In the heterogeneous hub represented by the lateral hypothalamus, neurons containing the orexin/hypocretin peptides play a key role in vigilance state transitions and wakefulness stability, energy homeostasis, and other functions relevant for motivated behaviors. Orexin neurons, which project widely to the neuraxis, are innervated by multiple extra- and intra-hypothalamic sources. A key property of the adaptive capacity of orexin neurons is represented by daily variations of activity, which is highest in the period of the animal’s activity and wakefulness. These sets of data are here reviewed. They concern the discharge profile during the sleep/wake cycle, spontaneous Fos induction, peptide synthesis and release reflected by immunostaining intensity and peptide levels in the cerebrospinal fluid as well as postsynaptic effects. At the synaptic level, adaptive capacity of orexin neurons subserved by remodeling of excitatory and inhibitory inputs has been shown in response to changes in the nutritional status and prolonged wakefulness. The present review wishes to highlight that synaptic plasticity in the wiring of orexin neurons also occurs in unperturbed conditions and could account for diurnal variations of orexin neuron activity. Data in zebrafish larvae have shown rhythmic changes in the density of inhibitory innervation of orexin dendrites in relation to vigilance states. Recent findings in mice have indicated a diurnal reorganization of the excitatory/inhibitory balance in the perisomatic innervation of orexin neurons. Taken together these sets of data point to “chronoconnectivity,” i.e., a synaptic rearrangement of inputs to orexin neurons over the course of the day in relation to sleep and wake states. This opens questions on the underlying circadian and homeostatic regulation and on the involved players at synaptic level, which could implicate dual transmitters, cytoskeletal rearrangements, hormonal regulation, as well as surrounding glial cells and extracellular matrix. Furthermore, the question arises of a “chronoconnectivity” in the wiring of other neuronal cell groups of the sleep-wake-regulatory network, many of which are characterized by variations of their firing rate during vigilance states. A wealth of studies has been stimulated by the discovery, two decades ago, of neurons of the lateral hypothalamus (LH) which synthesize the orexin/hypocretin peptides . InteresThese functions require capacity of adaptation to the internal milieu of the organism and to the external environment. Daily variations of activity, demonstrated for orexin neurons, are essential for such adaptation. At the synaptic level, adaptation should be subserved by plasticity. Seminal investigations have shown plastic synaptic changes in the excitatory wiring of orexin neurons after changes in food intake or prolonged wakefulness . Such fiThe orexin system and sources of inputs to orexin neurons are here first summarized. This is followed by a review of different lines of evidence pointing to daily variation of the activity of orexin neurons and to remodeling of their synaptic wiring in response to challenges. Dynamic changes in the synaptology of orexin neurons in basal conditions are then discussed. Altogether the findings point to daily variations in the excitatory/inhibitory wiring of orexin neurons as operational mode of these neurons inserted in a network that should meet daily demands.The orexin system has been the subject of authoritative reviews e.g.,  and onlyOrexin-A and orexin-B are also named hypocretin-1 and -2, and the double nomenclature is due to the designation of the molecules at the time of their discovery. These paired peptides are synthesized by neurons located in the perifornical area and LH . The oreOrexin-A and B are largely colocalized in the same neurons, as shown in the human  and rodeOrexin neurons represent a relatively small population: about 7,000 neurons in the rat brain  and 50,0OX1R and OX2R mRNAs suggests that these receptors enhance distinct physiological roles in diverse brain regions (Orexin actions are mediated by two membrane-bound G protein-coupled receptors: orexin-1 receptor (OX1R) and orexin-2 receptor (OX2R) . OX1R is regions .The study of the distribution of orexin fibers rapidly revealed that they establish a rich network within the LH and other hypothalamic regions, including the dorsomedial and ventromedial hypothalamic nuclei, arcuate nucleus, as well as the wake-promoting histaminergic tuberomammillary nucleus (TMN) . Orexin The orexin peptides were first recognized as regulators of feeding behavior  and orexOf note, orexin neurons link limbic regions and arousal-regulating regions, and the activation of orexin neurons by the limbic system could be instrumental in maintaining wakefulness during emotional arousal . In humaThe orexin system also regulates autonomic functions and the neuroendocrine axes, as well as plasticity mechanisms related to reward . FurtherFurthermore, orexin neurons innervate brain regions related to nociception, especially the periaqueductal gray , and plaThese broad range of functions has led to the definition of orexin neurons as “physiological integrators”  and “mulOrexin neurons are multitasking also concerning neurotransmitters. Orexin is excitatory  and a weIn early ultrastructural studies, orexin axon terminals in the TMN were found to establish asymmetric synapses , though There are also findings indicating that a subset of orexin neurons (about 10–20%) may release GABA, and that they exert a direct local inhibitory effect on neurons which contain melanin-concentrating hormone  . By singOrexin neurons also co-express other peptides (review in ad libitum) of a dense excitatory innervation of orexin cell bodies: immunofluorescence showed a 5:1 ratio of excitatory/inhibitory perisomatic input, and electron microscopy showed a ratio of about 2:1 of asymmetric versus symmetric contacts on dendrites. Excitatory synaptic currents were consistent with the prevalence of excitatory innervation of orexin neurons. Furthermore, dendro-dendritic interactions were prominent among orexin-immunopositive shafts and unidentified dendritic shafts of LH neurons. Since the receptor for the adipose hormone leptin is expressed at orexin neurons, a relationship between the levels of circulating leptin and the synaptic arrangement was hypothesized and verified with paradigms of fasting and re-feeding and leptin administration. The animals were maintained under a 12 h/12 h light/dark (LD) cycle. Although the time of sacrifice was not explicitly mentioned, on the basis of the experimental protocol (overnight food deprivation) the animals were presumably sacrificed at the end of the period of darkness.The normal synaptic organization of orexin neurons has been initially examined in intact mice used as controls of manipulations of food intake . This stThe predominantly excitatory synaptic organization of orexin neurons described in this study was defined as “unorthodox” compared to other neuronal cell types in the central nervous system , especiaThe sources of excitatory and inhibitory afferents to orexin neurons are diverse and represent a complex chapter of their connectivity and functional effects. Afferents have been investigated by methods with increasing selectivity and sensitivity: conventional tract tracing techniques, genetic tracing, neurochemical identification of boutons with multiple immunofluorescence, optogenetics. Altogether this wealth of data has shown that neural inputs originate from a variety of extra-hypothalamic sources, which do not always reciprocate orexin projections, and from intra-hypothalamic sources, including a dense network within the LH.TTC::GFP transgene) in orexin neurons  . GABAergIn addition, inhibitory functional glycine receptors and glycinergic synapses onto orexin neurons have been identified by electrophysiology and immuno-electron microscopy .Monoamine neurotransmitters such as dopamine, noradrenaline, and serotonin also regulate the state-dependent activities of orexin neurons through inhibition via their receptors, such as D1/D2- , alpha 2These cells are of special interest because they have been implicated in functions regulated also by orexin neurons, such as energy balance and sleep , autonomMelanin-concentrating hormone neurons are distributed over other areas besides the LH  but are Whole brain-mapping in mice has shown that afferents to MCH and orexin neuronal populations share many common sources, though it is not known whether the same cells innervate both peptidergic populations . MCH effcirca diem: recurring over a 24 h cycle) changes have been reported in the LH. For example, gene profiling in mice from tissue punches collected from VPLO, suprachiasmatic nucleus (SCN) and LH has shown a diurnal fluctuation in gene expression, with difference and overlap in these regions between day and night , it may not be surprising that diurnal , daily  and circadian , non-REM sleep , defined by neurophysiological parameters and, in particular electroencephalogram (EEG) recording. In model organisms such as the zebrafish, in which EEG cannot be recorded, sleep-like state is defined by behavioral criteria, based on the spontaneous occurrence of immobility with a circadian rhythm and under homeostatic control .It should be noted, however, that in many studies of molecular, cellular or electrophysiological features of orexin neurons the time of the day of the study  is not specified in the methodology, which therefore hampers a correlation with time of the day-dependent parameters.Most neuronal populations  in the sleep-wake network show diurnal variations in the profile of discharge that vary in strict dependency on the release of their neurotransmitter . The intFigures 1A,B).Consistently with a pivotal role of orexin neurons for arousal promotion and maintenance, in rats entrained under the LD cycle, single unit recordings across the sleep-wake cycle have revealed a precise profile of discharge . Thus, oThe same pattern of spiking was reported in rats  and in mfos, is a widely used marker of neuronal activity, induced by different stimuli . This was also documented when rats were maintained under constant darkness, indicating an endogenous oscillation . In addition, EEG recording showed a positive correlation between Fos expression in orexin neurons and the amount of wakefulness and a negative correlation with the amount of sleep (non-REM and REM sleep) in the 2 h preceding the animal’s sacrifice . A significantly greater number (24% increase) of orexin-immunopositive neurons, without significant changes in soma size, was found in undisturbed mice sacrificed during the dark phase compared to the light phase. The finding indicates that orexin expression does not reach the threshold for immunocytochemical detection in all orexin neurons during the phase of the animal’s rest. In contrast, there was no significant difference in the number of MCH-immunoreactive neurons between the animals in either phase, but a 15% increase in the soma size of MCH cells was found during the light phase compared to the dark phase  with a decrease during the day and an increase at night, in the preoptic/anterior hypothalamus and the pons, but not in other brain areas. Microdialysis and EEG (polysomnography) recordings showed a diurnal fluctuation of extracellular levels of orexin in the LH and medial thalamus of rats, with increase during the period of darkness and a decrease during the light period , falling then throughout the night . ProlongIn humans, the interpretation of findings on orexin levels in the CSF, which seem to increase during sleep, is complicated by the sampling times and by the methodology, represented by the lumbar puncture , due to which the temporal profile may differ, being delayed, from that of brain regions . It is iDiurnal variations of postsynaptic effects of orexin, especially relevant in the present context, have been studied in hippocampal subfields . In acutExperimental manipulations of the main functions in which orexin is involved, namely food intake and wake regulation, have pointed out a remarkable synaptic plasticity.The first data indicating that changes in the nutritional status are able to affect synaptic plasticity of orexin neurons and their connectivity derived from synaptology and electrophysiological studies during fasting and re-feeding in mice . Fastingob/ob mice and in mice subjected to high-fat diet (HFD), a model of diet-induced obesity  and to decrease during the subjective dark phase (rest period), highlighting a circadian control of the orexin synapse arrangement . To characterize excitatory and inhibitory terminals, the input to orexin cell bodies was visualized using the presynaptic marker synaptophysin combined with VGAT or VGluT2, as well as with each vesicular neurotransmitter transporter combined with the respective postsynaptic scaffold protein . A day/night reorganization of the balance between excitatory and inhibitory inputs to orexin cell bodies was observed, without a variation of their total number, with a predominance of inhibitory synapses during the day (about 75%) and a predominance of excitatory synapses during the night (about 70%) .The issue of the diurnal balance between excitatory and inhibitory inputs onto orexin somata in undisturbed mice has been recently examined in our laboratories . In thisThis set of data requires implementation. From the behavioral point of view, although the sleep and wake percentages in the hours preceding sacrifice showed very little interindividual variability in matched animals, vigilance state needs to be characterized in the same animals, and videorecording experiments are currently ongoing to this purpose. Furthermore, experiments under constant darkness need to be performed to verify an endogenous oscillation.At the subcellular level, excitatory and inhibitory inputs onto orexin neuron dendrites in the same paradigm remain to be examined. It should, however, be considered that the neuronal soma is the site of integration of all inputs and is a critical domain given the proximity to the site of action potential generation .At the synaptic level, electron microscopy is the gold standard for the visualization of synapses. Furthermore, although the contact with the postsynaptic scaffolding protein indicates a functional synapse , morpholEven considering these limitations, the above findings in mice show a time-of-day-dependent fluctuation in the perisomatic innervation of orexin neurons, and indicate a relationship of this process with sleep and wake predominance.Physiological functions are regulated by the strict interplay between the need of maintain an internal milieu  and the Evidence points to the daily fluctuation of orexin level in the CSF as an endogenous rhythm: this fluctuation was robustly maintained in rats entrained under dim red light  as well Concerning the complex issue of the relationships between orexin neurons and the biological clock, anterograde tracing data have suggested a polysynaptic link of the SCN with orexin neurons , consistInterestingly, findings on orexin level in the CSF of rats after different manipulations  have indA receptor (GABAAR) in orexin cell bodies after sleep deprivation in mice, confirmed by in vitro electrophysiology  and energy-related fine-tuning of synaptic inputs to orexin neurons, depending on CB1 expression at glutamatergic or GABAergic terminals. In this scenario, fasting-related reduction of leptin levels seems to control arousal by increasing the activity of orexin neurons, which are innervated by CB1-expressing excitatory inputs, and are a main source of the endocannabinoid 2-arachidonoylglycerol . Support“Chronoconnectivity” of orexin neuron wiring could potentially involve a timing-related regulation of neurons capable of glutamate and GABA co-release. This has not been examined in sources of orexin neuron innervation, but co-release of these fast neurotransmitters has been reported at other sites .Coexistence of VGAT and VGluT1 or VGluT2 has been reported in the hypothalamic periventricular nucleus , hippocaConsidering synaptic components besides the pre- and post-synaptic elements, it is now known that perisynaptic astrocytic sheaths are important local players, which has led to the concept of a tripartite synapse  and thatAstrocytes take part in the local network of regulation of orexin neuron activity within the LH . Knock-oIn their surveillance function, microglial cells continuously extend and retract their branches, contacting also synapses . DiurnalOn the other hand, ECM components are main players in synaptic plasticity phenomena, not only during development but also in the adult brain . The orgOverall, diurnal changes of glial cells and ECM in the LH in basal conditions await for further studies, which could reveal in this region an orchestra of multiple elements engaged in synaptic remodeling.The data here reviewed point to a daily fluctuation of excitatory and inhibitory inputs to orexin neurons as a feature of the logic of this vast network to face dynamic physiological demands. This envisages a “chronoconnectivity” which may involve different sets of inputs and may underlie diurnal fluctuation of neuronal firing and overall neuron activity.Such conceptual framework is different from the synaptic homeostasis processes hypothesized during sleep and wake , 2014. TOf note, changes of neuronal firing in different phases of the sleep-wake cycle is a property of many cell groups in the distributed network of sleep-wake regulation , as alsoIA wrote the first draft of the manuscript. FDG and LC contributed to various parts of the text and FDG prepared the figures. MB contributed to the theoretical framework and prepared the final manuscript version.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.


However, the long term effects that these cells may exert on the metastatic progression is not clear. One of the biggest challenges in the field is to distinguish between brain resident microglial cells and infiltrated bone-marrow derived macrophages. Part of this issue is the fact that both cell types share similar phenotypes. Current studies are based on the modulation of the immune response against cancer cells (immunotherapy). However, most of current clinical trials and newly developed drugs focus on the adaptive immune response . Additionally, the unique structure of the central nervous system with the presence of the blood-brain barrier have hindered a significant advance in novel therapies against brain metastasis. In this manuscript, we describe current advances in characterization of tumor-associated microglia and macrophages, the importance of microglia during the anti-cancerous response, and the future direction for the development of new strategies against this complex disease.The immune landscape in brain metastasis is a very heterogeneous framework. Amongst a broad plethora of cells within the tumor microenvironment, the presence of activated microglia has been perfectly described. The innate role of microglial cells is to detect and eliminate any insults that may disturb the regular behavior of the brain. As part of its defensive role, it releases pro- and anti-inflammatory cytokines that aim to modulate the inflammatory scenario at the metastatic  At the beginning of the last century, Currently, the use of pre-clinical models has helped us to delve into the importance of this unique brain cell type. Microglia are mostly present in the gray matter, particularly in the hippocampus, olfactory telencephalon, basal ganglia, and substantia nigra. The distribution of these cells in adult mouse brains fluctuates from 5% in the cortex to 12% in the substantia nigra . NeverthHowever, it is worth mentioning that metastatic colonization is a very inefficient process in which most cells die and only a minimal population survive and successfully establish metastases . The reaIn this manuscript, we aim at describing the complex biology of microglial cells within the CNS during metastasis progression and the importance of microglia and macrophages as future components of anti-metastatic therapies.Despite recent findings concerning the role of the glymphatic system in the CNS , the braIt has long been described that tumor cells share the same abilities as leukocytes to cross into the brain parenchyma . During Subsequently, during the development and growth phase of brain metastases, microglia show an impaired immune response evolving, it is thought, into a tumor-supportive phenotype . Microglloci has been clearly described. Our group has characterized such activation identifying a broad panel of proteins  that are highly upregulated in tumor-associated microglia together with other brain cell types  within the TME  and BMDMs (CD45high) in murine models, however, this does not seem to correlate with human brain tumor studies . RecentlThe innate role of the cells of the immune system is to guard, detect and respond to any insult that may disrupt the regular physiological equilibrium or homeostasis. However, as a result of their ability to take on an anti-inflammatory phenotype, microglia/BMDMs are able to advocate a pro-tumorigenic scenario during cancer and metastasis progression Immune cells with a pro-inflammatory phenotype have been described as exerting an anti-tumorigenic  effect whilst, in contrast, those with an anti-inflammatory profile  have shown tumor-supporting activity . The hisThe poor bioavailability in the brain of systemically successful treatments, and the impact of complex and variable environmental factors has reduced the efficacy of novel therapies in brain metastasis. In recent years, the inflammatory microenvironment has shown the potential for exploitation as both a prognostic and therapeutic tool in various malignancies. In the following section, we discuss the potential of microglia modulation as a novel therapeutic strategy in BM.Harnessing the body’s immune response to tumors has recently shown promise for the treatment of different cancer types. Current studies are focusing on the use of check-point blockade treatments not just to stop primary tumor progression, but also their metastasis to distant organs. The clinical success of specific immune checkpoint blockade, such as PD-1 and CTLA-4 (negative regulators of T-cell adaptive immune function), represent a promising advance in cancer immunotherapy development . An impoHowever, as previously described, microglia cells are also capable of immune suppressive functions. PD-L1 expression, the ligand of the inhibitory T-cell co-receptor PD1, has been detected on microglial cells . Via actAlthough most immunotherapies have been targeted to the adaptive immune response, recent studies have begun to focus on the innate immune response and macrophages in particular. Studies suggest that microglial cells manifest a pro-inflammatory phenotype during cancer progression, whilst BMDM play a more anti-inflammatory role . Most prThese promising results in pre-clinical studies could pave the way for future trials in which the immune response could be modulated into a more anti-tumorigenic state. However, we need to be cautious and consider the potential side effects that these drugs may provoke in healthy organs, as a consequence of the overstimulation of the immune system. Nevertheless, the substantial potential gains are likely to outweigh the possible negative side-effects, as has proven the case with both chemotherapies and current immunotherapies.Despite the slight improvement in patient life expectancy, the survival for BM patients is still measured just in months, reflecting, in large part, the complex nature of BM disease. Stereotactic radiotherapy and surgery are keystones for the management of many different subtypes of metastatic lesions in the CNS, but still yield relatively little benefit. However, combination of gold-standard therapies such as ionizing radiation with novel anti-tumorigenic drugs may lead the way to achieve better clinical results. For example, anti-CSF1-R therapies in combination with IR or anti-PD-1 are currently ongoing in primary brain tumors .Our group has described several cellular adhesion molecules specifically upregulated within the tumor-associated microglial population , 2016, afoci (Advanced treatments for brain metastasis patients consist of a combination of surgery, radiation and the administration of systemic chemotherapeutic drugs. However, these strategies entail a few limitations depending on location, total tumor mass, radio-resistant/sensitive histology, etc. Advances in systemically administered therapies have focused on overcoming these limitations, but the outcome has varied between no effect on metastatic burden, undesirable cognitive and physical side effects, and short transient response on tumor growth arrest with similarly unsuccessful results long-term . Part offoci  or to enfoci .At the same time, a lack of biological understanding of the long-term impact of newly designed drugs on different cell populations within the tumor microenvironment, including microglia, also contributes to the scarce improvement in BM patient outcome. Moreover, whilst the role of T cells, microglia and BMDM in brain metastasis progression has received some attention, the contributions of other immune cells, such as neutrophils, myeloid-derived suppressor cells and dendritic cells, have yet to be considered in any detail.Irrespective of the efficacy of novel targets in brain metastasis, it seems highly unlikely that single therapies will overcome this stage of cancer progression. Rather, combinations of gold-standard therapies such as radiotherapy with molecularly targeted brain-specific treatments against key components of the TME may afford increased life expectancy in patients suffering metastatic spread to the brain.Understanding the impact of immune check point blockade not just on T cell biology, but also on microglial behavior enable further enhancement of the global immune response and overcome the macrophage/microglia-induced tumor supportive scenario observed in BM. At the same time, transcriptomic sequencing of tumor-associated microglia during the course of brain metastasis may improve our understanding of the pathways that ultimately lead to the tumor supportive scenario.Current clinical trials in neurodegenerative disease aim to reduce the local inflammatory response targeting typical pro-inflammatory microglia biomarkers . In a brAll animal procedures were performed under the University of Oxford guidelines and approved by the UK Home Office.MS and NS participated in the bibliography search and the design, correction, and completion of this article.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. The handling Editor declared a shared affiliation, though no other collaboration, with one of the authors MS at the time of review.


Both reactive oxygen species (ROS) and reactive nitrogen species (RNS) are important signals in plants and key regulators of a variety of processes including metabolism, growth and development, response to abiotic and biotic stresses, solute transport, autophagy and programmed cell death (PCD). The reviews and original research in this special issue reflect a burgeoning area of investigation, and highlight the latest thinking on new roles and interactions of ROS and RNS, the basis of their specificity and flexibility, and emergent fields.2.–) as a substrate, research on reactive oxygen species (ROS) has grown exponentially. Next key findings were the identification of hydrogen peroxide (H2O2) as a by-product of aerobic metabolism  and discusses implications for photosynthetic light reactions, ROS production dynamics, antioxidant defence, and thiol-based redox regulation. In this sense, it draws attention to the issue that it appears impossible to utilize efficient C4 photosynthesis without understanding its exact redox needs, which will certainly be a topic of interest in the future.Although general mechanisms and dynamics of ROS production have been elucidated in plant cells under different environmental stimuli and at different developmental stages, we are far from a full picture. Reviews by se yield . The revSimilarly, In contrast, cis-regulatory elements, ROS-sensitive transcription factors and ROS-responsive transcripts. In addition, using cat2 mutants as a model system, they assess the impact of redox perturbations and oxidative stress on transcriptome adjustments and discuss how redox homeostasis can modify the various parts of the transcriptional machinery.Specificity in ROS signalling depends on the type, site, amount and duration of the ROS signature, as well as the ability to regulate gene expression in response to the perceived stimuli. As second messengers, besides induction of specific signals, ROS can act during the acquisition of cross-stress tolerance, which usually occurs during intense and prolonged exposure to oxidative stress. Orange gene (encoding a holdase chaperone protein) that is responsible for regulation of carotenoid homeostasis. Since carotenoids are indispensable for organisms that use oxygenic photosynthesis and are vital components for protection of photosynthetic machinery from oxidative damage, the authors discuss the rationale of increasing plant tolerance by controlling carotenoid biosynthesis via Orange.A final review related to ROS and oxidative stress, by + transport by ROS , which is also an important molecule involved in responses to abiotic stress and plant immunity (S-nitrosothiol (SNO) levels are addressed by S-nitrosylation via GSNO reductase and thioredoxin h5 (Trxh5) as being as important as generation of S-nitrosylation during RNS signalling. The final review on RNS in the special issue, by 2O2, NADPH, NO, peroxynitrite (ONOO–) and SNOs during fruit ripening. They follow a translational approach and discuss the use of basic research on ROS/RNS interaction to improve fruit yield and quality of pepper and tomato.The regulation of cellular metabolism and signalling via post-translational modifications such as As can be seen, new roles and interactions are being postulated for ROS and RNS, increasing our understanding of the underlying mechanisms allowing signalling, yet we still lack the fundamental knowledge as to how these reactive molecules act as such specific signals. To dissect both their specificity and their flexibility, recently attention has been given to their interactions at the molecular level, following technological advances. Another emerging area of research is the dissection and study of the roles of breakdown products as a result of biomolecule and ROS/RNS interaction. To conclude, it is of paramount importance to highlight the importance of physical interactions between different organelles to understand the specificity of the ROS/RNS signals, since the site of production and target for regulation must be in close proximity due to the reactive nature of these molecules.


Over the past few decades, antimicrobial resistance has emerged as an important threat to public health due to the global dissemination of multidrug-resistant strains from several bacterial species. This worrisome trend, in addition to the paucity of new antibiotics with novel mechanisms of action in the development pipeline, warrants the development of non-antimicrobial approaches to combating infection caused by these isolates. Monoclonal antibodies (mAbs) have emerged as highly effective molecules for the treatment of multiple diseases. However, in spite of the fact that antibodies play an important role in protective immunity against bacteria, only three mAb therapies have been approved for clinical use in the treatment of bacterial infections. In the present review, we briefly outline the therapeutic potential of mAbs in the treatment of bacterial diseases and discuss how their development can be facilitated when assisted by “omics” technologies and interpreted under a systems biology paradigm. Specifically, methods employing large genomic, transcriptomic, structural, and proteomic datasets allow for the rational identification of epitopes. Ideally, these include those that are present in the majority of circulating isolates, highly conserved at the amino acid level, surface-exposed, located on antigens essential for virulence, and expressed during critical stages of infection. Therefore, these knowledge-based approaches can contribute to the identification of high-value epitopes for the development of effective mAbs against challenging bacterial clones. Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.  high conservation between circulating strains, (b) expression during bacterial infection and/or colonization, (c) surface exposure in order to permit antibody binding, and (d) antigenically distinct compared to epitopes on human proteins and the normal human microbiota to prevent cross-reactivity.In the three aforementioned antibacterial mAbs, disease is prevented due to the neutralization of toxins via binding to highly conserved epitopes on toxin subunits. This approach is effective for anthrax and Streptococcus pneumoniae  may serve as raw material for elucidating high-value epitopes. Neisseria meningitidis  a net increment in the success rate of neutralization of a process by reducing the network tolerance of the pathogen; (2) lower chances of future immune evasion since several concerted epitope switches are exponentially more difficult to achieve than individual ones; (3) the possibility of designing complex blocking strategies concentrated on the same (pathogen siege) or sequential (pathogen exhaustion) stages of infection, thus applying a more comprehensive molecular view of the virulent process. Knowing this, the bottleneck in mAb development against bacteria may not lie in the experimental efficiency of mAb identification but in scaling processes required for the production of a mature pharmaceutical product.In contrast to cancer, rheumatologic diseases, and viral infections, the limited use of mAbs for MDR bacterial pathogens may be due to several technical and biological constraints. In this context, rational approaches based on large-scale data/systems biology methodologies may facilitate the identification of high-value epitopes for mAb development, perhaps in concert with traditionally used empirical strategies. The extreme challenge associated with finding ideal, immutable epitopes may support the development of mAb cocktails. This could require improvement in the efficiency  of mAb production, i.e., within a reasonable timeframe and at a reasonable cost, at the service of holistic paradigms that consider the molecular pathobiology of the targeted species. We envisage that the large data/systems biology combination will find its utility in RV approaches applied to mAb development as more information is collected.MM and AM-G planned the manuscript content, wrote the manuscript, and approved the final version.MM is a founding partner and shareholder in Vaxdyn, a biotechnology spin-off company developing vaccines and antibody-based therapies for antibiotic resistant infections caused by antibiotic resistant bacteria. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Editorial on the Research TopicMicroglia in Health and Disease: A Unique Immune Cell PopulationMicroglia are essential for the development and function of the adult brain. Their ontogeny, together with the absence of turnover from the periphery and the singular environment of the central nervous system (CNS), makes microglia a unique cell population compared with other tissue macrophages. Supporting this notion, recent transcriptional studies have revealed that microglia display specific gene expression signatures that are clearly distinct from other brain and peripheral immune cell populations in the healthy and diseased CNS .The unique properties and functions of microglial cells, such as their role in synaptic pruning or the exceptional capacity to scan the brain parenchyma and rapidly react to its perturbations, have emerged in recent years. In the coming years, understanding (i) how microglia acquire and maintain their unique profiles to fulfill distinct tasks in the healthy CNS and (ii) how these are altered in disease, will be essential to develop strategies to diagnose or treat CNS disorders with an immunological component.In this research topic, four original articles and four reviews cover several aspects of microglial biology, ranging from their origin and the functional role of microglia during development and lifespan, their molecular properties compared with other brain and peripheral immune cells to microglial phenotypes and functional states in neurodegenerative diseases and brain tumors.Menassa and Gomez-Nicola describe the various historical schools of thought that had debated the origin of human microglial cells. The data that have recently been accumulated on microglial dynamics in the developing human brain, together with the evidence obtained from rodent studies on the functional role of microglia during development, allowed them to identify limitations for the existing approaches in human studies.In particular, Savage et al. and colleagues focus on defining the distinctive ultrastructural features of microglia and the unique insights into their function that can be provided by electron microscopy. These techniques will further revolutionize the study of microglia across lifespan, brain regions, and contexts of health and disease.Ayana et al. conducted a meta-analysis of brain cells, using publicly available datasets, for deciphering microglia-specific expression profiles, thus identifying putative novel human microglia markers across lifespan and different brain regions.Sevenich summarizes recent developments in evaluating the distinct functions of brain-resident and recruited myeloid cells in neurodegenerative disorders and brain cancers, thus underlying disease- and cell-type-specific effector functions of microglia and macrophages.In her review, Walentynowicz et al. attempt to study glioma-associated microglia/macrophage functional states by identifying marker genes from different experimental models and clinical samples, revealing only a small set of common genes, thus reflecting divergent responses depending on specific clinical and experimental settings.Blank et al. suggests that early retinal microglia activation represents a first step in the pathological cascade of retinitis pigmentosa, which might initiate or accelerate photoreceptor degeneration, thus pointing out that activated microglial cells may be responsible for the onset or amplification of the degenerative process.A study by Zhou et al. show that the severity of retinal degeneration is related to microglia polarization. Specifically, the authors revealed that alpha-1 antitrypsin, a novel immunomodulatory agent in autoimmune diseases and transplantation, shifts microglia toward an anti-inflammatory phenotype along with a protective effect on retinal degeneration.In this line, Kounatidis and Chtarbanova review the recent findings obtained in the fruit fly (Drosophila melanogaster) on the contribution of glial innate immune pathways in lifespan and neurodegeneration. In particular, the NF-κB pathway and the phagocytic ability appear to be major contributors to lifespan, possibly through an immune–neuroendocrine axis.Finally, In conclusion, this research topic provides a comprehensive overview of our current understanding of several cellular and molecular mechanisms that make microglia a unique immune cell population within the healthy CNS as well as under inflammatory, neurodegenerative, and tumorigenic processes. Furthermore, an emerging key aspect of microglia is their heterogeneity across the CNS under homeostatic or pathological conditions. Established methodologies combining single-cell approaches, such as single-cell RNA sequencing, with functional screening of inferred cellular diversity, are starting to reveal microglia diversity. The application of single-cell techniques to phenotype homeostatic and activated microglia under different neurological disorders are currently opening new avenues to understand cellular and functional states of various subpopulations in a context-dependent manner, thus shedding more light on microglia unique features.AM wrote the manuscript. All the authors edited and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.


Within the cells which execute host immunity and inflammation, mitochondria play a critical regulatory role to support cell activation, pro-inflammatory gene expression, and propagation of inflammatory signaling In airway diseases such as asthma, the bronchial wall is the site of an aberrant inflammatory response, orchestrated by the cells of the immune system, and propagated within the bronchial epithelium and airway smooth muscle. Mitochondria feature prominently both in regulating the inflammation cascade and mediating tissue damage in the airway. This is evidenced by dysmorphic mitochondrial structure in airway Redox Biology, Hough et al. used a range of imaging, flow cytometric, and mitochondria labelling techniques to provide direct evidence for cell-cell trafficking of intact mitochondria in the human airway via exosomes, a subset of extracellular vesicles (EVs)  . The autIndeed, work in other models has revealed a number of ways in which mitochondria are exchanged between cells to accomplish diverse purposes The importance of studying mitochondrial physiology in human tissue cannot be overstated. Thus, the demonstration of intercellular mitochondrial transfer between immune cells in the human airway to circulating lymphocytes represents a powerful and highly translatable research tool. Recognizing the utility of novel methodologies for study of viable human mitochondria, our group has proposed and validated measurement of platelet bioenergetics as a biomarker in several human disease states, including asthma In summary, mitochondria play a central role in cell signaling in inflammatory diseases such as asthma. The investigation performed by Hough et al. provides direct evidence for cell-cell trafficking of intact mitochondria in the human airway, which is pertinent to the pathogenesis of asthma and potentially other airway inflammatory diseases, but also has wider implications for the study of human mitochondrial physiology.


Astringency is one of the most important components of fruit oral sensory quality. Astringency mainly comes from tannins and other polyphenolic compounds and causes the drying, roughening and puckering of the mouth epithelia attributed to the interaction between tannins and salivary proteins. There is growing interest in the study of fruit astringency because of the healthy properties of astringent substances found in fruit, including antibacterial, antiviral, anti-inflammatory, antioxidant, anticarcinogenic, antiallergenic, hepatoprotective, vasodilating and antithrombotic activities. This review will focus mainly on the relationship between tannin structure and the astringency sensation as well as the biosynthetic pathways of astringent substances in fruit and their regulatory mechanisms. Recently, the quality of fruits and vegetables has become increasingly important in people’s daily lives. Fruit quality can generally be divided into the following three components: the first is commercial quality, which includes the fruit’s outer appearance, fruit length and diameter; the second is fruit structural quality, for example, in terms of flesh thickness and cavity size; and the third is fruit sensory quality. Regarding oral sensory quality, there are six oral sensory characteristics of fruit: sourness, sweetness, bitterness, spiciness, aroma and astringency. For many people, the oral sensory properties of fruit have a great impact on the choice, acceptability and consumption of fruit. Sourness in fruits mainly comes from organic acids, such as citric acid, malic acid and tartaric acid as well as small amounts of oxalic acid, succinic acid and salicylic acid. Sucrose, fructose and glucose and their derivatives, such as sugar alcohols, are the main source of carbohydrate-related sweetness in fruit. Some amino acids, amines and other non-carbohydrates can also contribute to a sweet taste. Glycoside substances formed by sugar free radicals and aglycone, linked through glycosidic bonds, such as amygdalin, naringin and neohesperidin, are the cause of bitterness in fruits. The main spicy substances found in pepper fruits mostly consist of capsaicin. Fruit aroma is due to alcohols, esters, aldehydes, ketones and aromatic terpenes as well as other volatile substances . AstringThere is growing interest in the study of fruit astringency because of the healthy properties of astringent substances found in fruit including antibacterial, antiviral, anti-inflammatory, antioxidant, anticarcinogenic, antiallergenic, hepatoprotective and vasodilating . This reTannins are the key factor determining the degree of astringency in fruits. All astringency removal methods base on the reaction of acetaldehyde with soluble tannins forming an insoluble non-astringent compound .Combining tannins with saliva proteins results in precipitation and leads saliva to lose its lubricity, causes tongue epithelial tissues to contract, producing a dry feeling . This pret al. discovered that the greater the degree of polymerization of tannins, the higher the number of phenolic hydroxyl groups and the stronger the inhibition of free radicals [et al. studied 25 different types of tannins and other relevant compounds, 23 of which demonstrated antioxidant activity to differing degrees, depending on the location and number of phenolic hydroxyl groups [Tannins are polyphenols and their molecular structure contains a number of phenolic hydroxyl groups, and they can readily react with oxygen radicals. In addition, tannins can release a large amount of hydrogen ions that can combine with free oxygen radicals . Thereforadicals . Okuda el groups .The phenolic hydroxyls in tannins can interact with a variety of metal ions and cause an oxidation-reduction reaction, consequently reducing the metal ions from high valence to low valence and finally condensing them into a red-brown or brown quinone product. Reactions between tannins and metal ions reduce the absorption of calcium and iron by the human body. However, interaction of tannins and calcium outside of tissues can lower blood pressure .The molecular weight of tannins ranges from 500 to 3000 Daltons . In 1920et al. [There are several variables related to tannins that are highly correlated with the perception of astringency, such as their total concentration, average degree of polymerization (aDP) , subunitet al.  reportedin vitro studies have shown that complex polysaccharides can disrupt protein-tannin interactions through different mechanisms, such as via inhibiting protein-tannin interactions [Polysaccharide families clearly oppose the perception of astringency according to the results of a principal component analysis (PCAs), with the effect being stronger for mannoproteins and rhamnogalacturonan-II (RG-II). However, only polysaccharides rich in arabinose and galactose (PRAGs) were considered in a final fitted multiple linear regression (MLR) model, which explained 96.8% of the variability observed in the data. Oligosaccharides do not show clear opposition, revealing that the structure and size of carbohydrates are important for astringency perception. Mannose and galactose residues in the oligosaccharide fraction are positively related to astringency perception, most likely because their presence is due to the degradation of polysaccharides . Severalractions ,30 or inractions ,32, causractions ,34 or wiractions , and isoractions .Taking into account that all of the glycosyl residues found in the oligosaccharide fraction are also found in the polysaccharide fraction, it appears that the ability of carbohydrates to smooth out the perception of astringency is related to the size and tridimensional structure of the compounds. The mannose and galactose concentrations in the oligosaccharide fraction are positively related to astringency perception. This finding could be related to a decrease in the levels of mannoproteins and PRAGs and may not represent a direct effect of these glycoside residues on astringency perception. Commercial enzymatic preparations could play a role in the degradation of polysaccharides, leading to less protective oligosaccharides. A regression model constructed including the compositional variables and the perceived astringency allowed the authors to explain 96.8% of the variability observed in the data and to recognize the variables that were positively and negatively related to astringency perception .In grape skins, tannins can interact with proteins and cell wall polysaccharides. The mechanism of tannin-protein binding involves hydrogen bonding and hydrophobic interactions . Cell waet al. pointed out that the astringency of low to moderate tannin-wine is effected by acidity adjustments [et al. reported that astringency of alum was lowed equivalently by addition of equi-sour levels of lactic acid, citric acid or hydrochloric acid [Except for polysaccharides, the organic acids also have effect on astringency. Early in the last century, salivary volume, pH and protein composition were reported to change the flow rate, leding to the difference of the perceived intensity and duration of bitterness and astringency ,41. Guinustments . Hanna eric acid . Increasric acid . Coincidric acid . Beta-LGric acid . The astric acid .et al. [et al. [et al. [In grapes, tannins are mainly located in the seeds and stems but are also found in the skins, where they are reactive and easily extractible. Tannins accumulate during early developing stages then decrease continuously in grape skins ,49,50. Tet al.  reported [et al.  found th [et al. , while s [et al. . Peanut  [et al. . Plavac  [et al. . Peng-Mi [et al.  reported [et al. , pear [6 [et al. , peach [ [et al. , mango [ [et al. , pomegra [et al.  and quin [et al.  containsThe biosynthetic pathways of condensed tannins and other phenolic compounds have been verified in a variety of crops. Tannins are generated through the following three pathways: the shikimic acid pathway (for shikimic acid), the phenylpropanoid pathway and the flavonoid pathway. The anthocyanins and condensed tannins in flavonoid compounds are the main components responsible for pigment and astringency in fruits, and can be measured easily. Assessment of the route of synthesis has been the main model approach for studying associated gene expression and the regulation of plant secondary metabolism.PAL is a multi-gene family, and these genes control different metabolic pathways in different tissues.PAL represents the first step in primary and secondary metabolic pathways, carrying out the first step in the reaction of catalyzing phenylalanine . The conpara-position. trans-Cinnamic acid is catalyzed to 4-hydroxycinnamic acid (para-position-coumaric acid). C4H is a member of the cytochrome P450 monooxygenase superfamily. An enzyme that requires oxygen and depends on NADPH to function properly was first discovered in pea shoots by Russell and Conn [C4Hs of a variety of crops have been obtained, and the expression of C4Hs is closely related to the lignification of plants.The catalysis reaction of phenylalanine with C4H is the second step in the pathway , but it and Conn . Thus faFlavanones are catalyzed by flavanone hydroxylase to produce dihydroflavonol . Flavanoet al. described the three-dimensional structure of DFR in detail for the first time, in addition to studying the expression of the grape DFR in E. coli [et al. further obtained crystals by conjugating both DFR-NADP-myricetin and quercetin DFR-NADP and identified flavonols that might inhibit the activity of DFR based on analysis of three-dimensional structure [Dihydroflavonol reductase (DFR) belongs to an NADPH-dependent short-chain DFR superfamily. It is a single gene encoding a key enzyme in the biosynthesis of tannins . The ami E. coli , separattructure .DFR was first isolated in 1985, from corn (Zea mays) and snapdragon (Antirrhinum majus), through transposon tagging [et al. isolated the petunia (Petunia hybrida) DFR [DFR mutant gene from Antirrhinum majus as a probe. Employing homologous cloning methods, thus far, DFRs have been cloned from Arabidopsis , orchids (Bromheadia finlaysoniana), camellia (Camellia sinensis), tomato (Lycopersicon esculentum), rice (Oryza sativa) and other plants. Aida transferred DFRs to the ear of a blue pig using the Agrobacterium-mediated gene transfection method in 2000 [et al. firstly reported the expression of DFR of fruit flesh in vitro whose biochemical features might be very different from the ones of DFR of flowers or leaves as well as non-producing anthocyanin species [DFRs has been completed for many crops, thus providing the basis for further regulation of the content of tannins. Previous studies have shown that changes in light, temperature and other external factors could affect the expression of DFRs [ tagging . Subsequida) DFR  using a  in 2000 . Lo Pier species . In rece of DFRs .LAR has been cloned from apples, grapes, legume and other plants, and the enzyme encoded by LAR catalyzes colorless anthocyanin to form flavan-3-alcohol [The colorless anthocyanidin reductase gene belongs to the isoflavone reductase subfamily. It is a key structural gene involved in catalyzing the synthesis of condensed tannins . Flavan--alcohol ,75. In a-alcohol .LAR family play different roles in the flavonoid biosynthesis pathway and play space- and time-specific roles in different developmental stages. Colorless grape anthocyanin reductase genes regulate the type and accumulation of proanthocyanidins through their organization and spatial and temporal expression specificity [LAR [LAR is observed during the early stage of the fruit development and maturation periods in apple, while the lowest expression is observed during the middle period [LARs cloned from persimmon decreased gradually with maturing, but the content of condensed tannins increased in maturing persimmon [Different members of the cificity . The chaity [LAR , and thee period ,79. The ersimmon .Anthocyanin synthase (ANS), also referred to as anthocyanins dioxygenase (LDOX), belongs to the non-heme iron oxidase superfamily. It catalyzes leucocyanidin into colored anthocyanins , also knBAN in Arabidopsis and Medicago truncatula, was first reported in 2003. ANR may catalyze anthocyanins into epicatechins through coordination with NADPH in plants [ANR has been cloned from grapes [ANRs. There are five introns in the ANRs, and their location and number are conserved. Bogs et al. reported that ANR can be expressed in the developing seeds, peel, flowers and leaves of grape, but the expression levels are different in different tissues. Overexpression of ANR in Arabidopsis thaliana is related to the content of tannins. Compared with LAR, the expression level of ANR is lower or insignificant in various colors of apple skin [Anthocyanidin reductase (ANR), encoded by the anthocyanin reductase gene n plants   with cytokinin activity. The activity of F3H and DFR and catechin levels can be increased by spraying TDZ before harvesting [PALs in plants.Growth regulators play an important role in the process of the synthesis of astringent plant substances. Moalem-Beno rvesting . Auxin, MYB transcription factor, MYC family bHLH transcription factors and WD40 proteins.A transcription factor is a protein that combines with specific DNA sequences within eukaryotic promoters or a protein with a DNA binding domain. It can interact with specific cis-acting elements in the promoter region, and through interactions with other related proteins, can activate or inhibit transcription. Astringent substances have been found to be involved in the synthesis of regulatory factors, including the et al. reported that MYB is a large gene family. The transcription factors of this family are involved in different types of plant secondary metabolism (such as that of anthocyanins and tannins) as well as hormone and stress responses, cell differentiation, the cell cycle and organ morphogenesis. In Arabidopsis thaliana, the MYB transcription factor participates in the metabolic regulation of tannin, and the expression of the major associated structurally regulated genes, including DFR, ANS and ANR [VvMYB5a, VvMYB5b, VvMYBPA1, VvMYBPA2 and many other MYB transcription factors have been isolated from grapes. VvMYB5a is expressed mainly during the early development of the peel, pulp and seeds. Heterologous expression in tobacco revealed that the synthesis of anthocyanins, flavonols, tannins and lignins is influenced by VvMYB5a, indicating that VvMYB5a can regulate different branches of the phenylalanine metabolism pathway. VvMYB5b is mainly involved in the flavonoid pathway, and increases in the content of anthocyanins and tannins are caused by the overexpression of this gene in tobacco. The metabolism of tannins in grapes is specifically regulated by VvMYBPA1 and VvMYBPA2, which can activate the promoters of LAR, ANR and many structural genes in the flavonoid pathway. However, VvMYBPA1 and VvMYBPA2 do not bind to the UFGT promoter during the synthesis of celadon anthocyanin [MYB transcription factors have been isolated from persimmon, in which the expression of DkMyb4 and the expression patterns of DkF3'5'H and DkANR are very similar. The synthesis of tannins in persimmon is specifically regulated by DkMyb4. Heterologous expression in kiwifruit only causes the accumulation of tannins, without the accumulation of anthocyanins. Heterologous expression in kiwifruit also causes significant accumulation of tannins in persimmon calluses [DkMyb2 is involved in the metabolism of tannins in persimmon but does not cause continuous accumulation of tannins [Nesi  and ANR . VvMYB5ahocyanin . Five MYcalluses . Further tannins .bHLH transcription factors also belong to a multigene family. Different subfamilies play different roles in the growth and development, stress responses, signal transduction and regulation of the secondary metabolism of plants. bHLHs have been reported to participate in flavonoid metabolism or to be involved in the metabolism of tannins by controlling anthocyanin metabolism. The accumulation of anthocyanins in the peel and seeds is regulated by VvMYCl in grape. VvMYCl cannot activate the CHI, UFGT or ANR gene promoter by itself, but when it is co-transfected with MYB transcription factors, VvMYCl can significantly activate these three structural gene promoters [The romoters .MYB and bHLH transcription factors, there have been few studies conducted on WD40 in plants. The scope of the WD40 family of proteins is quite extensive and selective. These proteins can interact with different proteins in different physiological and biochemical processes. WDR1 and WDR2 proteins have been isolated from grapes and show heterologous overexpression in wild-type Arabidopsis. There is no difference in the accumulation of anthocyanins between VvWDR2-transgenic plants and control plants, while WDR1-transgenic plants exhibit high accumulation of anthocyanin in the stems and leaves [Compared with the d leaves .F3H was significantly inhibited in soybeans using the RNA interference technique, and the content of flavonoids in the transgenic plant was shown to be significantly increased. Additionally, when walnut was reverse transformed with the chalcone synthase gene (CHS) by Eluch, it resulted in a decrease in the accumulation of flavonoids in the transgenic plants. RNA interference technology will undoubtedly play an important role in the regulation of the content of astringent substances in fruits.RNA interference technology can be used to further test and verify the function of genes. For example, the expression of The synthesis of astringent substances is controlled by a variety of structural and regulatory genes. The study of structural and regulatory genes is not only beneficial for clarifying the mechanism underlying the development and regulation of astringency in fruit at the molecular levels but also provides an effective means for the genetic improvement and metabolic engineering of fruit astringency regulation. At present, there are still some problems including the fact that (a) in most crops, the astringent substances are tannins, but the genes that control catechins and epicatechins to generate tannins in the astringent substance metabolism pathway are still not clear; and (b) the expression of transcription factor genes can activate the coordinated expression of multiple genes in specific branches of metabolism, but the types and quantities of transcription factors related to the formation of astringency are not the same in different crops. Rapid progress has been made in the study of the genomics, proteomics, transcriptomics and metabolisms of plant crops, which will contribute technologically to solving the above problems. The cloning and functional identification of genes in the astringency metabolic pathway and their spatio-temporal expression patterns as well as tannin biosynthesis-related transcription factor genes must be considered in future work to finally make it possible to control fruit astringent substances quantitatively.


Vascular diseases constitute a global health issue due to the increasing number of cases of patients with these diseases. The pathogenesis of the majority of these diseases, including atherosclerosis and thrombosis, is complex and not yet fully understood. One of the major causes for their occurrence can be immune disorders resulting in the development of a chronic inflammation within the vessels. In recent years, studies have placed emphasis on the role of neutrophils in the development of these diseases, i.e., the discovery of neutrophil extracellular traps (NETs) demonstrated that the structures released by the cells may contribute to the enhancement of inflammatory reactions and cell damage. This article summarizes current knowledge on the role of NETs during atherosclerosis, thrombosis and small-vessel vasculitis, especially in antineutrophil cytoplasmic antibody (ANCA)-associated small-vessel vasculitis (AAV). Vascular diseases constitute a serious clinical, social and economic issue in the modern world. For many years, they have remained the major cause of mortality in developed countries. In recent years, studies have demonstrated that both innate and adaptive mechanisms of immune response are the basis for the majority of vascular diseases, including atherosclerosis and thrombosis  population of white blood system cells. Neutrophils are one of the first effector cells recruited at the inflammation site, where they constitute the first line of defense in the combat against a wide range of microorganisms. Moreover, neutrophils can affect the recruitment and activation of other immunocompetent cells, as well as participate in regenerative processes of tissues. For many years, the antipathogenic activity of neutrophils was linked solely to the elimination of pathogens through phagocytosis, including degranulation of antibacterial proteins and generation of reactive oxygen species (ROS) . As described, neutrophils play a significant role in both atherosclerosis pathogenesis as well as in the destabilization of atherosclerotic plaque -associated vasculitides (AAV), which are further divided into: microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA) and eosinophilic GPA (EGPA);immune-complex associated vasculitides, including: anti-glomerular basement membrane disease, cryoglobulinemic vasculitis, IgA vasculitis (Henoch-Schönlein) and hypocomplementemic urticarial vasculitis (anti-C1q vasculitis) Jennette .The AAV pathogenesis is complex and it is influenced by both genetic predispositions as well as environmental factors , leading to induction and excessive activation of lymphocytes and autoantibodies, contributing to tissue damage and predisposal to disease recrudescence  in the NETs. Kessenbrock et al.  noticed Propylthiouracil (PTU) is a drug with a proven impact on the development of AAV, which has been found to contribute to the induction of ANCA. Due to its thyrostatic action, PTU is administered to treat hyperthyroidism (Cao and Lin +) and B lymphocyte (CD19+) proliferation (Lange et al. The results of Söderberg et al.  showed tOf special interest appears the study conducted by Natorska et al. , who werIt has been known for a long time that thromboembolic complications accompany vasculitis (Tomasson et al. The literature reports cited in this study show that the recently discovered function of neutrophils, which is the formation of NETs, plays an important role in the development of vascular diseases, including atherosclerosis, thrombosis and SVV, which is confirmed by the elevated level of NET markers, including cfDNA and nucleosomes. According to numerous authors, an excessive amount of NETs formed within vessels may constitute a significant cause of chronic inflammation, which is one of the basic elements in the pathogenesis of these diseases. Therefore, inhibition of the generation of these structures may result in numerous favorable clinical effects for patients with vascular diseases. As has been hitherto observed, DNase I and the PAD4 inhibitor—Cl-amidine may influence the restriction of NET formation, thus protecting against the development of atherosclerosis and thrombosis however, these observations have been conducted so far only in animal experimental models.Numerous aspects of neutrophil hyperactivity and their function in vascular diseases are not entirely understood, thus it seems warranted to undertake studies aimed at the determination of the precise role of these cells, including the function of NETs in the pathogenesis of vascular diseases.Enhancement of knowledge of the phenomenon of NET formation, creating a physical barrier in vessels, could lead to the development of new treatment strategies and improvement of the quality of life of patients suffering from these diseases in the future.


With high treatment costs and limited capacity, decisions on which adult patients to treat with proton beam therapy (PBT) must be based on the relative value compared to the current standard of care. Cost-utility analyses (CUAs) are the gold-standard method for doing this. We aimed to appraise the methodology and quality of CUAs in this area.We performed a systematic review of the literature to identify CUA studies of PBT in adult disease using MEDLINE, EMBASE, EconLIT, NHS Economic Evaluation Database (NHS EED), Web of Science, and the Tufts Medical Center Cost-Effectiveness Analysis Registry from 1st January 2010 up to 6th June 2018. General characteristics, information relating to modelling approaches, and methodological quality were extracted and synthesized narratively.Seven PBT CUA studies in adult disease were identified. Without randomised controlled trials to inform the comparative effectiveness of PBT, studies used either results from one-armed studies, or dose-response models derived from radiobiological and epidemiological studies of PBT. Costing methods varied widely. The assessment of model quality highlighted a lack of transparency in the identification of model parameters, and absence of external validation of model outcomes. Furthermore, appropriate assessment of uncertainty was often deficient.In order to foster credibility, future CUA studies must be more systematic in their approach to evidence synthesis and expansive in their consideration of uncertainties in light of the lack of clinical evidence. Unlike traditional photon based radiotherapy, protons release most of their dose at the end of their range, limiting proximal and distal irradiation. PBT therefore has the potential to reduce unwanted irradiation of normal tissues, enabling higher treatment dose for better tumour control or greater normal tissue sparing to reduce treatment-related toxicities The costs of delivering PBT are significant, with up-front capital expenditure far greater than that of a photon unit With paediatric and skull base tumours already established indications for commissioning and reimbursement, uncertainty focuses on the use of remaining capacity for adult cancers, where comparative dose planning studies suggest benefit but little comparative clinical evidence as yet exists Given the limited capacity and higher costs, decisions on which adult patients to treat or evaluate in clinical trials should be based on comparisons of value against current best practice. This is typically performed through health economic evaluation (HEE). The gold standard for this is cost-utility analysis (CUA), involving the comparison of costs and effects of competing interventions with the latter expressed in quality-adjusted life years (QALYs) gained.A number of reviews have periodically looked at the CUA literature for PBT, highlighting results and discussing important limitations due to the lack of prospectively collected data 22.1A systematic search of the literature following the PRISMA guidelines Studies met the inclusion criteria if they consisted of a full CUA, defined as a HEE where results are expressed in terms of cost per QALY gained, comparing PBT with any comparators. With paediatric disease already commonly indicated for treatment, we restricted our review to adult disease where greater decision uncertainty lies. There were no restrictions on tumour site. Conference abstracts were not eligible for inclusion. The articles identified in the search were filtered for duplicates, before titles and abstracts were screened against the inclusion criteria. Potentially relevant studies underwent a full text review.2.2From each study we extracted data on their general characteristics, such as year of publication, country setting, population(s) assessed, interventions assessed, perspective, and the main results including the reported incremental cost-effectiveness ratio (ICER) of the intervention(s) using the original price year and currency. Further findings from sensitivity analyses were also extracted. These included results of probabilistic sensitivity analysis (PSA), in which model parameter values are randomly sampled from corresponding probability distributions with the model run repeatedly over the sampled parameters to create a distribution of results. The probability of being cost-effective can then be calculated for a given willingness-to-pay threshold. We also included results of any value of information analysis, which quantifies the potential gain from reducing uncertainty through further data collection.Characteristics of each decision analytic model were also extracted, including model type, health states representing the natural history of disease, structural assumptions, time horizon, cycle length, and discount rates. Finally, we extracted the key methodological approaches in each study regarding efficacy and costs of PBT, including cited sources of information. The extracted data were presented in tables alongside narrative synthesis.2.3A formal assessment of the quality of the studies was performed using the Philips checklist, a widely used framework for the assessment of decision analytical model-based cost-effectiveness studies 33.1The initial search of electronic databases found 1844 articles for review. Following removal of duplicates, full-text screening and assessment of eligibility, seven papers were identified for inclusion; two for head and neck cancer (HNC)  pneumonitis, oesophagitis, and irreversible dyspnoea for each of the compared interventions. Similarly, Moriaty et al. With randomised controlled trials to inform comparative effectiveness lacking, other approaches to estimate the effect of PBT were employed. Four studies An alternative approach, applying risk stratification through predictive dose–response models derived from radiobiological and epidemiological studies of photon radiotherapy outcomes, was employed by two studies Finally, in their base-case analysis Sher et al. Approaches and sources used to estimate proton related treatment costs ranged across studies in relation to their jurisdiction and perspective. Both Grutters et al. Sher et al. 3.3Also key in driving results are the characteristics and quality of the decision analytic models used. Model characteristics are summarised in Each of the seven studies used discrete-time cohort Markov models to simulate the natural history of disease. However, there was a general lack of explicit rationale for the disease model structure, with no references to the natural history of disease literature or other disease models. Despite this, structural assumptions were mostly transparent and justified. Just under half of the studies Systematic identification of disease progression parameters was performed in only two studies meta-analysed results from systematically identified single-armed studies, Parthan et al. As previously mentioned, four studies Utilities were found to be poorly reported in three studies. Moriaty et al. Whilst all studies considered some form of uncertainty, none addressed all, and no justification was given for their absence. Parameter uncertainty was assessed using either one-way sensitivity analysis Finally, in regards to external consistency, calibration of natural history of disease outputs against independent data was performed in only one study 4Although several reviews of the PBT HEE literature have been performed, none have looked in-depth at the methodological approaches to modelling costs and effects, nor assessed the quality of the models. Even within the wider radiation oncology literature, reviews of HEE study quality have been performed infrequently Our appraisal of model quality using the Philips checklist found limitations in most of the seven identified studies in terms of their transparency (clarity in the description and assumptions of the model and identification of model inputs) and validity . Transparency was hindered by a lack of systematic methods or even an explanation for the identification of model parameters, such as transition probabilities, health state costs, and utilities. Although not a guarantor of a model’s internal validity, transparency of model input choices is a key requirement of credibility, allowing decision makers to accurately assess the merits of the study and any biases introduced through selective choice of inputs A specific problem posed in the HEE of PBT is the well-documented lack of prospectively collected comparative data, especially from randomised controlled trials Furthermore, well conducted quantification of parameter uncertainty though PSA can be harnessed to infer the value and prioritisation of future data collection through value of information analysis. Uncertainty in results leads to the risk of making a suboptimal treatment decision, incurring a loss in health and healthcare resources compared to the optimal choice. Reducing decision uncertainty through further data collection therefore has quantifiable value by increasing the probability of making the optimal treatment decision. Less than half of our identified studies A limitation of our review is its dependence on what was reported within the identified papers and any supplements: we had insufficient time or resource to contact individual authors to seek clarification. The Philips checklist has an element of subjectivity arising from individual interpretation of checklist items, and had resources permitted, secondary assessment would have been performed on all papers.In conclusion, our review indicates that lack of transparency or external validation were key areas for improvement in future CUAs. The full reporting of uncertainty should be undertaken, ideally through PSA, which will also allow for the application of more advanced HEE methods to inform data collection and future research. The fast pace of developments in PBT will hopefully lead to a greater frequency of CUAs, and our review will hopefully provide direction on where their quality should be improved.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper


Resistance to digestion by digestive proteases represents a critical property of many food allergens. Recently, a harmonized INFOGEST protocol was proposed for solid food digestion. The protocol proposes digestion conditions suitable for all kinds of solid and liquid foods. However, peanuts, as a lipid-rich food, represent a challenge for downstream analyses of the digestome. This is particularly reflected in the methodological difficulties in analyzing proteins and peptides in the presence of lipids. Therefore, the removal of the lipids seems to be a prerequisite for the downstream analysis of digestomes of lipid-rich foods. Here, we aimed to compare the digestomes of raw and thermally treated (boiled and roasted) peanuts, resulting from the INFOGEST digestion protocol for solid food, upon defatting the digests in two different manners. The most reproducible results of peanut digests were obtained in downstream analyses on TCA/acetone defatting. Unfortunately, defatting, even with an optimized TCA/acetone procedure, leads to the loss of proteins and peptides. The results of our study reveal that different thermal treatments of peanuts affect protein extraction and gastric/gastrointestinal digestion. Roasting of peanuts seems to enhance the extraction of proteins during intestinal digestion to a notable extent. The increased intestinal digestion is a consequence of the delayed extraction of thermally treated peanut proteins, which are poorly soluble in acidic gastric digestion juice but are easily extracted when the pH of the media is raised as in the subsequent intestinal phase of the digestion. Thermal processing of peanuts impaired the gastrointestinal digestion of the peanut proteins, especially in the case of roasted samples. Arachis hypogaea L.) are considered a valuable source of proteins, lipids, dietary fiber, vitamins, minerals, phenolic compounds, and phytosterols. Despite their economic importance as a protein source, the peanut is renowned to frequently provoke allergic reactions in sensitive individuals. Among the sixteen known peanut allergens, Ara h 1 is responsible for 35–95% of peanut-allergic patients, and Ara h 2 is responsible for 95% of allergic reaction in peanut-allergic patients . Ho. Ho22]. Ara h 2 is seen in the SDS-PAGE as a doublet of bands of 17 kDa and 19 kDa , and we Peanut conglutins  are pepsin-digestion resistant not only due to their conserved globular structure but also due to the protective features of the lipid-rich peanut matrix. This specificity of the peanut matrix results in the delayed extraction of the proteins and makes conglutins unavailable for digestion by pepsin at the early onset of gastric digestion. Our results are completely in line with widely accepted knowledge on Ara h 6 as an extremely stable and proteolytically-resistant protein since this protein is detected in all control and gastric-digested samples of raw, boiled, and roasted peanuts. In contrast to gastric digestion, intestinal digestion of Ara h 2 and Ara h 6 by pancreatin results in significant degradation of these two proteins.We also aimed to characterize the gastric digestion of thermally treated peanuts over a 2 h time course. Our focus was on peanut proteins of higher molecular weights, such as Ara h 1 and Ara h 3. In raw peanuts, these two proteins were digested by pepsin in between 60 and 90 min . At thosThe SDS-PAGE profiles of pellets of control and pepsin-digested raw, boiled, and roasted peanuts are shown in A thorough assessment of the gastrointestinal digestion efficacy solely by the densitometry analysis of SDS-PAGE profiles is prone to erroneous estimations, due to the possible overlapping of the different protein fragments at the same position in the gel. Moreover, this is of great importance when assessing the digestion efficacy of the proteins of lower molecular weights, since the presence of the fragments derived from the proteins of higher molecular weights in the same position in the gel could cause false readings, and higher quantities could be assigned to the lower molecular-weight protein.Therefore, we have applied a pilot assessment using high-resolution tandem mass spectrometry and the PEAKS Suite X software’s post-translational modification search engine (PEAKS PTM) to assess the pepsin digestion efficacy of raw peanuts. This approach is, unlike the label-free in solution approach, only semi-quantitative and these results should be taken with caution. However, we have compared the amount of each protein or allergen to matched gel bands A. MoreovThe intact form of Ara h 1 (65 kDa) was partially digested and its digestion pattern is given in Regarding the digestion fate of the major allergen Ara h 1, the accumulation of Ara h 1-derived fragments occurs upon pepsin action. More specifically, Ara h 1-derived fragments were detected in bands nine (approximately 29–33 kDa range) and six (approximately 22–24 kDa range) of the digested sample, while Ara h 1-derived fragments were completely absent in the control within the corresponding regions. In contrast to Ara h 1, which is resistant to pepsin digestion to a decent extent, Ara h 3 was almost completely digested by pepsin, even in the case of band six where it seems to be unaffected by pepsin A. In thiAdditionally, we succeeded in identifying the pepsin proteolytic sites by comparing homologs N-terminal fragments of the acidic Ara h 3 subunit found in the 25–45 kDa region, and they included peptide bonds between the following amino acids: aspartic acid and asparagine, glycine and glycine, alanine and glutamine, and glutamine and glycine B. In addResistance to gastric digestion and limited proteolysis in the intestinal tract are characteristics of a typical food allergen. Moreover, the assessment of the digestibility of foods should be tested in the most realistic conditions possible; that is, in the presence of the food matrix, which is a prerequisite to properly judging a protein’s digestion stability. Additionally, analyzing the effects of thermal treatments on the extraction efficacy and digestibility of peanut proteins will provide information on processing conditions that may increase the allergenicity of peanuts. The lack of strict compliance with the in vitro digestion protocols and lack of detailed instructions for post-digestion defatting procedures enables unwanted diversification of data on peanut allergen gastrointestinal digestion stability/resistance. According to our study, a single, non-repetitive TCA/acetone defatting/protein precipitation protocol is proposed as a method of choice over h-hexane defatting. Inclusion of gastric lipase, as proposed already in the novel INFOGEST 2.0 protocol, might help in avoiding the defatting steps, which lead to the loss of the proteins.The solubility of proteins and extraction efficacy, as observed in digested and control samples, is profoundly altered upon the thermal processing of peanuts. The thermal treatment of peanuts decreased the solubility and extraction efficacy of the peanut proteins in the acidic buffer of the simulated gastric juice.The extraction of peanut proteins with higher molecular masses, Ara h 1 and Ara h 3 upon the thermal treatment, was impaired in simulated gastric juices, most probably due to aggregation.The extraction of peanut proteins in intestinal conditions  had the opposite trend observed in the gastric phase; the proteins from raw peanuts were extracted to the lowest extent, while the extraction of proteins from roasted peanuts was favored in these conditions.Thermal treatments of peanuts impaired the gastrointestinal digestion of the peanut proteins, especially in the case of roasted samples.


Cerebellar hemorrhage (CBH) represents the most commonly acquired lesion of the posterior fossa in the neonatal period. We aimed to perform a systematic review and meta-analysis of studies exploring the perinatal risk factors and neurological outcome of CBH in preterm infants. A comprehensive literature search was conducted using PubMed/MEDLINE and EMBASE. Studies were included if they examined preterm infants and reported primary data on maternal, obstetric, or perinatal characteristics, and/or outcomes of infants with and without CBH. A random-effects model was used to calculate mean differences (MD), odds ratios (OR), and 95% confidence intervals (CI). We found 231 potentially relevant studies, of which 15 met the inclusion criteria . Meta-analysis could not demonstrate a significant association between CBH and multiple gestation, chorioamnionitis, pre-eclampsia, placental abruption, use of antenatal corticosteroids, mode of delivery, or infant sex. Infants with CBH had a significantly lower gestational age  and birth weight , and significantly higher rates of intubation at birth, hypotension, patent ductus arteriosus, intraventricular hemorrhage, sepsis, necrotizing enterocolitis, and bronchopulmonary dysplasia. CBH was significantly associated with delayed mental  and psychomotor  development, and higher rates of cerebral palsy . In conclusion, the present meta-analysis shows that the youngest and sickest preterm infants are at higher risk of developing CBH. Our results highlight the multifactorial nature of CBH and reinforce the idea that cerebellar injury in very preterm newborns has important neurodevelopmental consequences among survivors. In the last decade, imaging of the posterior fossa in preterm infants has gained increased attention. This has been due to the growing awareness of the occurrence of cerebellar injury Volpe,  and the During the third trimester of pregnancy, the cerebellum undergoes a rapid and precisely programmed growth resulting in a 3.5-fold increase in volume and a 30-fold increase in surface area   and EMBASE for relevant studies. We searched from the inception of the databases until May 2017. The strategy for EMBASE was “exp cerebellum hemorrhage/ AND exp prematurity/,” in addition to related free text terms . We used a similar strategy for PubMED, with the MeSH terms for “cerebellar hemorrhage” and “neonatal prematurity” in addition to related free text terms. We reviewed reference lists of relevant articles and reviews for additional studies. We did not exclude studies based on language, and studies were translated where necessary.We included studies that compared infants with CBH to infants without CBH and reported on risk factors for and/or outcomes of CBH. We included studies which only included preterm (<37 weeks) and low birth weight  infants. Studies without data on a control group were excluded. Two reviewers  screened studies for inclusion independently on title and abstract. In case of disagreement, the study was included and reevaluated in the second round of inclusion. The second round of inclusion was based on full-text screening, and discrepancies between reviewers were resolved through discussion or in consultation with a third reviewer (EV).Using a predefined worksheet, one researcher (YA) extracted data from included studies. We extracted the following data from each study: citation information, location of study, study period, primary objective, criteria for inclusion/exclusion of infants, definitions used for CBH, baseline characteristics, risk factors and outcomes . Two researchers  checked extracted data for accuracy and completeness. We resolved discrepancies through discussion and by consulting the primary report.We used the Newcastle-Ottawa Scale (NOS) for cohort or case control studies to assess the methodological quality of included studies . We calculated the odds ratio (OR) and 95% confidence intervals (CI) for dichotomous outcomes from the data extracted from the studies. We calculated the mean difference (MD) and 95% CI for continuous outcomes. We used the method of Wan and colleagues  and for not clearly defining CBH (k = 3). We did not carry out an analysis of publication bias for any of the included outcomes due to the low number of studies per outcome (k < 15).The quality assessment for each study according to the NOS is shown in Infants with CBH had significantly lower GA  and high (3–4) grade IVH, NEC, sepsis, and BPD. In addition, our meta-analysis confirms the findings of previous systematic reviews   playing a role in the pathogenesis of CBH.Maternal administration of corticosteroids in case of anticipated preterm delivery reduces neonatal mortality and morbidity, including IVH , and studies reported on different risk factors and outcomes. Consequently, we probably lacked the power to find some associations. Secondly, studies showed heterogeneity in inclusion criteria, as well as in definition and assessment of CBH, risk factors, and outcomes, and 6 studies out of 15 did not control for confounding factors. Some of the included studies did not have the investigation of CBH as primary aim. In addition, it should be noted that only 2 studies exclusively included extremely preterm infants (GA < 28 weeks) (O'Shea et al., Our meta-analysis has some important limitations that hamper the practical application of the results. Firstly, and as mentioned above, we were only able to include a limited number of studies (The present meta-analysis highlights the multifactorial nature of CBH and reinforces the idea that cerebellar injury in very preterm newborns has important neurodevelopmental consequences among survivors. However, further research is warranted to understand the complex relationship between hemorrhagic cerebellar injury and its influence on neurodevelopmental outcome (Brossard-Racine et al., EV-M selected studies for inclusion, carried out data collection, carried out statistical analyses, assessed methodological quality, contributed to interpretation of results, drafted part of the initial manuscript, and reviewed and revised the manuscript. MF contributed to the design of the study, the statistical analysis and interpretation of results, supervised data collection, drafted part of the initial manuscript, and reviewed and revised the manuscript. YA selected studies for inclusion, carried out data collection, contributed to statistical analyses and interpretation of results, and reviewed and revised the manuscript. SP checked extracted data for accuracy and completeness, contributed to interpretation of results, and reviewed and revised the manuscript. GC contributed to interpretation of results and reviewed and revised the manuscript. FM contributed to interpretation of results and reviewed and revised the manuscript. EV conceptualized and designed the study, supervised the search and selection of studies, supervised data collection, assessed methodological quality, contributed to statistical analyses and interpretation of results, and reviewed and revised the manuscript. All authors approved the final manuscript as submitted.MF and FM co-authored one of the studies included in the review and provided additional data. The remaining 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.