regarding light spectrum and temperature cycles, should be also considered with care, particularly during early embryo and larval development as they may have long lasting irreversible effects. Light contamination at night should be particularly avoided, providing fish with a “melatonin friendly” environment.FS-V, JL-O, and LV provided the figures. All authors contributed equally in the writing and revision of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Zika virus recently re-emerged and caused global outbreaks mainly in Central Africa, Southeast Asia, the Pacific Islands and in Central and South America. Even though there is a declining trend, the virus continues to spread throughout different geographical regions of the world. Since its re-emergence in 2015, massive advances have been made regarding our understanding of clinical manifestations, epidemiology, genetic diversity, genomic structure and potential therapeutic intervention strategies. Nevertheless, treatment remains a challenge as there is no licensed effective therapy available. This review focuses on the recent advances regarding research models, as well as available experimental tools that can be used for the identification and characterization of potential antiviral targets and therapeutic intervention strategies. Aedes mosquitos followed by the identification of the first infected human case in Uganda in 1962 [Zika virus (ZIKV) was first isolated in 1947 in the Zika forest in Uganda from an infected rhesus macaque monkey [ in 1962 ,4,5. Sin in 1962 , which w in 1962 ,11,12. S in 1962 . In addi in 1962 ), sexual in 1962  and via  in 1962 ,17. Rece in 1962 . Further in 1962 ) includiZIKV infection is in most cases asymptomatic or can lead to mild, self-limiting symptoms including rash, fever, joint pain, as well as conjunctivitis . InfrequZIKV belongs to the genus flavivirus within the Flaviviridae family. It has a positive-sense single-stranded RNA genome of 10.8 kb, containing a single open reading frame, flanked by a 5′ and 3′ untranslated region (UTR), encoding for three structural and seven nonstructural proteins, which are co- and post-translationally processed by viral and host proteases . The thrAfter binding to specific receptors on the host cell, the virus is internalized by clathrin-mediated endocytosis. The low pH within the endosomes triggers viral uncoating and release of the viral genome into the cytosol . The genSeveral therapeutic approaches that target the different steps of the viral replication cycle  have beeThe first prerequisite with respect to the development of antiviral compounds is the availability of in vitro systems including virus isolates and/or recombinant constructs and robust in vitro cellular models .Clinical viral isolates are the best representatives of the currently circulating ZIKV strains. Among ZIKV strains, there are two distinct lineages described, the African and the Asian lineages . PhylogeEscherichia coli. This cDNA can be modified and manipulated as required before genomic RNA is obtained via in vitro transcription and subsequent virus recovery upon transfection of suitable cells [E. coli and may contain viral proteins with bacterial toxicity expressed through cryptic bacterial promoters, which hamper the propagation of the viral cDNA via bacterial plasmids  copy of an RNA virus is usually stably incorporated into a plasmid vector and amplified in le cells . Generatiewed in ,77). Neviewed in ,79, or tiewed in , usage oiewed in  and sepaiewed in ,83. Furtiewed in  based oniewed in ,86,87. Fiewed in  based oniewed in ,90. Anotromoters . All theromoters ,93, featCompared to full-length infectious clones, ZIKV replicons offer an alternative for the identification and characterization of antiviral compounds. Subgenomic replicons contain the genetic elements crucial for viral replication, but lacking the structural genes required for assembly and release of infectious progeny virus . This reCell culture models are crucial tools to study the molecular mechanisms of ZIKV life cycle and to develop and identify therapeutics. ZIKV has been shown to display a broad tissue tropism with several cell surface receptors facilitating ZIKV entry. These include receptor tyrosine kinase family members like AXL and Tyro-3, as well as the innate immune receptor DC-SIGN and the receptor TIM-1, also known as hepatitis A virus cellular receptor 1 (HAVcr-1) (reviewed in ). HoweveCell lines can be used as a platform to produce high-titer ZIKV stocks, but also to study various aspects of virus replication, including replication kinetics, susceptibility and, to a certain extent, immune response. Especially for the identification and initial characterization of antivirals, they offer a solid basis and have been used to test several approaches. One of the most commonly-used cell lines in ZIKV research is the Vero cell line derived from epithelial kidney cells from African green monkeys. These cells are deficient in type I interferon (IFN) production, making them suitable to yield high virus titers . Other cIn contrast to cell lines which often differ genetically and phenotypically from their parental tissue, primary cells have a low population doubling level and therefore more closely recapitulate the physiological conditions observed in vivo . PrimaryWhen screening for anti-ZIKV therapeutics targeting host factors with a protective effect on the human neuronal tissue, the use of primary human cell-based systems is challenging for ethical and technical reasons. In addition, in the context of congenital neurotropic ZIKV infection, there is currently no robust in vivo system modelling early human brain development. These obstacles can be bypassed using elaborated in vitro systems exploiting the possibilities offered by human embryonic stem cells (ESCs) and/or human induced pluripotent stem cells (iPSCs) (collectively PSCs) . PSCs prIn addition to testing specific compounds that have been described to inhibit related viruses or have been shown to possess a known antiviral activity, unbiased screening approaches offer an alternative to identify new antiviral compounds based on libraries containing hundreds of bioactive molecules. Several of these libraries contain FDA-approved drugs and clinical investigational agents, which have shown good safety and pharmacokinetics in vivo, thereby facilitating drug-repurposing strategies. Drug repurposing plays a very important role, given that a considerable risk of an infection exists during pregnancy, which can complicate medical intervention. Identification of antiviral drugs that are already approved during pregnancy could greatly accelerate the availability of therapeutic interventions.Different in vitro screening approaches exist, which are usually based on an automated readout, mostly microscopy- or luminescence-based. This readout can be either based on virus yield reduction (VYR) or on cytopathic effect (CPE) reduction . VYR assComputational in silico approaches have proven to be powerful tools in predicting drug targets and the evaluation of treatment strategies. In general, these in silico systems use computational models to describe macromolecule–ligand interactions. Structure-based approaches mimic the binding of the putatively active ligand within the relevant binding site and are therefore frequently able to facilitate further chemical optimization of the compound . StructuIn vitro models greatly facilitate the initial screening and identification of new antivirals. However, they do not fully reflect the complexity and physiology of living organisms. To this end, several in vivo models have been developed to study virus pathogenesis and to test new drugs and vaccines .−/−) or the transcription factors Irf3, Irf5 and Irf7  in 129 (A129) or C57BL/6 background have been shown to be highly susceptible to ZIKV infection and to develop severe diseases following peripheral inoculation, including development of neurological disease and death [−/−-deficient A129 mice [+/−) fetuses, which better resembled the immune status of human fetuses, with ZIKV maternal inoculation resulting in fetal demise that was associated with ZIKV infection of the placenta and fetal brain [−/−- or Stat2−/−-deficient mice has also been shown to facilitate ZIKV infection and spreading [−/− mice with ZIKV and subsequent treatment with potential antiviral compounds can lead to decreased viral burden in various tissues, reduced pathology, as well as reduced or delayed morbidity and mortality [Mouse models are widely used to study various aspects of virus infection in vivo, such as pathogenesis and transmission. Several mouse models have been developed to study ZIKV infection . Very eand death ,189,190.nd death ,176. Dou129 mice ,191. Fural brain . Besidespreading ,193. Furpreading ,195,196.preading . Recentlortality ,202,203.ortality , and in ortality ,206,207.NHPs seem to be natural hosts for ZIKV and are believed to represent the major animal reservoir . ZIKV waSeveral other animal models have been investigated for the study of ZIKV infections, including guinea pigs, cotton-rats and rabbits, all of which failed to show clinical signs of infection after intracerebral inoculation with the prototype African ZIKV strain MR766 . HoweverThese studies show that the host range of ZIKV might be broader than originally anticipated, and different animal models might be useful for the study of different aspects of ZIKV infections. However, the value of these alternative animal models for the characterization and development of antivirals and therapeutics still needs to be fully explored.In recent years, significant advances were made regarding our understanding of ZIKV-associated morbidity in human. Consequently, significant progress was achieved regarding the availability of research models and new experimental tools for the identification of therapeutic and antiviral strategies against ZIKV. However, treatment remains a challenge. Given that treatment needs to be safe and efficient especially in the context of pregnancy further complicates drug development. In addition, a declining trend in ZIKV infections could limit clinical trials. As there is no licensed effective therapy available yet, further efforts are required in order to find potential drug candidates with ideally a wide spectrum of action against flaviviruses found in similar geographical regions with often overlapping symptoms. Furthermore, assuming the broad cellular tropism and the different routes of ZIKV transmission of ZIKV, a variety of models should be applied in order to validate a promising drug. Ultimately, beyond the potential clinical benefits, the knowledge derived from the screening of anti-ZIKV drugs using relevant models will reveal new insights into the understanding of the molecular mechanisms of ZIKV-associated morbidity and greatly contribute to increase our knowledge on flavivirus biology.


Lower-extremity arterial disease (LEAD) is a major endemic disease with an alarming increased prevalence worldwide. It is a common and severe condition with excess risk of major cardiovascular events and death. It also leads to a high rate of lower-limb adverse events and non-traumatic amputation. The American Diabetes Association recommends a widespread medical history and clinical examination to screen for LEAD. The ankle brachial index (ABI) is the first non-invasive tool recommended to diagnose LEAD although its variable performance in patients with diabetes. The performance of ABI is particularly affected by the presence of peripheral neuropathy, medial arterial calcification, and incompressible arteries. There is no strong evidence today to support an alternative test for LEAD diagnosis in these conditions. The management of LEAD requires a strict control of cardiovascular risk factors including diabetes, hypertension, and dyslipidaemia. The benefit of intensive versus standard glucose control on the risk of LEAD has not been clearly established. Antihypertensive, lipid-lowering, and antiplatelet agents are obviously worthfull to reduce major cardiovascular adverse events, but few randomised controlled trials (RCTs) have evaluated the benefits of these treatments in terms of LEAD and its related adverse events. Smoking cessation, physical activity, supervised walking rehabilitation and healthy diet are also crucial in LEAD management. Several advances have been achieved in endovascular and surgical revascularization procedures, with obvious improvement in LEAD management. The revascularization strategy should take into account several factors including anatomical localizations of lesions, medical history of each patients and operator experience. Further studies, especially RCTs, are needed to evaluate the interest of different therapeutic strategies on the occurrence and progression of LEAD and its related adverse events in patients with diabetes. Lower extremity arterial disease (LEAD) is a major manifestation of systemic atherosclerosis with severe associated cardiovascular, lower limb and functional complications. It results from a partial or complete obstruction of one or more lower limb arteries. The first known presentation of lower-limb vascular disease was reported in 1831 in a horse that had a lameness thought to be due to femoral artery occlusion of the posterior limb. Similar symptom was described few years later in humans and characterized as an intermittent claudication (IC). Further investigations showed that IC was linked to muscle ischemia, induced by walking, and considered it as a clinical manifestation of LEAD. During the last decades, a large body of data have reported that LEAD was associated with increased risk of non-traumatic lower limb amputation (LLA), cardiovascular disease (CVD), and mortality –6. Yet, LEAD affects over 200 millions people worldwide, including 40 millions living in Europe . It is 2LEAD is one of the major causes of diabetic foot. It was present in 49% of patients with diabetic foot in the EURODIALE study, and one-third of participants had both LEAD and infection . DiabetiMicrovascular disease, mainly macroalbuminuria and diabetic retinopathy, have been shown to be independent risk factors for LEAD , 16. FurIntermittent claudication results from a diminished inflow of oxygen due to a reduced blood flow in the lower limbs during physical activity, which is a consequence of stenosis or obstruction of an artery irrigating the skeletal muscle . Many meEndothelial cells play an important role in vascular biology based on their strategic location between blood and tissues. They secrete many paracrine factors in the vascular wall and its lumen. In pathological setting, endothelial dysfunction induces structural, hemodynamic, and functional vascular abnormalities, altering blood vessels reactivity and relaxation, and generating atherosclerosis . EndotheLEAD has clearly been identified as a common manifestation of atherosclerosis of the large vessels . HoweverThe American Diabetes Association (ADA) recommends Table  an initiAnkle–brachial index has emerged as the relatively simple, non-invasive, and inexpensive tool for LEAD diagnosis . The ADAThe Doppler ultrasound exam is an imaging method with a good LEAD diagnosis performance (sensitivity 93% and specificity 97%) . It is aThe computed tomography angiography, magnetic resonance angiography and angiography permit a precise topographic diagnosis and are often performed in the pre-operative work-up when large arterial vessels are involved. The topography of LEAD is usually categorized as proximal  and distal lesions . The distal localisation has been shown to be more common than the proximal one in patients with diabetes .The critical limb ischemia (CLI) is defined as the presence of ischaemic chronic rest pain (> 2 weeks) typically in the forefoot with or without ischaemic lesions or gangrene due to arterial occlusive disease. It is considered as the last stage of LEAD spectrum, with excessively high risk for CVD and death . The CLIThe management of LEAD in patients with diabetes requires a multidisciplinary team including endocrinologist, vascular surgeon, infectious disease specialist, radiologist, rehabilitation doctor, nurse, and podiatrist. Despite the high macrovascular risk, the based-evidence prevention therapies remain underused in diabetic patients with LEAD compared to their counterparts with coronary or cerebrovascular disease , 58. TheEpidemiological studies and RCTs showed the efficiency of intensive blood glucose control in the reduction of the development and progression of long-term microvascular complications  in patients with diabetes –61. HoweThe PROspective pioglitAzone Clinical Trial In macroVascular Events (PROactive) trial showed a non-significant association between use of pioglitazone, an agonist of peroxisome proliferator-activated receptor γ (PPAR γ), and a higher risk of leg revascularization, compared with placebo . Howeverhttps://www.easd.org/programme-glance.html). The pathophysiological mechanisms likely to explain the high risk of LLA associated with canagliflozin in the CANVAS trial have not yet been established. It remains unclear if the risk of amputation is a class effect for all SGLT-2 inhibitors. While some studies suggested association with different SGLT-2 inhibitors and increased risk of LLA , bu, bu84], The European Society of Cardiology (ESC) and the European Society for Vascular Surgery (ESVS) recommended targeting serum low-density lipoprotein cholesterol (LDL-C) less than 1.8 mmol/L (< 70 mg/dL) or decreased by ≥ 50% if the initial value is between 1.8 and 3.5 mmol/L (70 and 135 mg/dL) for all patients with LEAD (Table The use of Fenofibrate failed to reduce macrovascular events in participants with type 2 diabetes in the Action to Control Cardiovascular Risk in Diabetes (ACCORD) and the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) trials , 100. HoDespite the availability of effective drug therapies that reduce LDL-cholesterol, CVD remains an important cause of mortality and morbidity. Therefore, additional LDL-cholesterol reduction may be warranted, especially for patients who are unresponsive to, or unable to take, existing LDL-cholesterol reducing therapies. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serin protease with effect on the LDL receptor cycle leading to its degradation and therefore inhibition of continuing LDL-cholesterol clearance from the blood. This path is the target of newly developed lipid-lowering drugs, PCSK9 inhibitors, monoclonal antibodies leading to further LDL-cholesterol decrease, with reducing CVD risk, but not cardiovascular or all-cause mortality . The FurAntiplatelet drugs are advised in all patients with symptomatic LEAD or having undergone previous vascular revascularization, to reduce both CVD and peripheral vascular event. The ESC and the ESVS guidelines did not recommend antiplatelet therapy in subjects with asymptomatic LEAD Table  90]. In. In90]. The meta-analyses from the Antithrombotic Trialists Collaboration group showed that aspirin  was protective in different high vascular risk populations, including those with LEAD . HoweverNo evidence exists for any benefit related to a dual antiplatelet therapy in patients with LEAD. In the post hoc analyses of the Clopidogrel for High Atherothrombotic Risk and Ischemic Stabilization, Management, and Avoidance (CHARISMA) trial, dual therapy (clopidogrel and aspirin) did not provide further vascular protection over aspirin alone in LEAD patients (36% with diabetes), except for the risk of myocardial infarction and hospitalization for ischaemic events . This moAnticoagulation strategy is currently advisable in the presence of its traditional indication , although new drugs have provided encouraging findings for LEAD-related events. The cardiovascular outcomes for people using anticoagulation strategies (COMPASS) trial showed that rivaroxaban, an oral factor Xa inhibitor, plus aspirin was associated with fewer adverse cardiovascular events, but more major bleeding events versus aspirin alone . The rivThe Steno2 trial has compared a multifactorial intervention versus conventional treatment in patients with type 2 diabetes and microalbuminuria . During Some vasodilator agents may be used to relieve intermittent claudication and increase walking distance in patients with LEAD. The cilostazol, a selective inhibitor of the phosphodiesterase III, is the most studied drug; its benefit is modest with no evidence for vascular protection. In a comprehensive meta-analyses, cilostazol improved walking distance with no relevant cardiovascular effect or improvement in quality of life . This vaSome data suggested a relationship between circulating levels of growth factors and the development of LEAD . The theSome studies have tested stem cell therapy in patients with LEAD with encouraging results, although the lack of definitive evidences. A recent systematic review of the literature and a meta-analysis have shown that autologous cell therapy reduced the risk of LLA and rest pain, improved wound healing, and increased ABI and TcPO2 in patients with LEAD who were ineligible for surgical or percutaneous revascularization . InteresRemote ischaemic conditioning (RIC), involving repeated applications of short periods of limb ischemia over days or weeks, may improve endothelial function, skin microcirculation, and regulates the inflammatory response. Some data suggested that repeat RIC may boost healing of ischaemic diabetic foot .Tobacco smoking including second-hand smoke has been highlighted as one of the three leading risk factors for global disease burden worldwide . It is aThe exercise training improved walking ability, and distances, as well as physical function, vitality and general health . HoweverHealthy diet might help to achieve and maintain body weight goals, reach individualized glycaemic, blood pressure, and lipid targets, and delay or prevent diabetic complications, especially microvascular disease . PreviouContrasting observations were reported in terms of perioperative outcomes in patients with diabetes undergoing revascularization –144. RecLEAD is one of the most severe conditions seen in patients with diabetes. It leads to excess risk of death, CVD, and limb loss, and is responsible for disabilities and an important socio-economical burden. The diagnosis strategy has been better codified now focusing in patients with evocative symptoms of LEAD, far from the worthless and expensive universal screening in asymptomatic patients. Anti-diabetic, anti-hypertensive, lipid-lowering and antiplatelet medications may improve the cardiovascular prognosis of patients with LEAD, but few has been done to test their benefits to reduce the occurrence and the progression of LEAD as well as lower-extremities adverse events. High quality studies are required to advance the knowledge in pathophysiology and natural history of LEAD, and to evaluate different aspects of its management.


Drosophila melanogaster, sparked interest in using this model organism to dissect the mechanisms that govern gut (patho) physiology in humans. In 2007, the finding of the intestinal stem cell lineage, followed by the development of tools available for its manipulation in vivo, helped to elucidate the structural organization and functions of the fly intestine and its similarity with mammalian gastrointestinal systems. To date, studies of the Drosophila gut have already helped to shed light on a broad range of biological questions regarding stem cells and their niches, interorgan communication, immunity and immunometabolism, making the Drosophila a promising model organism for human enteric studies. This review summarizes our current knowledge of the structure and functions of the Drosophila melanogaster intestine, asserting its validity as an emerging model system to study gut physiology, regeneration, immune defenses and host-microbiota interactions. In all metazoans, the intestinal tract is an essential organ to integrate nutritional signaling, hormonal cues and immunometabolic networks. The dysregulation of intestinal epithelium functions can impact organism physiology and, in humans, leads to devastating and complex diseases, such as inflammatory bowel diseases, intestinal cancers, and obesity. Two decades ago, the discovery of an immune response in the intestine of the genetic model system,  The structure and function of the gastrointestinal (GI) tract are broadly conserved in metazoans . The GI The GI absorbs and processes ingested food to fulfill the energy demands of an organism’s development, reproduction, and survival . The GI The intestinal epithelium is the single cell layer that form the lining of the intestine of the GI.The intestinal epithelium has defense mechanisms to detect and eliminate invading pathogens. The intestinal epithelial cells (IECs) deploy several immune response strategies such as innate immune receptor families that recognize specific microbe-associated molecular patterns (MAMPs) and in turn activate cellular immune signaling strategies that include the production of microbicidal reactive anionic species and other antimicrobial molecules such as antimicrobial peptides ,11,12. TOverall, genetic and environmental factors  can affect intestinal epithelial function, stability, and homeostasis and lead to severe pathologies. Dissecting the signaling pathways that modulate metabolic signaling, host–microbial interactions and stress responses in the gut epithelium is, therefore, pivotal for understanding how enteric health is achieved and maintained. Studies of model organisms have served as a great resource to untangle the complex mechanisms that determine the health of the intestinal epithelium in humans. Drosophila genome has been fully sequenced [Drosophila genetic, developmental, and biochemical blueprints for maintenance of gut epithelial stability and homeostasis are largely conserved between flies and vertebrates . Here, Drosophila gut consists of a simple epithelium surrounded by visceral muscles, nerves, and the trachea. The epithelium of the Drosophila adult intestine is subdivided along the anteroposterior axis into the foregut, midgut, and hindgut  and the hindgut [1, duodenum 103, jejunum 104, ileum 107, colon 1012 cells per gram) as the pH of the region decreases [Helicobacteraceae and Lactobacillaceae, whereas the colon strictly supports anaerobic bacteria such as Lachnospiraceae, Bacteroidaceae, and Prevotellaceae [Drosophila gut does not have an anoxic portion, unlike the human gut; therefore, the bacterial communities found in the Drosophila gut are aero-tolerant species and represent a limit of this model for studies of host-microbiota interactions in humans. The most commonly found bacterial species in Drosophila are Lactobacillaceae and Acetobacteraceae [Different intestinal segments carry out unique functions, and many physicochemical parameters vary accordingly, such as pH and, oxygen concentration. For example, both nutrient breakdown and enzymatic activity are strongly affected by the physicochemical properties of the lumen . Specifi hindgut ,32. Thes hindgut ,34. Thusecreases . The uppteraceae ,37,38, wteraceae ,39,40, aDrosophila and humans. The crypt-like niche structures of the human small intestine and one or more carbohydrate-rich barriers, such as the secreted mucus layer [The intestinal epithelium is in constant contact with the external environment, but the ISCs are partially isolated and protected from it in both us layer , provideus layer . Additious layer . Drosophila, the intestinal epithelium is covered by a semi-permeable structure composed of chitin and proteins, the peritrophic membrane (PM), as well as an additional thin mucus layer. The Drosophila’s mucus layer has been identified only recently thanks to the development of a mucosubstance staining method (periodic acid Schiff) [Drosophila genes have been annotated as mucin-like proteins [Drosophila does not have a lamina propria (LP), a large layer of connective tissue that contains resident innate immune cell populations such as dendritic cells, macrophages, eosinophils and mast cells, and adaptive immune cells [Drosophila lacks adaptive immunity and resident immune cell populations, these aspects of the host’s immunity defenses cannot be studied in the Drosophila model system. Both PM and the LP selectively allow molecules to pass through and come into contact with ECs without potentially damaging them [Drosophila PM and the human LP and mucus determines which microbial components or metabolites can come into contact with ECs.In  Schiff) . Over 30proteins , but theproteins . These lproteins ,45  in Drosophila) and Notch signaling pathway [Drosophila intestinal epithelium. Studying these conserved signaling pathways using the Drosophila gut model system has provided insight into the precise mechanisms by which these signaling pathways are activated within different ECs to maintain epithelial health.In the intestinal epithelium of young adult fections , the profections . The act pathway . AlteratDrosophila. Notably, in humans, Wg/Wnt dysfunction is attributed to the development of over 80% of human colorectal carcinomas [Drosophila midgut  can be tightly regulated to dissect the key factors of the diet–microbiota–gut axis that affect organism health. This would be very important to translate the effect of the diet–microbiota interaction to study specific human pathologies. For instance, neurological disorders from the autism spectrum are concomitant with changes in the microbiome and enterocolitis [Acetobacter pomorum, provides thiamine to its host. Moreover, endogenous A. pomorum is such a main source of thiamine that flies were capable of developing on a thiamine-free diet [Comparative studies on the transcriptome of germ-free and conventionally reared  the gut ,79, prov the gut . The Drohe diet, , this paocolitis ; howeverocolitis . In thisree diet .Drosophila intestine as a model to study the bacterial microbiota has helped to define the plasticity, flexibility, and mutual benefits of host–bacteria interactions within the gut and its use will continue to help in the future to understand how the microbiota can affect different aspects of an organism’s physiology such as growth, maturation, immune and stress responses, stem cell activity, metabolic reprogramming, and disease susceptibility.In conclusion, the use of the Drosophila melanogaster ingests potential pathogenic microbes during each meal. To defend against these pathogens, Drosophila has several constitutive as well as inducible levels of defense. Resistance and resilience to microbes are two critical aspects of a host’s defense [Drosophila, resistance to pathogens is mediated by two inducible immune response pathways: the reactive oxygen species (ROS)-generating Dual oxidase (DUOX) pathway and the antimicrobial peptide-producing immune deficiency (Imd) pathway (see the next section) [As in all mammals,  defense . In Drossection) . Resiliesection) . Drosophila and humans, the fruit fly has been successfully used to study infections from over 40 human pathogens, to investigate pathogen virulence factors, and how the host’s innate immune system detects and combats these microbes  to Vibrio cholerae pathogenesis and assess consequences on host viability [V. cholerae pathogenicity is dependent on T6SS and host–commensal interactions [Due to the well-conserved mechanisms behind resistance and resilience in del, see ). It appysiology . For exaiability . This inractions . Drosophila model revealed that Pseudomonas aeruginosa, a major agent of lethal infections in cystic and burn wound patients, modulates the local host defense responses in a tissue-dependent manner and may contribute to epithelial inflammation and cancer [Drosophila studies show that there is an inverse correlation between biofilm formation [P. aeruginosa virulence [Similarly, work in a d cancer . Moreoveormation  and acutirulence .Drosophila model system led to the discovery of a rapid and evolutionarily conserved defense response to pore-forming toxins in the gut [Drosophila intestines exposed to hemolysin, a pore-forming toxin secreted by Serratia marcescens, exhibit epithelial thinning followed by a recovery of their initial thickness once the infection is cleared. A few hours post-infection, ECs expel most of their cytoplasm and organelles, although the cells do not lyse [Drosophila model, epithelial thinning appears to be a fast and efficient recovery mechanism to combat intestinal infections with pore-forming toxins in mice [Moreover, studies using the  the gut . Drosophnot lyse . Consist in mice , suggest in mice . This pr in mice . Interes in mice , suggestGiven that the intestinal epithelium acts as a barrier against the external environment, it consistently enters into contact with bacteria that can be beneficial or harmful to the host. Therefore, epithelial cells have developed mechanisms to discriminate between beneficial and pathogenic microbes, thus tolerating the proliferation of symbiotic microorganisms while mounting immune responses towards invading pathogens.Drosophila as an immune elicitor [meso-diaminopimelic acid-type PGN (Dap-PGN), while Gram-positive bacteria have a L-lysine-type PGN (Lys-PGN). Peptidoglycan (PGN) was the first bacterial component characterized in elicitor . PGN is elicitor . TypicalPGN synthesis is highly coordinated with the process of cell wall remodeling to ensure growth and cell division. After each round of cell division, PGN fragments are released into the surrounding medium under the action of hydrolase enzymes . Some PGIn both vertebrates and invertebrates, PGN detection is mediated by the PeptidoGlycan Recognition Receptors family (PGRP), which is characterized by a functional domain that is approximately 165 amino acids in length at the C-terminus position and is denoted the PGRP domain . Drosophila, each type of PGN (Lys or Dap) will activate a specific Nuclear factor κB (NF-κB)-dependent signaling cascade [Drosophila Toll pathway is activated upon recognition of Lys-PGN and triggers an immune response in many Drosophila cells, but it is not activated within ECs [Drosophila) to induce the production of antimicrobial peptides, which kill invading pathogens.In  cascade  althoughthin ECs . In contthin ECs . The Imdthin ECs . Once acEscherichia coli phagocytosis [Studies using genetic, biochemical, and cellular biology experiments revealed that two PGRP receptors can detect at least two forms of PGN . Peptidoocytosis ,102,103.ocytosis . In vivoBox 1Drosophila husbandry can be controlled and implanted to minimize variation during bacterial infection or host–bacteria interaction studies:Several stringent parameters in Age: Typically, 4–14-day-old adults are used since the final steps of gut maturation take place during the first 4 days after eclosion [eclosion , while aeclosion .Gender and mating status: Sex-specific differences have been observed in biological processes such as immune and metabolic pathways [Drosophila model presents a difference in gut plasticity between females and males [pathways ,108,109 pathways ,111. As nd males , controlPopulation size: As ethical considerations do not restrict Drosophilists, a sufficiently large numbers of individuals can and should be scored. Standard density cohorts have been determined according to physiological assay [al assay ,112.Diet: As observed in the human microbiota, the Drosophila gut microbiota can be shaped by the diet [Drosophila, in which the exact composition and concentration of every ingredient is known and can be manipulated, is available [the diet . Moreovethe diet ,114. A hvailable  and is available .Microbes and immune elicitors: The Drosophila model can be used as the model of 40 infection conditions observed in humans [n humans . Both hun humans ,117,118.Time and dose of inoculation: Standard culture conditions, typical dose, and usual route of infection are available for infection study [on study . For hoson study  or stablon study  flies wiTemperature: The temperature influences biochemical reactions and biological processes of all living organisms [Drosophila studies are 18, 25 and 29 °C and flies can be maintained at a constant temperature or exposed at transient temperature treatments. The optimal condition for Drosophila physiology is 25 °C; lower and higher temperatures will be considered as stressful conditions for the animal physiology [rganisms ,112. Theysiology ,120. Thunucleotide-binding oligomerization domain (NOD) receptors  while PGRP-LE works in the posterior domains (R3–R5) and both receptors function in R2 . Even ifsurvival ,129. To survival . Bordetella pertussis is the specific cytotoxic factor that induces damage to ciliated epithelial cells and inhibits neutrophil function [Streptococcus pneumoniae, and Staphylococcus aureus [Experimental evidence demonstrates that PGN acts as an elicitor across eukaryotes . PGN is function . Locallyfunction . The fors aureus . The PGNs aureus ,137.Drosophila and humans, Drosophila is an excellent model organism to characterize novel PGN signaling functions related to innate immune surveillance, activation, and tolerance. Despite the fact that Drosophila shares several similarities with the mammalian detection system of PGN, future investigation is required to dissect how Drosophila activates and modulates intestinal inflammation in response to PGN. However, to identify unexplored immunoregulatory strategies in mammals, we also need to consider the limitation of the system. The PGN-mediated induction of immune signaling in Drosophila intestines is limited to the NF-κB-mediated responses through the PGRP receptor family, such as PGRP-LE, a NOD-like receptor in ECs , which acts as a first line of defense to combat the uracil-secreting bacteria [2O2 production represents a fast innate immune response strategy in both Drosophila and mammals to combat pathogens; however, a concurrent elimination of the residual H2O2 is required to protect the host from tissue damage and inflammation [2O2 can lead to chronic epithelial inflammatory diseases [2O2, DUOX activity needs to be finely regulated to avoid chronic inflammation, which results in EC apoptosis and oxidative damage. Genetic studies in Drosophila allowed researchers to determine the signaling pathways that regulate the activation of DUOX. DUOX enzymatic activation is regulated by Hedgehog (Hh) signaling, which triggers phospholipase Cb (PLCβ)-Ca2+, which in turn controls the DUOX-dependent production of bactericide H2O2 [However, the biological significance behind uracil secretion remains to be elucidated. It has been proposed that uracil secretion may be part of the stringent response, which helps bacteria to adapt and survive the stressful conditions preceding stationary phase. Notably, uracil synthesized through the UMP/uracil biosynthesis pathway is critical for proper biofilm formation, which acts as an important virulence factor for numerous bacterial species . Therefobacteria ,145,146.diseases . To previde H2O2 . MoreoveDrosophila. Notably, DUOX orthologues are present in the mouse and humans and have roles in innate immune responses, including intestinal epithelial immunity [Despite the extensive characterization of the DUOX pathway, host receptors that detect uracil and are responsible for DUOX activation remain unknown. Preliminary genetic screens revealed that multiple G-protein coupled receptors (GPCRs) might be involved in DUOX activation during gut–bacterial interactions. Further investigation of these GPCRs and their respective ligands will provide a more precise mechanism by which the host detects uracil and eliminates microbes via DUOX regulation in immunity , yet theimmunity . Drosophila. DUOX activation is mainly required as a source of H2O2 that is needed to catalyze the oxidative conversion of thiocyanate (SCN−) to hypothiocyanite (OSCN−) by the enzyme lactoperoxidase  pathway, and the mammalian target of rapamycin pathway (mTor) [Drosophila as they do in humans. Notably, the activation of insulin signaling down-regulates the expression of immune genes, whereas the inactivation of insulin signaling induces immune genes expression and increases resistance to infection [Drosophila model presents some limitations that must be taken into consideration in each study. Drosophila does not have lymphocytes [The tebrates ,164. Simy (mTor) . For exanfection . The Drophocytes , immune phocytes ,169. Drosophila, cachexia-like phenotypes can be observed after oral infection with pathogens [Drosophila and the human suggests that the use of the Drosophila model will help to further define immunometabolic pathways in humans.In humans, acute and chronic inflammation can induce cachexia, which causes progressive weight loss due to an atypical increase in energy consumption associated with defects in carbohydrate, lipid, and protein metabolism . Similarathogens . This phathogens . This coDrosophila model system as an emerging model system to study intestinal physiology in health and disease, we will report the recent contributions to the field of immunometabolism coming from studies carried out in the Drosophila gut model. Since the gut is a central metabolic  and immune responsive tissue , it is a valid organ to study the cross-talks between metabolism and immunity. Since this review focuses on the contribution of the Drosophila gut. This metabolic reprogramming is required for DUOX-dependent antimicrobial responses and host resistance against invading pathogens. The study by Lee et al. highlights the extensive metabolic modulation that intestinal bacteria can induce at the site of infection. Recent work from Lee et al. demonstrated that bacterial uracil triggers metabolic reprogramming in ECs to induce DUOX activity and ROS production . AuthorsDrosophila model system may contribute to unraveling a previously unexplored model that indicates that intestinal bacteria can also modulate host metabolism at the systemic level [Acetobacter induces systemic insulin signaling activation that is necessary for host developmental and metabolic homeostasis [V. cholerae infection, pathogens and host cells compete for the Acetobacter-derived acetate [V. cholerae will adsorb acetate more efficiently than the host, and therefore cause systemic insulin signaling inactivation that triggers a cachexia-like phenotype [Hence, studies using the ic level . For exaeostasis . However acetate . V. cholhenotype . SCFAs hhenotype .Emerging evidence demonstrating the complexity of host–bacteria interactions raises questions regarding how these finely tuned pathways are integrated into IECs to maintain enteric stability and homeostasis. Peroxisomes are ubiquitous organelles conserved across the breadth of eukaryotes and govern essential metabolic reactions such as cellular redox homeostasis and lipid oxidation ,176. RecDrosophila as well as to promote survival and development of the organism [Drosophila, the authors generated flies with dysfunctional peroxisomes in ECs, allowing them to study the cell-specific role of peroxisomes in ECs. ECs with dysfunctional peroxisomes present an accumulation of non-esterified free fatty acids and high amounts of hydrogen peroxide, which becomes exacerbated in Drosophila challenged with the opportunistic but not lethal bacterium Erwinia carotovora carotovora (Ecc15). These unbalanced metabolic conditions cause the induction of Tor kinase-dependent autophagy, which leads to dysplasia of the intestinal epithelium and increased apoptosis of ECs. Moreover, the lack of functional peroxisomes in ECs induces dysbiosis and compromises the immune response, effectively reducing an organism’s survival upon microbial pathogenic stress. The work by Di Cara et al. suggests that peroxisomes, act as hubs that coordinate responses from stress, metabolic, and immune signaling pathways to maintain enteric health and beneficial interactions between commensals and the host. The same group reported that peroxisomes are required to maintain intestinal epithelium homeostasis and renewal in organism  . Therefore, Drosophila melanogaster is undoubtfully an established and powerful model organism with strong potential to increasingly contribute to the elucidation of the cellular mechanisms that govern intestinal health and host–microbial interactions and is a valuable genetic platform to accelerate further discoveries, which can then be validated in mammalian models and humans.Notably, defenses . These i


During its reproductive phase, rice is susceptible to heat stress. Heat events will occur at all stages during the reproductive phase of rice as a result of global warming. Moreover, rice yield traits respond differently to heat stress during panicle initiation, flowering and grain filling. The reduction in the number of spikelets per panicle of heat-stressed plants is due to the attenuated differentiation of secondary branches and their attached florets as well as the promotion of their degradation during the panicle-initiation stage but is not affected by heat stress thereafter. Spikelet sterility as a result of heat stress is attributed not only to physiological abnormalities in the reproductive organs during the flowering stage but also to structural and morphological abnormalities in reproductive organs during the panicle-initiation stage. The reduced grain weight of heat-stressed plants is due to a reduction in nonstructural carbohydrates, undeveloped vascular bundles, and a reduction in glume size during the panicle-initiation stage, while a shortened grain-filling duration, reduced grain-filling rate, and decreased grain width contribute to reduced grain weight during the grain-filling stage. Thus, screening and breeding rice varieties that have comprehensive tolerance to heat stress at all time points during their reproductive stage may be possible to withstand unpredictable heat events in the future. The responses of yield traits to heat stress are regulated by phytohormone levels, which are determined by phytohormone homeostasis. Currently, the biosynthesis and transport of phytohormones are the key processes that determine phytohormone levels in and grain yield of rice under heat stress. Studies on phytohormone homeostatic responses are needed to further reveal the key processes that determine phytohormone levels under heat conditions. Human activities have triggered climate change, which is manifested as global warming. Global climate change represents one of the most serious challenges faced by humans . Crop prRice plants are vulnerable to heat stress, especially during the reproductive phase . Heat stPhytohormones play an important role in coordinating the response of yield traits to heat stress in rice. For example, cytokinin (CTK) and abscisic acid (ABA) modulate floret differentiation and the number of spikelets per panicle and indole-3-acetic acid (IAA) and gibberellin (GA) are thought to be involved in regulating the development of reproductive organs and activities involving pollination and fertilization and thus spikelet fertility. IAA, GA, ABA and CTK mediate grain weight under heat . HoweverResearch papers published both in English and in Chinese were searched from the Web of Science and the China National Knowledge Infrastructure  from 1950 to 2018. There were six search terms queried as follows: “rice AND high temperature/heat/warming” and “high temperature/heat/warming AND phytohormone”. An initial search resulted in 26,618 articles, which were reduced to 8,813 by limiting research to plant science, agronomy, cell biology, physiology, biology, developmental biology and environmental science. We focused mainly on the physiological and molecular aspects involving phytohormone homeostasis in rice varieties in response to heat stress. We then examined the article titles, abstracts and key words to judge their relevance, after which 2,115 articles were considered relevant. There were 15 studies in other crop species  and in Arabidopsis that were highly relevant to the topic of this review, which were also included.Heat stress is defined as an increase in temperature beyond a critical threshold and for a certain period of time resulting in irreversible damage to plant growth and development . Heat inHeat stress reduces the number of spikelets per panicle during the panicle-initiation stage, but the number of spikelets per panicle does not decreased when heat events occur after anthesis . The redHeat stress during the early reproductive stage induces panicle enclosure, which manifests as a partial panicle trapped within the flag leaf sheath . In the + and Ca2+)  , carbohynd Ca2+)  and regund Ca2+) , as wellnd Ca2+) , which cNotably, anther dehiscence is the initial step of pollination and is highly susceptible to heat stress; thus, anther dehiscence was suggested to be a selective marker for screening heat tolerance . HoweverIn summary, spikelet sterility induced by heat stress can be attributed primarily to physiological abnormalities in the reproductive organs during flowering and the associated mechanisms of the effects of heat stress during the early reproductive phase differ, which is mainly attributed to structural and morphological abnormalities of reproductive organs .Rice grain weight is determined by multiplying grain volume by grain plumpness. Heat stress during the reproductive phase adversely affects grain volume and grain plumpness . Small gHeat stress reduces CTK contents. Reductions in CTK contents in the young panicles of heat-stressed rice plants are due to the following mechanisms: (i) reductions in the long-distance transport of CTKs from the roots to the shoots; (ii) inhibition of CTK biosynthesis via decreased biosynthesis of related enzymes (isopentenylation of adenosine phosphate by isopentenyltransferases (IPTs), cytochrome P450 mono-oxygenase (CYP735A) and LONELY GUY (LOG)); and (iii) increases in the catabolism of CTKs due to increased activity of CTK metabolism-related enzymes (cytokinin oxidase/dehydrogenase (CKX)) and CTK glycosylation , 2016. AThe number of spikelets per panicle is affected mainly by heat stress during panicle initiation . ChangesYUCCA, leading to anther indehiscence and reduced pollen vigor that could be rescued by applications of exogenous IAA , YUC flavin monooxygenase (YUC) genes, and UDP-glucosyltransferase 74b1 (UGT74B1) (GH3) in rice  and the transport of IAA  during heat stress in rice is limited. Monitoring isotope-labelled 14C-IAA in root tissues of pea revealed that heat stress reduced IAA transport; however, the sensitivity of the diffusion component (the IAA-influx/efflux gene) to temperature and IAA concentration has not been determined  , and red in rice , which p in rice . Howevertermined . In summtermined .GA20ox1, GA20ox2, GA20ox3) and GA3-oxidase genes , which regulate the late steps of the GA biosynthetic pathway, are suppressed by heat stress (OsCPS1), ent-kaurene synthase (OsKS1), ent-Kaurene oxidase (OsKO2) and ent-kaurenoic acid oxidase (OsKAO), have seldom been investigated in plants under heat stress conditions. Information concerning the responses of the catabolism-related genes  and transport-related genes (OsNPF and OsSWEET) to heat stress is also limited. According to existing results, heat stress suppresses the biosynthesis and promotes the deactivation of GA, but few studies have documented the effects of heat on the catabolism and transport of GA in rice plants (Heat stress reduces the content of GAs in young panicles of rice varieties . The GA t stress . Howevere plants .YUC) and transport genes  in rice under high temperature . In aerial organs, heat exposure increased the ABA content by upregulating ABA biosynthesis-related genes, including the zeaxanthin epoxidase genes  and the 9-cis-epoxycarotenoid dioxygenase genes , but downregulated expression of the ABA 8′-hydroxylase gene CYP707A  The number of spikelets per panicle is reduced by heat stress during panicle initiation but is not affected by heat stress during flowering and grain filling. In addition, the reduction in the number of spikelets per panicle in heat-stressed plants is due to the attenuated differentiation of secondary branches and their attached florets as well as the promotion of their degradation during panicle-initiation stage. (ii) Spikelet sterility induced by heat stress can be attributed primarily to physiological abnormalities in the reproductive organs during the flowering stage but also can be attributed to structural and morphological abnormalities in reproductive organs during the panicle-initiation stage. (iii) Last, the relatively low grain weight caused by heat stress during panicle initiation is due to a reduction in nonstructural carbohydrates, undeveloped vascular bundles and a reduction in grain length and width, while a shortened grain-filling duration, reduced grain-filling rate and decreased grain width affect grain weight when heat stress occurs during the grain-filling stage. Different responses of yield traits to heat stress have been identified in rice during the panicle-initiation, flowering and grain-filling stages. Although many heat-tolerant rice varieties have been identified, most of these varieties endure heat injury only during specific reproductive stages, mainly during flowering. To cope with unpredictable heat events in the future, we should screen and breed rice varieties with comprehensive tolerance to heat stress throughout the entire reproductive phase.The physiological and molecular mechanisms of the effects of heat on rice grain yield have drawn much attention. Under heat conditions, CTK and ABA modulate the number of spikelets per panicle; IAA and GA are thought to be involved in spikelet fertility; and IAA, GA, ABA and CTK mediate grain weight. The responses of yield traits to heat stress are regulated by phytohormone levels, which are determined by phytohormone homeostasis. Currently, biosynthesis and transport are purported to be the key processes that determine phytohormone levels in and final grain yields of rice under heat stress. Studies on phytohormone homeostatic responses are needed to further reveal the key processes that determine phytohormone levels under heat conditions.


An increasing number of migrants have fled armed conflict, persecution and deteriorating living conditions, many of whom have also endured risky migration journeys to reach Europe. Despite this, little is known about the perinatal health of migrant women who are particularly vulnerable, such as refugees, asylum-seekers, and undocumented migrants, and their access to perinatal care in the host country.Using the Swedish Pregnancy Register, we analyzed indicators of perinatal health and health care usage in 31 897 migrant women from the top five refugee countries of origin between 2014 and 2017. We also compared them to native-born Swedish women.Compared to Swedish-born women, migrant women from Syria, Iraq, Somali, Eritrea and Afghanistan had higher risks of poor self-rated health, gestational diabetes, stillbirth and infants with low birthweight. Within the migrant population, asylum-seekers and undocumented migrants had a higher risk of poor maternal self-rated health than refugee women with residency, with an adjusted risk ratio (RR) of 1.84 and 95% confidence interval (95% CI) of 1.72–1.97. They also had a higher risk of preterm birth , inadequate antenatal care  and missed postpartum care visits .Refugee, asylum-seeking and undocumented migrant women were vulnerable during pregnancy and childbirth. Living without residence permits negatively affected self-rated health, pregnancy and birth outcomes in asylum-seekers and undocumented migrants. Pregnant migrant women’s special needs should be addressed by those involved in the asylum reception process and by health care providers. Tens of millions of refugees have been trying to find security outside their country of origin because of armed conflicts, persecution and poverty. The overwhelming majority of the world’s refugees resettle in low-income and middle-income countries in Africa and Asia,,Refugee women are a vulnerable group at risk for sexual and gender-based violence before and during migration, and even after arrival in the destination country,,In Sweden, all women are guaranteed by law to have equal access to critical care during pregnancy, birth and the postpartum period, regardless of immigration status.Asylum-seekers apply for refugee status upon arrival to the host nation. Refugees with an approved refugee status in Sweden have permanent or extendable temporary residence lasting at least 13 monthsPerinatal care is free for all women living in Sweden, regardless of their residency status. A temporary number is used for perinatal care for women without a permanent personal identification number. Approximately 99% of the births in Sweden take place in 46 delivery hospitals, each with their own catchment area and local antenatal units.The new national Swedish Pregnancy Register provides comprehensive information on each pregnancy, collected from the first antenatal health care visit to the postpartum visit. Electronic medical records are linked with data collected by midwives at the first antenatal visit and the postpartum visit, including the mother’s country of birth, education and self-rated health.N = 286 870) which comprised the top five countries of origin for asylum-seekers in Sweden from 2014 to 2016.All 394 343 singleton births registered in the Swedish Pregnancy Register in 2014–17 were eligible for study inclusion . We exclWe used maternal country of birth to identify the migrant population, including refugees, asylum-seekers and undocumented migrants. Women from the top five asylum-seeker countries of origin who had a unique personal identification number were classified as refugees. Women who only had a temporary number in the register were classified in the group of asylum-seekers and undocumented refugees.Information on parity, education level, smoking and alcohol consumption and self-rated health prior to pregnancy were collected at the first antenatal care visit. Self-reported health was rated as very good, good, neither poor nor good, poor, very poor and do not know. Women who provided any answer apart from very good or good were classified as having poor self-rated health prior to pregnancy. Maternal body mass index (BMI) was derived from body height and weight at the first antenatal care visit and maternal age at delivery was retrieved from the medical records.We examined the relevant International Classification of Diseases, Tenth Edition codes. Pre-eclampsia (codes O14 and O15), gestational diabetes (code O24) and III–IV degree anal sphincter tears , which involves the anal sphincter and can be associated with maternal morbidity such as faecal incontinence, were retrieved from maternal diagnosis in antenatal care and at discharge from the delivery hospital. Severe postpartum haemorrhage was defined as having a blood loss of more than 1000 ml, according to the medical records. Maternal death was defined with code O95, O96 or O97 or an infant diagnosis of P016 indicating maternal death.in vitro fertilization, when applicable, ultrasound scans or the last menstrual period when an ultrasound estimation was not available. Preterm birth before 37 gestational weeks was primarily based on gestational age using the same information. Preterm births that were only defined by the estimated gestational age on ultrasound were also compared to avoid differences due to the method of gestational age estimation. Small for gestational age was defined as a birthweight below two standard deviations for gestational age, according to the sex-specific Swedish standard curve of foetal growth.Stillbirths were directly retrieved from the medical records. Gestational age at birth was estimated from the date of The number of antenatal care visits and ultrasound exams were retrieved from medical records. Having fewer than nine visits was regarded as below the recommended number of antenatal care visits. Having no ultrasound screening or ultrasound estimated gestational age was coded as having no ultrasound screening. We calculated the expected number of antenatal visits based on the Swedish antenatal care programme.Medical records also provided information on the use of professional translators during antenatal care, abnormal cardiotocography (CTG) tracing on admission to the delivery ward, the mode of delivery and the date of the postpartum visit. Mode of delivery was split into four categories: non-instrumental or instrumental vaginal deliveries and planned or unplanned Caesarean sections. If there was no date recorded for the postpartum visit, it was defined as a missing visit. The way postpartum visits were organized may have differed between different maternity units, as some book them during late pregnancy, while others ask the woman to call after birth and book the visit herself, which may be difficult for refugees who are unfamiliar with the health system or face language barriers. Unfortunately, the data did not allow us to differentiate between the type of maternity units, which is not associated with individual characteristics or quality of care to our knowledge. Having no recorded postpartum visit was classified as a less adequate use of postpartum care.Logistic regression models were used to analyze binary outcomes and multinomial logistic regression models were used for categorical outcomes, such as mode of delivery. Adjusted risk ratios (RRs) and risk differences (RDs) were then calculated. The confidence intervals were estimated by delta-method standard errors.We first described the perinatal health of all migrants in this study, by comparing their perinatal health outcomes to the Swedish-born women. We reported the observed risks and mean birthweights of term births in both groups along with the relative risks and RDs between them, adjusting for maternal age and parity in quadratic forms.We further estimated the effect of not having residency on perinatal health by comparing the asylum-seekers and undocumented migrants to refugees. Maternal education, age and parity in quadratic forms, calendar year of delivery and country of origin were adjusted in all models. Additional adjustments for maternal BMI categories and heights, in continuous forms, were made to analyze the risk of adverse maternal and foetal outcomes measured during delivery.N = 7356), restricting the analysis to outcomes measured during or shortly after delivery. Due to missed information on maternal country of birth, they were not included in our study population. Undocumented migrants might be over-represented in this population.Due to large mass migration during a relatively short period, a sensitivity analysis was conducted for Syrian women, to avoid confounding of the measures by duration of residence. We also analysed the outcomes of births with no information on antenatal care and no personal identification numbers, who might have presented only at delivery care  and all data analyses with STATA® version IC 14 (64 bit) .All data management was performed using SASWe evaluated records of 286 870 women who gave singleton births, 88.9% of whom were Swedish-born, 10.4% who were refugees and 0.7% who were asylum-seekers and undocumented migrants. Migrant and Swedish women had similar risks of having abnormal CTG tracing at admission to the delivery ward. Maternal diagnoses of pre-eclampsia, preterm birth and severe postpartum haemorrhage were less common in migrant women than Swedish women. We cannot make statistical inference on maternal death, due to its rarity (data not shown).Refugees and asylum-seekers, including undocumented migrants, had similar risk of pre-eclampsia, gestational diabetes and III–IV degree anal sphincter tears. However, asylum-seeking and undocumented migrant women were less likely to have a severe postpartum haemorrhage . A sensitivity analysis of Syrian women showed similar findings for maternal and infant outcomes among asylum-seekers and undocumented migrants compared to refugees (data not shown).N = 675). They show similar risks of adverse birth outcomes as the asylum-seekers and undocumented migrants included in the study population.This study shows that from 2014 to 2017 pregnant refugee women in Sweden had higher risks of poor self-rated health, gestational diabetes and severe anal sphincter tears, as well as higher risks of adverse infant outcomes including stillbirth, low birthweight, small for gestational age and low Apgar score than Swedish-born women. Asylum-seeking and undocumented migrant women were particularly vulnerable, with higher risks of poor maternal self-rated health, preterm birth and missed perinatal visits than refugee women with established residency.Our results suggest that legal residency status had a potent influence on the perinatal health of refugee women and their infants. Previous studies have demonstrated the connection between maternal psychosocial stress and preterm birth.Pregnant refugee and asylum-seeking women, including undocumented migrants, in European countries have the greatest needs for special attention and care from maternal health services. The International Organization for Migration (IOM) surveys conducted in 2017 demonstrated alarmingly high rates of rape and sexual exploitation among asylum-seeking women en route to Europe.,,,,However, our study also indicates that equal rights to maternity care, which exists in Sweden, is not sufficient to guarantee equal access to care. Missed antenatal visits and ultrasound screening may be partially due to a later arrival in the destination country (Sweden); however, the increased risk of missing postnatal care visits clearly indicates lower care usage and missed opportunities to improve maternity care. There may also be barriers to the delivery of maternity care, including language and more subtle barriers related to cultural differences in belief systems and being less familiar with preventative care. Medically trained interpreters and cultural mediators can facilitate communication between midwives, society and refugee women.,,Pre-eclampsia, preterm birth and severe postpartum haemorrhage were less common in migrant women than in Swedish women. The result on pre-eclampsia is consistent with a previous Swedish study and a systematic review of pregnancy-related hypertensive disorder in migrant populations.The Swedish Pregnancy Register provided a unique opportunity to investigate the perinatal health of refugee women in Sweden, where free health care regardless of residency status is guaranteed. To the authors’ knowledge, this was the first population-based study to compare the impact of legal residency on perinatal health by comparing refugees with asylum-seekers and undocumented migrants. The findings may not be directly generalized to other contexts and setting, but provide valuable information for future policy-making in response to forced migration.Duration of residence could not be included in the analysis, given the unknown date of arriving in Sweden. Comparison of asylum-seekers and refugees from Syria (who migrated more recently) showed results consistent with those of the main analysis. Longer duration of residency may also be associated with higher risk of preterm birth.This Swedish study showed that asylum-seeking and undocumented migrant women had poorer self-rated health before giving birth than refugee women, and also received less perinatal maternity care. They also had a higher risk of preterm birth than refugee women. More effective asylum procedures and greater intervention by health care providers, in partnership with other public service sectors, are critical for improving the pregnancy outcomes of forcibly displaced and newly resettled women.ckz120_Supplementary_DataClick here for additional data file.