Transcriptomics provides valuable data for functional annotations of genes, the discovery of biomarkers, and quantitative assessment of responses to challenges. Meta-analysis of Nofima’s Atlantic salmon microarray database was performed for the selection of genes that have shown strong and reproducible expression changes. Using data from 127 experiments including 6440 microarrays, four transcription modules (TM) were identified with a total of 902 annotated genes: 161 virus responsive genes – VRG (activated with five viruses and poly I:C), genes that responded to three pathogenic bacteria (523 up and 33 down-regulated genes), inflammation not caused by infections – wounds, melanized foci in skeletal muscle and exposure to PAMP (180 up and 72 down-regulated genes), and stress by exercise, crowding and cortisol implants (33 genes). To assist the selection of gene markers, genes in each TM were ranked according to the scale of expression changes. In terms of functional annotations, association with diseases and stress was unknown or not reflected in public databases for a large part of genes, including several genes with the highest ranks. A set of multifunctional genes was discovered. Cholesterol 25-hydroxylase was present in all TM and 22 genes, including most differentially expressed matrix metalloproteinases 9 and 13 were assigned to three TMs. The meta-analysis has improved understanding of the defense strategies in Atlantic salmon. VRG have demonstrated equal or similar responses to RNA , and DNA (gill pox) viruses, injection of bacterial DNA (plasmid) and exposure of cells to PAMP (CpG and gardiquimod) and relatively low sensitivity to inflammation and bacteria. Genes of the highest rank show preferential expression in erythrocytes. This group includes multigene families  and many paralogs. Of pathogen recognition receptors, only RNA helicases have shown strong expression changes. Most VRG (82%) are effectors with a preponderance of ubiquitin-related genes, GTPases, and genes of nucleotide metabolism. Many VRG have unknown roles. The identification of TMs makes possible quantification of responses and assessment of their interactions. Based on this, we are able to separate pathogen-specific responses from general inflammation and stress. Salmo salar L.) genome (https://www.ensembl.org/index.html). However, functional annotations lag far behind the genomes sequencing, assembly, and identification of genes. Migrating the annotations of putative homologues is an efficient and straightforward approach, but it comes with limitations and challenges. The roles of many genes remain unknown even in the best-studied species and the presumed homology and structural similarities do not always imply preservation of functions in vertebrate evolution. Many teleost fish-specific genes remain completely unexplored, despite their active participation in various processes and responses. Transcriptome data is a valuable source of functional annotations, which still hasn’t realized the potential. The main conditions for the association between a gene and a trait or biological process are the reproducibility of responses and the magnitude of differential expression, and meta-analysis of data obtained with RNA-seq and DNA microarrays is the most appropriate strategy for approaching this problem. In addition to the annotation of genes, such a strategy works for the discovery of marker genes. Despite the rapid development of high-throughput methods, qPCR analyses of single genes or small diagnostic sets remain important. Candidate marker genes are often selected based on general knowledge of their roles without detailed information on the expression profiles. Meta-analyses facilitate the discovery of genes with strong and reproducible responses. Identification of co-regulated genes, which we refer to as transcription modules (TMs) enhances the reliability of diagnostics and reduces the risk of misinterpretation of findings. TMs include genes with various functions co-activated under specific conditions, and their composition adds to the understanding of biological processes, and strategies of adaptation and defense.Since its inception at the beginning of the new millennium, transcriptomics has continued to play an essential role in fish biology and aquaculture research. The assembly and publication of the Atlantic salmon  genome  have enhin vivo and in vitro studies of fish responses to viral and bacterial pathogens, inflammation, wound healing, and stress have reached a critical mass, which makes reliable identifications of TMs possible. The genes were ranked and divided into groups according to the scale of expression changes. Both specialized and multifunctional genes were identified. TMs enable the quantitative assessment of complex transcriptome responses and their separation into biologically meaningful components. Identification and ranking by the stability and magnitude of differential expression aids in selection of candidate marker genes for diagnostics. The first practical outcome of this activity was the development of multi-gene expression assay for assessment of the immune status of Atlantic salmon smolts and growers  and contrasts – expression differences, such as differences between the treatment and control groups, time-points, developmental stages etc. Contrasts from exemplary studies with large gene expression changes served as material for the search for co-regulated genes. Microarray data  were also used for the assessment of tissue distribution of transcripts. We used experiments that were published or submitted to NCBI GEO Omnibus (Nofima’s bioinformatic pipeline STARS  is used -Virus responsive genes (VRG). The research included five pathogens (four RNA and one DNA virus): salmonid alphavirus (SAV) causing pancreas disease (PD) , piscineMoritella viscosa, the aetiological agent of winter-ulcer disease . We have performed challenges with three bacteria:  disease , Tenaciberosions , 22, andsalmonis , causing-Inflammation responsive genes (INFL). The main data sets were time-course of wound healing in skin and melanized foci in skeletal muscle. In addition, we included exposures of primary cultures of head kidney leukocytes to PAMP – TLR agonists CpG oligodeoxynucleotides and gardiquimod, and analyses of skeletal muscle injected with bacterial DNA (plasmids).-Stress responsive genes (STR). A suite of genes was activated in the heart and spleen after acute swimming exercise, in the skin after crowding, and in the skin of salmon with cortisol implants.2-expression ratios were first averaged across trials and then between trials with the same pathogen or treatment. In several trials with few contrasts and high variance, medians were calculated instead of means. Genes that showed significant differential expression in several independent trials and higher than 2-fold mean expression changes were included in TMs. To rank the genes, the magnitude of expression changes in each TM was divided into three  or two (STR) equal intervals. The list of genes with ranks is in p < 0.05).The meta analyses were performed in three stages. In trials with bacterial and viral challenges, infected tissues with strong expression changes were selected (tissue that do not harbor pathogens show only weak unspecific responses). The logcholesterol 25-hydroxylase a, was included in all TMs, though its responses to viruses were much greater than to other challenges. This enzyme, which is induced with infection and inflammation  and two immune regulators (irf8 and nfkb inhibitor) can orchestrate expression changes of multiple genes. Of acute phase proteins, the structure and functions of serum amyloid a are well conserved in all vertebrates , regulators of cell adhesion plasminogen activator inhibitor and e-selectin and chaperone hsp90 alpha. The immune role of perilipin, a protein of lipid droplets, to our knowledge, has not been reported in fish. Enrichment analysis was performed in four TMs. Most part of the enriched GO terms and KEGG pathways are associated with the immune and related systems , and encompass communication, signal transduction, cell differentiation and migration, humoral and cellular effectors , lgp2, and dhx58 have consistently demonstrated strong transcription responses to viruses. Signal transducers include irf1, irf3, irf7 (2 genes), stat1 (4 genes), and the lesser-known srk2. It should be noted that fewer terms are enriched in VRG than in BACT and INFL , VRG with the highest rank in Atlantic salmon showing strong responses to viruses in different fish species, including phylogenetically distant sturgeons  or have not been reported in association with salmon diseases, for example, cell adhesion molecule ceacam20 (irg1 (aconitate decarboxylase)  and sugar  metabolism and regulator of blood pressure angiotensinogen. il-6 and socs2, genes with anti-inflammatory action, may enhance inflammation. The similarity of inflammatory responses caused by bacterial infections and non-pathogenic factors is suggested with enrichment of the same functional categories and pathways , lectins and Ig receptor, ROS producing neutrophil cytosolic factors, enzymes of polyamine and nitric oxide metabolism, matrix metalloproteinases and mmp inhibitor. In addition to mmp 9 and 13, strong expression changes were shown by the fish-specific collagenase-3 like enzyme. Wound up-regulated several VRG . The roles of highly ranked cytidine deaminase and myelin-associated glycoprotein in inflammation remain unknown. Overall, pathogen-free inflammatory responses included fewer genes than those caused by disease-causing bacteria, but the changes in expression were comparable in scale.BACT includes genes involved in stable and reproducible responses to bacterial pathogens that are manifested at high infection loads. Expression changes comparable with those induced with ceacam20  and reguoxylase) . Both ene)  and many genes generally do not show responses at the transcription level. The identification of TM significantly enhances this approach by providing groups of genes, the responses and co-regulation of which have been confirmed on a large experimental basis. Pure cases are rare, and as a rule, transcriptome responses include several components with different contributions , 49. JudThe potential of transcriptome data for the functional annotation of genes and discovery of biomarkers is obvious, and rapid progress in this area can be expected. The reported approach is based on the search of groups of co-regulated Atlantic salmon genes referred to as transcription modules – TMs. Our meta-analyses have revealed two types of TM. The first type includes gene sets showing consistently high correlation of expression profiles most likely due to transcriptional co-regulation, examples are Atlantic salmon myofiber proteins co-regulated with proteins of calcium and glucose metabolism, pituitary hormones, immunoglobulins, and genes involved in steroid biosynthesis (not included in this paper). The number of such groups is small and each gene is firmly anchored to only one TM. Here, we present genes that have shown differential expression under certain conditions, where each member of the TM can be relatively independent of the rest and belong to several groups. We focused on responses to bacterial and viral pathogens, inflammatory agents, and stress, due to the high importance of these issues for aquaculture and the large amount of data produced by our group. A similar search can be implemented for any biological process and trait, and the only requirement is a set of high-quality transcriptome data. In our approach, the main priority is the consistency of expression changes – each gene in the TM must show differential expression in several independent trials under the standard thresholds. The results of transcriptome analysis in similar studies, as a rule, do not coincide in detail, and even if the main trends are stable, the deviations can be large. This does not mean a weakness in the design of experiments and analysis. The instability is largely determined with the complexity and diversity of transcriptome responses combined with an imbalance between the number of measurements (genes) and the number of animals, known as the multiplicity problem. There can be several different scenarios for responding to a challenge in a population. A small sampling group can highlight genes or groups of genes that are affected in some individuals but will not overcome statistical thresholds of significance in a representative comparison of populations. Deviations between experiments can also be caused by the difference in the genetic background, age and physiological condition of fish, season, the severity of infections and damages and many other factors. Both annotations and diagnostics are based on stable responses and therefore, a meta-analysis of independent studies is essential.M. viscosa. The stress component is clearly seen in all treatments including the viral and bacterial infections and inflammation caused by non-pathogenic factors. Identification of TM enhances diagnostics. In relation to transcriptomic data, differential expression of genes representing a significant portion of TM substantially increases the confidence of conclusions and ranking of genes provides additional validation: upregulation of the core set is strong evidence. TMs allow quantification and comparison of the magnitude of responses. TMs also serve as a basis for developing biomarkers and diagnostic sets of genes for qPCR or multigene assays, such as our assay of immune competence  and Nofima . SA was supported with a grant from I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry .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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Daily rhythm of melatonin synchronizes the body to the light/dark environmental cycle. Several hypotheses have been raised to understand the intersections between melatonin and depression, in which changes in rest-activity and sleep patterns are prominent. This review describes key experimental and clinical evidence that link melatonin with the etiopathology and symptomatology of depressive states, its role in the follow up of therapeutic response to antidepressants, as well as the clinical evidence of melatonin as MDD treatment. Melatonin, as an internal temporal cue contributing to circadian organization and best studied in the context of circadian misalignment, is also implicated in neuroplasticity. The monoaminergic systems that underly MDD and melatonin production overlap. In addition, the urinary metabolite 6-sulfatoxymelatonin (aMT6) has been proposed as biomarker for antidepressant responders, by revealing whether the blockage of noradrenaline uptake has taken place within 24 h from the first antidepressant dose. Even though animal models show benefits from melatonin supplementation on depressive-like behavior, clinical evidence is inconsistent vis-à-vis prophylactic or therapeutic benefits of melatonin or melatonin agonists in depression. We argue that the study of melatonin in MDD or other psychiatric disorders must take into account the specificities of melatonin as an integrating molecule, inextricably linked to entrainment, metabolism, immunity, neurotransmission, and cell homeostasis. Melatonin (N-acetyl-5-methoxytryptamine) is an amphiphilic indoleamine conserved from unicellular organisms to plants and animals. In mammals, pineal and extra-pineal synthesis were described . DarknesBiological rhythms and melatonin represent recent frameworks in the investigation of the etiology, prognostic improvement, and treatment approach in the clinical course of depression. The somatic, cognitive, and affective symptoms of depressive states that occur in association with neurochemical/hormonal imbalance, may result, among other factors, from the misalignment of biological rhythms –5. A sumConsidering the cyclic nature of mood disorders, the shared monoaminergic involvement, the data from seasonal depression, and the marked sleep and circadian changes in depressive disorders, several hypotheses have been raised to understand the intersections between melatonin with depression. Experimental and clinical studies were designed to understand possible pathophysiological connections and devise therapeutic strategies to optimize the pharmacological management of depressive patients. This review aims to discuss the interconnections of melatonin production pathways and its physiological actions with the etiopathology and clinical approaches to depression. We will initially describe the framework of depression relevant for the understanding its association with biological rhythms and melatonin. We proceed by discussing the role of melatonin as an internal temporal cue, its production and physiological actions, and its role as a neurohormone sharing common neuro-endocrine inputs with the pathways associated with depression. Finally, we describe the current knowledge from clinical research in the field, including the use of exogenous melatonin and urinary 6-sulfatoxymelatonin as treatment and response biomarker, respectively.Depression is a term with multiple meanings. As a state, a depressive mood may be an adaptive stress response . DepressClinical response is usually noticeable after 2–5 weeks –13. The The difficulties in modeling depression and neuropsychiatric disorders in experimental non-human species may, in part, be attributed to the variability of depressive symptoms , and the sheer impossibility to reproduce in non-human animals some important features, such as guilt and suicidality. This somehow mirrors the challenges of Psychiatry as a field in categorizing debilitating disorders (with overlapping symptoms and varied underlying mechanisms) as opposed to normal behavior. Chronic stress, maternal deprivation, and olfactory bulbectomy are among the models that present higher degrees of face validity , predictive validity , and construct validity . The majority of animal models only replicate anhedonia , which is certainly one of the core symptoms of depression. The widely used tail suspension and forced swimming tests have no pathophysiological or undisputed behavioral manifestations equivalent to those seen in depression but possess some degree of translation predictability. It is of utmost importance to select the model appropriate for a given question, and interpret results considering specific limitations.Arvicanthis niloticus), a diurnal rodent, exposed to short photoperiod (5 h light/19 h dark) for 6 weeks presented reduced saccharin preference, higher immobility time in the forced swim test, and no changes on time spent in the light side of the dark/light box in comparison to controls  in mice suprachiasmatic nucleus (SCN) led to increased escape latencies and number of escape failures in the learned helplessness paradigm, induced higher immobility time in the tail suspension test, and decreased the time spent in the light compartment of the light/dark test. These results indicate that disrupting central SCN rhythms causes helplessness, behavioral despair, and anxiety-like behavior. The experiment supports a causal relationship because environmental light-dark cycles and light input pathways were unchanged. Therefore, results cannot be explained by light affecting depressive-like behavior through other pathways  by the aromatic l-amino acid decarboxylase (AADC). Serotonin is acetylated by the phosphorylated arylalkylamine N-acetyltransferase (P-AANAT), forming N-acetylserotonin (NAS), which is converted to melatonin by N-acetylserotonin O-methyltransferase (ASMT). The melatonin pineal daily rhythm is determined by the conversion of 5HT to NAS under sympathetic control . EnvironThe pineal gland is a circumventricular organ that monitors the level of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) in the cerebrospinal fluid (CSF) and peripheral circulation. In pinealocytes, the activation of the NFκB pathway by toll-like receptors and TNFRs inhibits AANAT-gene transcription both in diurnal and nocturnal animals. Cortisol or corticosterone at concentrations compatible with arousal (but not the ones found in models of chronic unpredictable mild stress) potentiate melatonin synthesis, while concentrations compatible with immune suppression blocks it. In immune-competent cells PAMPs and DAMPs promote the synthesis of melatonin by activating the transcription of the gene that codifies AANAT. This process is also dependent on the binding of NFkB dimers to the gene promoter, but it is not an immediate effect. Activation of PAMPs or DAMPs receptors promote the nuclear translocation of the NFkB dimer (p50/p65) that leads to the synthesis of the subunit (cRel) necessary for activating AANAT transcription. The induction of melatonin synthesis in rodents and human macrophages by the transcription factor is a hallmark in inflammatory responses and inflammation –46. The During the mounting of an innate immune response melatonin plays various roles: the reduction of nocturnal melatonin leads to the mobilization of leukocytes from the bone marrow to the blood, and from the blood to the site of lesion; the synthesis of melatonin by macrophages/microglia increases its phagocytic ability and reduces the oxidative stress participating in the recovery phase of the inflammatory response . The retOver the past decades, studies reported neuroinflammatory responses in a series of neurodegenerative and psychiatric disorders. Intense research has been undertaken to determine the critical elements of such responses and putative therapies for their suppression. Some reports associate MDD to increased levels of cytokines including TNFα, IL-6, and IL-1β, as well as a reduction in complement C3 –49. IncrMelatonin effects triggered by a large range of concentrations (pM to mM) are mediated by mechanisms of action with different grades of sensitivity. The effects of exogenous vs. endogenous melatonin, as well as the concentration reached by intracrine/autocrine, paracrine and hormonal sources, depend on the melatonin bioavailability and the mechanisms that mediate each production source. In a concentration range compatible with the nocturnal surge (pM to nM), melatonin orchestrates a plethora of physiological functions by activating GPCRs receptors (MT1 and MT2) localized in plasmatic, nuclei and mitochondrial membranes. At much higher concentrations, melatonin may act as an electron donor, promoting a receptor free non-specific antioxidant response . Other nMelatonin receptors MT1 and MT2 couple to Gi/o and beta-arrestins and depending on the context also couple to Gq . The seceigen-zeitgeber action  or seasonal effect resulting from these sources. A detailed review on the physiological actions of melatonin was composed by Cipolla-Neto and Amaral  and bloodstream. As a hormone that gains the bloodstream at night in the dark, melatonin is an internal temporal cue that has immediate interactions with its molecular effectors as well as prospective effects, since it primes physiological responses that will take place hours after its peak. The duration, timing, and cyclic-nature of pineal melatonin production, as well as its seasonal variation according to photoperiod changes, contribute to the temporal organization of physiological phenomena into circadian and circannual timescales . Finallyd Amaral .Since the report of two pharmacological agents in the late 1950s, the pathophysiology of MDD has been linked to a depletion in monoaminergic neurotransmitters, such as noradrenaline, serotonin and dopamine . InitialSince melatonin production occurs from serotonin after stimulation of adrenoreceptors (see section What Is the Pathway of Melatonin Production? above), it has been hypothesized that a disturbance in melatonin secretion could be present in the acute phase of depressive illness, and possibly be related to its pathophysiology. Moreover, melatonin controls dopamine signaling in the forebrain, hypothalamic and hippocampal areas –63. Of nSeveral clinical studies tried to identify alterations in absolute levels of melatonin as a possible maker of depressive states; considering the above detailed physiological regulation of melatonin, this was a misleading proposition, and the evidence is at best controversial. While some earlier studies reported lower nocturnal serum or saliva levels of melatonin in depressed individuals, others reported no differences, or even higher levels compared to controls . One stuThough the immediate mechanisms of action of current antidepressants are well-understood, the delay in clinical outcome conveys the gap of knowledge on the complete pathways that ultimately determine the clinical response. This delayed response shifted theories toward the neuroplastic hypothesis, whereby reduced levels of neurotrophic factors result in atrophic morphological changes, especially at the synaptic level . Animal Melatonin exerts neuroprotective actions by counteracting the NMDA-mediated excitotoxic effects of glutamate, including the impaired BDNF signaling and cell death . ExogenoBiomarkers are easily accessible molecules that discriminate the presence or absence of a disease or predict treatment response. They are based on genomic, proteomic, functional, and structural features that characterize the disorders. “Diagnostic biomarkers” identifiable at any stage of the illness are often measured before treatment. The term “treatment biomarkers” refers either to baseline values that predict how effective treatments may be and guide therapeutic choice, or short-term variation between baseline and an early phase in the course of therapy .A promising group of “treatment biomarkers” for SNRI/SSRI search for consistent changes in monoamines neuronal reuptake inhibition associated with subsequent mood changes . To measure the blockage of neuronal monoamine transporters directly, it would be necessary to assess changes in an immediate molecular product on the postsynaptic level that directly results from the neurotransmitter release (see section What Is the Pathway of Melatonin Production?). We proposed the assessment of 6-sulfatoxymelatonin aMT6s,  to detecn = 12) or antidepressants  for 8 weeks. Both placebo and SNRIs groups showed improvement in depressive symptoms but only the group treated with the drug showed a significant increase in aMT6 urinary excretion in the first urine of the day ethyl]acetamide) first reported in 1992, is by far the best studied. This synthetic molecule has high affinity for MT1 and MT2 receptors, but also present moderate affinity for the serotonin receptor  placebo , 97 and  placebo , with noThe knowledge of clinical use of melatonin is growing, with evidence supporting the prescription of exogenous melatonin for a few clinical situations. In the case of adult chronic insomnia, a few studies show modest benefits for sleep onset, sleep latency and total sleep time, although clinical significance is still questionable , 102. FoCircadian Rhythm Sleep-Wake Disorders are a set of clinical conditions to which melatonin are classically indicated. For sleep disturbances in shift workers, the readjustment to nighttime sleep following a night work shift or the promotion of desired daytime sleep can be reached with doses of no more than 1–3 mg about 30 min prior to the desired sleep onset , 106. InIn conclusion, the evidence supporting the clinical use of melatonin should be analyzed very carefully. A potential therapeutic effect of melatonin for mood disorders can only be expected if compatible with the physiological regulation of melatonin detailed in this review. Pineal melatonin is a neuro-endocrine message of darkness that is physiologically secreted in humans after a few minutes in the dark, peaking shortly after, serving as an internal temporal cue to cells, tissues, and systems. Extra-pineal production of melatonin happens in a constitutive manner, and on-demand production or suppression may also be regulated by levels of glucocorticoids, and different stages of an inflammatory response triggered by immune-competent cells. Hence, the optimization of potential therapeutic uses of melatonin in psychiatric disorders must encompass the specificities of an integrative molecule inextricably linked to entrainment, metabolism, immunity, neurotransmission, and cell homeostasis.Localizing phenomena, dissecting underlying mechanisms, and decomposing systems to understand their functioning have been helpful strategies in building knowledge in Biology. The downside of tackling questions in such a manner is that one might overlook how interconnected systems are, and thus lose perspective of the whole. Therein lies the challenge of decomposing complex regulatory structures like the timekeeping and melatonergic systems.Pineal melatonin is synthesized in the absence of light  under sympathetic control. Its production relies on the neurotransmitter norepinephrine and requires serotonin as precursor, monoamines linked to depression. The rhythmic production of pineal melatonin is a message of darkness to the body, which aids in synchronizing oscillations in physiological functions and behaviors. Nevertheless, melatonin may be implicated in the pathophysiology of depressive mood for its chronobiotic effects and the various immunological processes in which it is involved. A growing number of studies highlight the relevance of exploring the potential of melatonin extra-pineal synthesis, as well as the regulation of pineal melatonin production apart from the circadian light stimuli. Therefore, the study of melatonin in the context of depression must acknowledge its regulatory effect via chronobiotic (endocrine), paracrine and autocrine functions. It is crucial to acknowledge that detectable circulating levels of melatonin are a reflection of its amplitude and phase of secretion.per se.Studies of the pharmacodynamics of exogenous melatonin or melatonin agonists should always anticipate distinct clinical outputs depending on the route of administration, dosage, and timing, as these substances would potentially represent (or interfere with) different physiological actions. The pleiotropic actions through which melatonin might affect mood include the role of melatonin as an internal temporal cue, as a neurohormone in close relation with the monoaminergic system, and its indirect effects on depression via the immune and stress response systems. In this context, the resulting effects of melatonin actions on mood must be understood as a more complex and multifactorial pattern of systemic regulation, rather than an “antidepressant” effect AT and EE reviewed and edited the final manuscript. MH was responsible for funding acquisition. All authors participated in the conceptualization and the writing of the original draft.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 current intense logging activities induce rapid, adverse effects on our ecosystems and climate. Here we review large old trees with a focus on ecosystem preservation, climate issues, and therapeutic potential. We found that old forests continue to sequester carbon and fix nitrogen. Old trees control below-ground conditions that are essential for tree regeneration. Old forests create micro-climates that slow global warming and are irreplaceable habitats for many endangered species. Old trees produce phytochemicals with many biomedical properties. Old trees also host particular fungi with untapped medicinal potential, including the Agarikon, Fomitopsis officinalis, which is currently being tested against the coronavirus disease 2019 (COVID-19). Large old trees are an important part of our combined cultural heritage, providing people with aesthetic, symbolic, religious, and historical cues. Bringing their numerous environmental, oceanic, ecological, therapeutic, and socio-cultural benefits to the fore, and learning to appreciate old trees in a holistic manner could contribute to halting the worldwide decline of old-growth forests.Old forests containing ancient trees are essential ecosystems for life on earth. Mechanisms that happen both deep in the root systems and in the highest canopies ensure the viability of our planet. Old forests fix large quantities of atmospheric CO Cyanophyta  of North America being reduced to an inferior retrograde status, both stunted and twisted . WWF Canada has also just released a detailed report, submitted to the Global Biogeochemical Cycles, which demonstrates that the soil organic carbon of the great boreal forests was widely underestimated. It also supports the carbon storage capacity of large old trees from the old-growth forests of the Pacific Maritimes eco-zones  trees, in particular, demonstrates that most of its stand-level biological nitrogen fixation occurs within epiphytic bryophyte systems and lichen populations which house N-fixing cyanobacteria (Lindo and Whiteley Abies grandis) forests in northwestern Montana, showed that N-fixing foliose lichens were much more abundant in the older forests than in the younger, with certain species being completely absent from the newer stands of trees , is intrinsically linked to that of the old-growth forests of British Columbia where it makes its home. Many other vertebrates and vascular plant species are also at risk of extinction or extirpation due largely to logging activities in the woods of British Columbia (Yezerinac and Moola Rhyacotriton olympicus), the Del Norte salamander (Plethodon elongatus), and the tailed frog (Ascaphidae) were all found to rely primarily on these particular eco-systems for their survival (Welsh Ponginae) in southeast Asia and the lead-beaters possum (Gymnobelideus leadbeateri) in south-east Australia  which we have previously mentioned. A similar fate also befell the North-American white pine (Pinus strobus). Being particularly susceptible to the loss of genetic diversity due to logging, most white pine which have been planted as second-growth are stunted, only reaching a height of 25 m, half of what their older relatives can grow to be  forests of Great Britain also demonstrated how large older trees create ideal nurseries for younger growth to flourish . In order to slow the progression of the novel coronavirus SARS-CoV-2 and to prevent new such pathogens from making their appearance in the future, the part that old-growth forests play in preserving our health must be factored into forest management plans  are shown together with those of other isolated compounds endowed with anti-inflammatory (eburicoic acid) and antimicrobial , the Madrone (Arbutus menziesii), and the Western Hemlock (Tsuga heterophylla) out of the old-growth forests of Southern Vancouver Island by Saanich and Cowichan Coast Salish people who also rely heavily on the western red-cedar (Thuja plicata) for wood and fibers  and nitrogen (15N) in the soil and litter surrounding these mother trees act as markers for the spatial and temporal extent of their below-ground beneficial effects. When looking at their benefits to human health, apart from their ability to filter the air, produce medicinal volatile organic compounds, and the medicinal and culinary uses of their various parts, large old trees have a unique cellular structure which makes them ideal hosts for endogenous fungi which in turn are sources of specific phytochemicals with therapeutic properties. Also important to our mental and spiritual wellbeing, old trees are part of our human identity and cultural heritage, presenting us with aesthetic, symbolic, religious, and historical cues. In many cultures, particularly large trees are in fact treated with reverence. All in all, we are convinced that looking at the big picture, at all the numerous benefits provided by large old trees in terms of environmental, oceanic, ecological, therapeutic, and social-cultural impacts, could contribute to the arrest of their worldwide decline. Indeed, never has the phrase “to see the wood for the trees” been more relevant. We have sadly fragmented trees into specific economic or ecological values while forgetting that they are so much more than the sum of their parts. Perhaps the saying should now be that we have failed to see the trees for the wood.Large old trees are organisms of great importance in the field of forest ecosystems as they are able to fix large quantities of atmosphere gases, produce oxygen and create local habitats which are unique and are not comparable to younger stands of trees. Nevertheless, climate change and the ensuing frequent forest fires, as well as logging, continue to have a global impact on the forest’s matriarchs Fig. , while n


In the majority of joint diseases, changes in the organization of the synovial architecture appear early. Synovial tissue analysis might provide useful information for the diagnosis, especially in atypical and rare joint disorders, and might have a value in case of undifferentiated inflammatory arthritis, by improving disease classification. After patient selection, it is crucial to address the dialogue between the clinician and the pathologist for adequately handling the sample, allowing identifying histological patterns depending on the clinical suspicion. Moreover, synovial tissue analysis gives insight into disease progression helping patient stratification, by working as an actionable and mechanistic biomarker. Finally, it contributes to an understanding of joint disease pathogenesis holding promise for identifying new synovial biomarkers and developing new therapeutic strategies. All of the indications mentioned above are not so far from being investigated in everyday clinical practice in tertiary referral hospitals, thanks to the great feasibility and safety of old and more recent techniques such as ultrasound-guided needle biopsy and needle arthroscopy. Thus, even in rheumatology clinical practice, pathobiology might be a key component in the management and treatment decision-making process. This review aims to examine some essential and crucial points regarding why, when, where, and how to perform a synovial biopsy in clinical practice and research settings and what information you might expect after a proper patient selection. Changes in the organization of the synovial architecture are evident in the majority of joint diseases. Thus, the synovium has been studied at the macroscopic, microscopic, and molecular levels as it is an important determinant for the understanding of the biology of the joint and the etiopathogenesis of several joint diseases . In rheuThe synovium is a complex tissue composed of different cell types including tissue-resident macrophages, fibroblasts, and endothelial cells, as well as blood vessels, lymphatic vessels, and nerves . The hisWhen pathology comes in, the normal architecture of the synovial membrane may be disrupted leading to alterations of the lining thickness, stromal cell density, and inflammatory infiltrate.As in many joint diseases, the changes mentioned above occur early, and synovial tissue analysis might provide useful information for the diagnosis, especially in the case of atypical and rare joint disorders, and might have a supportive value in case of undifferentiated inflammatory arthritis, by improving disease classification. Moreover, it gives insight into disease progression helping patient stratification, a process in constant evolution. Finally, it contributes to an understanding of joint disease pathogenesis holding promise for the identification of new synovial biomarkers and the development of new therapeutic strategies .This review aims to examine some essential and crucial points regarding why, when, where, and how to perform a synovial biopsy in clinical practice and research settings and what information you might expect after a proper patient selection. Given the breadth of the matter, we focus only on those aspects that are of the most interest to the rheumatologist.The synovium is involved in all chronic inflammatory arthropathies. Although in routine clinical practice synovial biopsy is not mandatory for most diagnoses of inflammatory arthritis ; in some circumstances it becomes irreplaceable . Indeed,Moreover, a synovial biopsy can be complementary to the synovial fluid analysis. Comparative studies concerning the accuracy of the same diagnostic procedures  on synovial fluid and synovial biopsy are not abundant but underline the utility of the two analysis, also in consideration of the possibility of false-negative results of both procedures –6.A synovial biopsy is often performed for research purposes for example in RA patients; synovium histological and molecular alterations are considered a target for identifying new biomarkers to help rheumatologists tailor their clinical and therapeutic decision according to patient characteristics . RecentlFinally, in the case of refractory synovitis , arthroscopic synovectomy could be a viable strategy to reduce local and persistent inflammation .In rheumatology clinical practice, thanks to the great feasibility and safety of ultrasound-guided needle biopsy and needle arthroscopy, pathobiology may become a key component in the management and treatment decision-making process . For cliClassification of early undifferentiated arthritis. Since an early diagnosis and treatment of chronic inflammatory arthritis are linked to better long-term outcomes in terms of prevention of irreversible structural damage, nowadays the number of undifferentiated arthritis defined as inflammatory arthritis not satisfying classification criteria for RA  inflammatory arthritis (mostly RA) (- stly RA) . This fistly RA) , 25. Furstly RA) . Previoustly RA) , but subIdentification of predictors of treatment response and outcome. Thanks to advances in molecular biology on synovial tissue, more recent studies have focused their attention on potential predictive synovial biomarkers of response to therapy. To date, unfortunately, we do not have tools to identify patients who are likely to benefit from a specific therapy. Dennis et al. identified four histological pathotypes confirmed by molecular analysis of gene-expression profile on synovial tissue in patients with RA: lymphoid phenotype characterized by enrichment of genes related to B cells and plasmablasts, and T lymphocyte activation and differentiation and antigen presentation; myeloid phenotype characterized by M1 monocyte signature with abundance of NFKB-dependent cytokines such as TNF-alpha and IL1-beta; low-inflammatory phenotype; and fibroid phenotype characterized by genes related to fibroblast and osteoclast/osteoblast regulation, and angiogenesis. In this study, the myeloid phenotype (associated with the circulating marker ICAM1) was more represented in the group of anti-TNF responder patients compared to the lymphoid pathotype (associated with the circulating marker CXCL13), which was more represented in IL6 inhibitor responders  positive synovitis at ultrasound evaluation , a significant result was achieved considering the reduction in the number of sublining CD68+ macrophages as a marker of the effectiveness of treatment independent of the primary mechanism of action , where synovial biopsy and culture are required for the diagnosis and for selecting the right treatment regimen given the spread existence of multidrug-resistance bacteria. A detailed history should consider the following: previous TB exposure, living/traveling in endemic areas, concomitant HIV infection, previous trauma causing direct tissue inoculation, and concomitant TNF inhibitor therapy. These forms of arthritis follow a chronic course preferentially involving large joints  and rarely associate with constitutional symptoms or pulmonary findings , 52. FunParasites  are usually mentioned as causative agents of rheumatologic disorders mainly due to immune-mediated mechanisms like reactive arthritis. However, sometimes symptoms are directly related to their infiltration of musculoskeletal structures such as in dracunculiasis, strongyloidiasis , filariaWhipple disease is another challenge that deserves to be mentioned. In 75% of cases, gastroenteric symptoms are preceded by a seronegative oligo- or polyarthritis with a relapsing course , and wheArthritis represents one of the well-known late-stage complications of Lyme disease, especially in the United States. After having investigated prior tick exposure, hazard occupation or hobbies , or previous cutaneous manifestation, the clinician will combine serology and PCR-based testing for B. burgdorferi DNA in the synovial fluid to confirm the diagnosis . HoweverKeeping in mind the plethora of the aforementioned microorganisms and their relative hints, once the clinical suspicion arises, the dialogue opens up with the infectiologists, microbiologists, and pathologists to manage properly the analysis of the synovial tissue with their respective culture medium and expected histologic findings .Amidst deposition diseases, there are few which may manifest as monoarthritis, occasionally resembling aggressive osteoarthritis. Ochronosis is an autosomal recessive disorder where the homogentisic acid oxidation products are in excess and therefore deposit in the connective tissue, making it stiffened and brittle, ultimately leading to ochronotic arthropathy. Suspicion should arise if a patient in its fourth decade of life starts having back pain and subsequently knee pain  with radiological findings showing knee osteoarthritis and wafer-like disc calcification with a reduction of intervertebral spaces in the spine. The clinical examination may reveal deposits of bluish or brownish pigment in the ear cartilage and sclerae .Hemochromatosis arthropathy, where iron in excess deposits in the synovial tissue, may virtually involve any joint. The most reported symptoms resemble osteoarthritis and less often recurrent synovitis. Clinical clues comprise the involvement of the second and third metacarpophalangeal joints with their corresponding radiographic findings (hook-like osteophytes), chondrocalcinosis, abnormal liver enzymes, and hyperferritinemia .In the context of monoarthritis, histology remains the gold standard to characterize the nature of proliferative lesions . HoweverAfter evaluating challenging disease, it is worth mentioning crystal arthropathies, which are supposedly a straight diagnosis. When clinical, instrumental, and synovial fluid analyses are not conclusive, and some uncertainty remains, it must be kept in mind that to indentify crystals under polarizing microscopy, the synovial tissue needs to be processed with absolute alcohol, which is not usually done in the routine analysis, because other fixatives dissolve monosodium urate crystals .Synovial biopsy may be also useful in rheumatic polyarticular disorders –68. OsteLikewise, multicentric reticulohistiocytosis mimics RA, progressing up to a mutilans phenotype. It mainly affects women of Caucasian origin in their fourth decade. While laboratory findings are hardly helpful or specific, except by excluding other etiologies, few clinical hints are the involvement of the distal interphalangeal joints, the appearance of papulonodular skin lesions especially affecting the face and hands, and in 25% of cases the association with neoplasia .Finally yet importantly, among the infectious agents, leprosy is one of the diseases where biopsy remains of key importance to demonstrate the presence of the bacilli in the joint. Arthritis in leprosy may be polymorphic, including acute or chronic polyarthritis, septic arthritis, and Charcot's arthropathy. Clinical clues comprise skin lesions and symptoms suggestive of motor-sensory neuropathy in the context of an endemic area .Sarcoidosis is well-known for being polymorphic, with myriads of different musculoskeletal manifestation: acute arthritis (Lofgren syndrome with symmetric hilar adenopathy and erythema nodosum), chronic symmetric, medium to large joint oligoarthritis , up to Jaccoud arthropathy and dactylitis. During the diagnostic management, X-rays could show bone involvement, equally various with different patterns of bone lesions . The diagnosis is always challenging, and synovia is one of the precious target tissues that can contribute to it .Synovial biopsy is an invasive procedure; thus, the results expected have to be relevant and informative. All joints are not the same and vary greatly in their vulnerability to different rheumatic diseases. Thus, the choice of the joint to biopsy is crucial and should be guided based on the rheumatologist's purposes.For example, synovial tissue analysis might be instrumental for the differential diagnosis between rheumatic and non-rheumatic conditions (see paragraph above) of mono-arthritis. In this context, the affected joint should be chosen.By contrast, in patients with RA, the choice of the joint where synovial biopsy is performed might be based on the published literature .Concerning the inter-joint heterogeneity, in the same patient, it has been demonstrated that synovial samples, taken at the same time, from an active joint are generally representative of other inflamed joints , 73. In In the same patient with RA, a comparison between synovial biopsies in clinically involved and non-involved knee showed that a considerable degree of histological changes, mainly hyperplasia of the synovial lining layer, was present in the uninvolved joint, although changes were less severe than those observed in active joints , 73.Of note, intra-joint variability has also been documented as inflammatory mediators might be differently expressed in different locations of the same joint. In particular, tissue samples from sites close to the cartilage-pannus junction showed a higher level of inflammatory biomarkers that might be underestimated by analyzing specimens from other joint sites –77. AlthThus, it is still a matter of debate on the best location from which to obtain synovial tissue samples within a given joint. Recently, an international expert consensus stated that a minimum of four synovial tissue specimens from small joints and six from large joints have to be retrieved for reproducible research studies –81, whilSynovial tissue samples can be retrieved by using different techniques  1). Tis. Tis1). Blind-needle methods were described in 1950 by Polley et al. with the use of 5-mm-large needles, resulting in a practical but invasive procedure for the modern standards considering that the needle size is similar to the new arthroscopy instrument size. In the years, new and thinner needles have been introduced in the market, simplifying the histologic investigation of articular rheumatic diseases and allowing to perform these procedures without significant pain for the patients, reducing the risk of post-procedural complications . Parker Using a blind-needle biopsy technique, the clinicians achieve the procedure without a direct or indirect view, and it is not always possible to have an adequate tissue sample, especially in joints lacking a significant effusion. Some authors described a technique of synovial biopsy under fluoroscopy visualization with a semiautomatic needle. This technique allows the performance of multisite biopsies such as in the hips, shoulders, elbows, ankles, and wrists but requires a complex setting, exposing the patients to x-ray irradiation. Performing harvesting with the aid of ultrasounds could combine the low morbidity of a needle biopsy and the instrumental support's accuracy without ionizing radiation exposure. In recent years, many authors have described good results of ultrasound-guided needle biopsy , 44. TheArthroscopic-assisted synovial biopsy is a surgical technique in which the tissue is harvested under the direct view of the suspected pathological area's region, dramatically reducing the risk of mistakes. The technique is a standard joint arthroscopy, with a second portal required to harvest the material of interest using specific instrumentation. This approach's advantages are obtaining more significant macroscopic evaluation pieces, with better sampling from interest areas. Also, arthroscopic synovial biopsy techniques allow biopsies from sites adjacent to the cartilage . This arA new impressive field of research is needle arthroscopy, where clinicians can use in local anesthesia small modern devices, which permits an exploration of the joint in an outpatient setting and obtain tissue samples under direct view. This well-tolerated procedure allows good macroscopic evaluation of synovial inflammation and selective sampling of the synovial membrane .Finally, surgeons can obtain samples of synovial tissue during an open surgical procedure as a total joint replacement. This technique permits obtaining a relevant quantity of tissue and can help obtain synovial tissue useful for clinical and histological studies in rheumatic patients.Within the past decades, several considerable advances have been made in synovial tissue research. Synovial tissue represents the target tissue of many rheumatic and non-rheumatic diseases, and its analysis is crucial in the assessment of many infective, malignancy, and infiltrative disorders. Retrieving synovial tissue samples of good quality using affordable and safe methods from large and small joints is now a realistic desirable objective. Thus, clinical practice pathobiology might be a key component in the management and treatment decision-making process, even in rheumatology.FI, LC, IS, and RCo writing manuscript. RCa and PR revising manuscript. All authors contributed to the article and approved the submitted 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Inflammatory bowel disease is a chronic and recurring inflammatory condition of the gastrointestinal tract encompassing ulcerative colitis and Crohn's disease. Although the pathogenesis of inflammatory bowel disease remains to be fully elucidated, environmental factors such as diet are believed to play a pivotal role in the onset and management of inflammatory bowel disease. Diet is thought to play an essential role in intestinal inflammation due to its regulatory effects on the microbiota, gut immune system, and epithelial barrier function. Although the evidence remains insufficient to draw firm conclusions on the role of specific dietary components in gastrointestinal diseases, studies have suggested that a Western diet with high intakes of total fats, omega-6 fatty acids, and meat have been associated with intestinal inflammation and relapse of inflammatory bowel disease. In contrast to a Western diet, plant-based diets often result in a reduced intake of total fats and meats and an increased intake of plant fibers which may contribute to reduced intestinal inflammation. This review critically examines the influence of plant-based dietary components on the clinical disease course of inflammatory bowel disease. Furthermore, this review discusses the benefits and possible limitations of plant-derived dietary components in the treatment of inflammatory bowel disease while addressing the principal type of disease and the anatomic site of inflammation within the gastrointestinal tract. Finally, this review points out important directions for future research on the role of diet in inflammatory bowel disease. A better understanding of the role of diet and intestinal inflammation may pave the way for novel dietary interventions and specific foods- or food supplements, which can support the treatment of inflammatory bowel disease. Crohn's disease (CD) and ulcerative colitis (UC) are chronic relapsing inflammatory bowel diseases (IBD) , 2. AlthAdequate dietary protein consumption plays an essential role in IBD patients because of the increased risk of disease-related malnutrition found in these patients , 17. DepThe quality of a dietary protein is primarily characterized according to its content of EAAs . It is oAlthough greater consumption of plant-based protein sources may fulfill the daily EAA requirement, excessive consumption of protein including its potentially limited digestibility may result in increased accessibility of peptides and AAs for microbial fermentation in the large intestine . In addi2S), aromatic compounds , polyamines including ammonia, and nitric oxide (NO), and studies have found a higher fecal concentration of several of these metabolites  in IBD patients compared to healthy individuals   . These ogy of UC , 35. Altgy of UC , H2S mayonocytes . Despiteonocytes . In a pronocytes . Similaronocytes . The sugonocytes . In factonocytes . Moreoveonocytes . Hence, Although there may be several detrimental effects of over-eating protein on intestinal homeostasis, caution must be drawn before neglecting proteins derived from plant foods despite increased requirements to ensure sufficient EAA. Several factors influence the rate of protein fermentation, such as the substrate availability , pH, and the ratio between carbohydrates and proteins within the diet. As described by Yao et al. , consumpTo sum up, IBD patients may have an increased protein requirement depending on their disease status (active vs. remission). Adequate amounts of plant-derived protein may provide adequate amounts of AA and EAA essential for MPS and preservation of lean mass, which is particularly relevant to IBD patients as these patients are often at increased risk of malnutrition. However, high intakes of dietary protein may contribute to alterations of the gut microbiota and colonic inflammation by producing harmful metabolites. These effects may be attributed to animal-derived protein due to its high content of sulfur—and aromatic-containing AAs. Further, the potentially detrimental effects of increased protein consumption may, at least in part, be attenuated due to the coexisting consumption of various NDCs often found in plant-protein sources. However, increased consumption of plant-based foods to ensure proper intake of EAAs may not be feasible in all IBD patients due to IBD-related symptoms such as anorexia. In fact, at present, little is known about the role of proteins originating from plants in regard to actual measures of disease activity and nutritional status of IBD patients, as most of the research seemingly has been conducted in other populations or using animal protein. Thus, the role of plant-derived protein on IBD-related outcomes  remains to be determined.Two factors determine the role of lipid nutrition in health and disease: (1) the composition and (2) the total amount of fat in the diet . Fatty aThe quantitatively most important lipid component in the human diet is the triglyceride (TG) fraction, which accounts for more than 95% of the dietary fat. The dietary fatty acid composition of this fraction is highly dependent on the origin of the food, i.e., in general, the fatty acid composition of animal-derived food items consists mostly of SFAs, while plant-derived foods are rich in unsaturated fatty acids. With regard to IBD, it is generally said that people who eat more fruits and vegetables have a lower risk of IBD, while people who eat fewer fruits and vegetables and more animal fats and sugar have a higher risk of IBD . The sugC. rodentium challenged mice despite increased n-3 PUFAs in intestinal tissues and increased caecal concentration of SCFAs (with proposed anti-inflammatory effects)  . Other seffects) . When useffects) . Dietarythe diet .To sum up, it remains to be elucidated if dietary plant-derived fatty acids would be beneficial in relation to disease activity in UC and CD patients. It seems most likely that reducing the SFA content and the level of n-6 fatty acids (relatively to n-3 fatty acids) in the diet by consumption of plant-based foods potentially could minimize disease activity via the incorporation of n-3 acids into the host membrane phospholipids or due to their influence on the intestinal microbiota. However, plant sources rich in n-3 fatty acids do not contain EPA and DHA, and although controversy exists regarding the influence of these longer-chained n-3 fatty acids on disease activity, a dietary change from an animal-based diet toward a diet primarily based on plant foods should not include omission of fish or other marine foods as the metabolism of n-3 fatty acids into anti-inflammatory longer chained fatty acids from plant sources are limited.Plant-based foods represent a major source of dietary carbohydrates in the human diet, with a wide range of physiological properties important for human health . Many caAlthough NDCs are generally considered dietary fibers, NDCs represent a heterogeneous group of carbohydrates of varying chemical structures with varying composition and branching patterns , 77. ThiF. prausnitzii and Clostridium cluster IV, XIVa, XVIII , only limited weak evidence of the role of NDCs on disease activity was found  . The intcapacity , 113. Inogenesis . Consumpogenesis . In addin et al.  describen et al. , 115. Thn et al. . The antn et al. . Furthern et al. . SimilarWhile many studies have addressed the impact of polyphenols in relation to gut health of monogastrics, less studies have addressed their impact on human health and diseases [see for instance Cardona et al. ]. Most sIn clinical studies investigating the efficacy of polyphenols in IBD patients, promising results of polyphenols and phenolic compounds such as anthocyanin, resveratrol, epigallocatechin-3-gallate including foods rich in polyphenols  on various markers of inflammation, IBD-related symptoms and measures of disease activity have also been reported, although the number of studies is limited .Despite the limited number of intervention studies investigating the efficacy of polyphenols in IBD patients, particular interest has been devoted to the hydrophobic polyphenolic compound curcumin, and to date, curcumin may be the most investigated polyphenol in IBD patients due to its potentially clinical therapeutic anti-inflammatory and immunoregulatory effects .Therefore, the results of clinical studies investigating the efficacy of curcumin on measures of disease activity in IBD patients are summarized down below.Curcuma Longa and is the principal constituent of turmeric found in several common spices , oral capsule curcumin was used with a dosage ranging from 450 mg to 3 g/day  and food processing methods such as cooking and boiling. Thus, the actual intake of polyphenols may vary considerably according to these factors. Moreover, the bioavailability of polyphenols may be limited. As only a limited number of studies have investigated the efficacy of polyphenols in IBD patients, the optimal safe dose and efficacy of these bioactive substances still need to be determined based on adequate powered clinical studies. Similarly, although clinical studies of curcumin in UC patients with promising results exist, the number of studies is limited. Likewise, the optimal dose and administration route still need to be determined. Furthermore, the efficacy of curcumin on disease activity in CD patients need to be elucidated. Thus, large RCT studies of curcumin in UC and CD patients are required to determine its efficacy.Vitamins are organic micronutrients mainly synthesized by plants and do not provide energy. Vitamins are essential nutrients and humans are capable of synthesizing certain vitamins, while other vitamins cannot be synthesized by humans. Therefore, diets must supply the body with small amounts of vitamins daily to maintain the metabolic functions of the cells. It has often been stated that “healthy eating” would not require additional vitamin supplementation. However, the fact is that the majority of e.g., Americans do not follow a healthy eating pattern . Some viTo summarize, IBD patients may be at risk of developing deficiency for certain vitamins, i.e., vitamin D deficiency. Similarly, deficiencies of vitamin B12 and other B-vitamins are highly prevalent in IBD patients. While there is insufficient evidence to support the use of vitamins to induce or maintain remission in UC and CD, plant-based dietary habits may require vitamin supplementation of especially vitamin B12 and vitamin D.As discussed in this review, plant-based dietary components may impact various physiological mechanisms of intestinal inflammation. Therefore, the central question is whether it is possible to support and maintain clinical remission of IBD by adopting a plant-based eating pattern? Plant-based foods are major sources of NDCs and phytochemicals that are believed to interact with intestinal microbiota and exert direct influence on the gut immune system through several mechanisms related to the increased production of immunoregulatory postbiotics and downregulation of proinflammatory responses. That said, the clinical effects of NDCs and phytochemicals on induction and maintenance of disease activity in IBD patients remains inconclusive. Hence, more research is needed to determine whether these dietary components reduce disease activity in IBD patients. Although plant-based diets provide essential micronutrients, consuming a diet primarily based on plant foods may result in inadequate intakes of vitamin B12 and other micronutrients depending on the strictness of the diet, the nutritional status of the patient, and seasonal effects (regarding vitamin D status). Thus, vitamin supplementation may be needed. Consumption of plant-based diets, may also contribute to an increased dietary protein intake to ensure sufficient coverage of AAs and EAAs and the avoidance of malnutrition. Increased consumption of dietary protein may increase the amount of protein that reaches the colon, resulting in the production of several metabolites that may have detrimental effects on the gut and enhance intestinal inflammatory processes. However, whether plant and animal protein sources associate differently with IBD, and the course of disease, remains presently unknown. Furthermore, consuming foods primarily originating from plants may result in a decreased intake of SFAs and a more optimal ratio of n-6 fatty acids to n-3 fatty acids, which may decrease inflammatory responses and reduce disease activity in IBD patients. However, plant sources of n-3-fatty do not encompass EPA and DHA that are believed to exert anti-inflammatory properties. That said, most of the research on the role of EPA and DHA on disease activity has been conducted using fish oil with inconclusive results. Therefore, more research is needed to elucidate the role of these fatty acids and other fatty acids on disease activity in IBD patients. In the interim, adopting a diet primarily based on plant foods should not exclude fish and marine foods. To sum up, whether it is possible to induce and maintain clinical remission of IBD is not a simple “yes or no” question. Although the clinical effects of plant-based foods on disease activity need to be investigated further, clearly, plant-derived foods contribute with several food components and nutrients that may possess the ability to reduce and prevent intestinal inflammation. However, plant-based diets may also have drawbacks  that need to be addressed. Moreover, IBD patients represent a diverse group of patients with distinct disease characteristics, of which the site of inflammation may be of utmost importance to consider with respect to the nutritional composition of the diet, and especially the specific role of bioactive components and their association with the locally present microbiome see . BesidesCA, MH, HR, and CL: conceptualization and writing—review and editing. CA and CL: writing—original draft preparation. MH, HR, and CL: supervision. All authors have read and agreed to the published version of the manuscript. The authors contributed equally to the preparation of this manuscript.This research was supported by the Nutrition Board at Aalborg University Hospital, which provided funding for CA during his affiliation as a research assistant at the Department of Clinical Medicine, Faculty of Medicine, Aalborg University, Aalborg, Denmark.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A comprehensive experimental investigation of the wear behaviour of coated spur polymer gears made of POM is performed in this study. The three physical vapour deposition (PVD) coatings investigated were aluminium (Al), chromium (Cr), and chromium nitrite (CrN). Al was deposited in three process steps: By plasma activation, metallisation of Al by the magnetron sputtering process, and by plasma polymerisation. Cr deposition was performed in only one step, namely, the metallization of Cr by the magnetron sputtering process. The deposition of CrN was carried out in two steps: the first involved the metallization of Cr by the magnetron sputtering process while the second step, vapour deposition, involved the reactive metallisation of Cr with nitrogen, also by the magnetron sputtering process. The gears were tested on an in-house developed testing rig for different torques  and rotational speed of 1000 rpm. The duration of the experiments was set to 13 h, when the tooth thickness, and, consequently, the wear of the tooth flank was recorded. The experimental results showed that the influence of metallisation with aluminium, chromium, and chromium nitrite surface coatings on the wear behaviour of the analysed polymer gear is not significant. This is probably due to the fact that the analysed coatings were, in all cases, very thin (less than 500 nm), and therefore did not influence the wear resistance significantly. In that respect, an additional testing using thicker coatings should be applied in the further research work. Polymer gears are used widely in many engineering applications, such as office appliances, mechatronic devices, household facilities, computer and laboratory equipment, medical instruments, etc. ,2,3,4. TBecause gears are key machine elements in many engineering applications, the proper estimation of load capacity against failures under given loading conditions is crucial when dimensioning the gear drives. In the case of polymer gears, the standardised procedure according to the VDI 2736  is usualRecently, many researchers have been investigating different types of polymer gears in respect to the failure modes as described above. Because polymer gears usually run in dry operating conditions (without lubrication), the high contact friction and, consequently, high degree of wear, may be the main reason for the short service life of a gear drive, especially in medium to high power transmission applications. Singh et al.  investigPolymer gears are often used in applications in which lubricants cannot be used, such as food processing machines and office equipment . HoweverPhysical vapour deposition (PVD) is another technique which may be used to improve the wear resistance of contacting mechanical elements. As presented by Baptista et al. , the PVDThe most commonly used PVD-method is magnetron sputtering. This is a process of material vaporisation by bombarding the target material with high-energy ions. The process takes place in a vacuum chamber containing an inert gas, the substrate, and the coating material ,41. MagnTo improve the final characteristics of PVD-coatings, some additional technologies are often used with combinations of magnetron sputtering : plasma An experimental investigation of the wear behaviour of PVD-coated spur polymer gears made of POM is performed in this study. Three PVD-coatings investigated were aluminium (Al), chromium (Cr), and chromium nitrite (CrN). The deposition process of the analysed coatings is described briefly in In the proposed experimental study, three different coatings were prepared on the polymer gears made of POM. Aluminium (Al) coating was applied through a plasma activation process, followed by metallisation of the aluminium through a magnetron sputtering process, and, finally, a plasma polymerisation step. Chromium metallisation by the magnetron sputtering process was used for the chromium (Cr) coating. The third coating of chromium nitride (CrN) was prepared in two steps, namely, the metallisation of chromium by the magnetron sputtering process, and, finally, the step of reactive metallisation of chromium and nitrogen by the magnetron sputtering process. The process parameters of all three analysed coatings are shown in The polymer gear specimens made of POM were machine cut from extruded bars using a hobbing process , which was equipped with a Berkovich diamond indenter. The indentation instrument was controlled by an electromagnetic drive loading system with a high-precision coil and a permanent magnet. The tests were carried out with increasing loads. One series of tests was carried from 1 to 10 mN, and the other from 10 to 100 mN. In both cases, the loading time, unloading time and time at the maximum load were 10 s. A precision saw, Buehler Isomet 1000 , and a diamond blade designed for polymeric materials were used to cut the gear samples. The samples were cut at the edge of the gears in the cross-section and longitudinal directions. For the microscopy of the uncoated polymer substrate, we used the Environmental Scanning Electron Microscope , that allows analysing the electrically and thermally non-conductive materials. We used two detectors, an LFD (Large Field Detector) for secondary electrons and gaseous BSE for backscattered electrons. The pressure in the chamber was 60 Pa. For the surface and cross-section of the coated samples, we used high resolution scanning electron microscopy , equipped with an energy dispersive spectrometer .The gears were tested on an in-house developed testing rig, as shown in Before the test began, the tested gears were weighed on a technical balance, Mettler Toledo AX 204 SI 01 05 02, with a weighing accuracy up to 0.1 mg. Furthermore, the tooth thickness was measured along the entire circumference of the gear using a Mitutoyo Absolute dial gage with a roller of diameter 5 mm see . During −4 mN s−1) up to 200 nm of the indentation depth. A sharp change occurred on the indentation curves during loading, when an indenter penetrated through the coating and entered into the substrate. The previous characterisation of the morphology and thickness of the coating was performed by SEM see . The coaIt is clear that the obtained coating thicknesses were in the range between 8 to 40 nm. This is in good agreement with the experimental studies by Baptista et al.  and FerrInitially, the POM-polymer gears without coating were tested at torques 16, 20, 24 and 30 Nm. The experimental results are shown in Furthermore, all three groups of coated polymer gears  were tested at the torque 16 Nm. It is clear from In the subsequent experimental testing, the Al- and Cr-coated polymer gears were tested at the torque 20 Nm. The experimental results shown in It is obvious from In general, the influence of the analysed metal coatings on the wear behaviour of POM polymer spur gears is small and does not reduce the wear significantly. Namely, the thickness of the analysed coatings was, in all cases, very thin (less than 500 nm), and did not influence the wear behaviour significantly.If we compare the three analysed coatings, the Cr- and CrN-coatings have a little more beneficial effect compared to the Al-coating.The further study should be focused on the wear evaluation of coated polymer gears, where multilayer (thicker) Al, Cr and CrN coatings will be considered. Furthermore, the additional scratch tests should be performed for the appropriate adhesion evaluation of the analysed coatings. Therefore, more accurate conclusions could be made as to whether metal coatings reduce the wear of POM gears.The further study should also have to consider the measurement of the coefficient of friction for all three analysed surface coatings. Based on such measurements, the tribological behaviour could be analysed and evaluated critically for both dry and lubricated contact of meshing gears.An experimental investigation of the wear behaviour of coated spur polymer gears made of POM was performed in the presented study. Three different coatings were prepared and analysed regarding the possible wear reduction of meshing gears: aluminium (Al) coating, chromium (Cr) coating and chromium nitride (CrN) coating. Based on the obtained experimental results, the following conclusions can be made:


Community mobility is an individual’s movement outside the home. It is essential for the completion of many instrumental activities of daily living, such as shopping and healthcare, and promotes physical function, social engagement, independent living, and quality of life. Mobility research often focuses on gait speed measured in clinical settings, a critical but not sufficient determinant of community mobility. Here we present four talks that assess community mobility and its determinants using novel methodologies to enhance our understanding of how to maintain independence in older ages. First, Andrea Rosso presents characteristics of individuals with the strongest associations between environmental walkability, as assessed by virtual audits, and walking. Second, Kyle Moored demonstrates associations of self-reported fatigability with life space among older men, independent of their physical functioning. Breanna Crane introduces GPS-based objective measures of community mobility and their associations with cognitive and physical function of older adults. Finally, Pam Dunlap presents results of a randomized clinical trial of a physical therapy intervention to improve walking in older adults on subjective and objective measures of life space. These talks will provide a better understanding of the factors related to community mobility, introduce attendees to novel methodologies in the assessment of both community mobility and risk factors associated with the loss of community mobility, and demonstrate approaches to improve community mobility in at-risk older adults. The discussant, Jana Hirsch, will provide perspectives on how these data inform our current view of community mobility and will lead a discussion with the audience.


Breastfeeding is the single, most cost-effective intervention to reduce worldwide child mortality. Women empowerment interventions have positive impacts on child and maternal nutritional, and health status. Women’s employment and economic participation in Ethiopia have shown progress over the past three decades. However, consistent evidence indicated that maternal employment is often negatively associated with optimal breastfeeding in Ethiopia. The existence and enforcement of breastfeeding law, arrangement, and support in the workplace have vital roles in protecting employed mothers’ ability and right to breastfeed upon return to work from maternity leave. This commentary compared the breastfeeding laws, policies, and arrangements in Ethiopia with international standards, recommendations, and evidence-based practices.Public legislations of Ethiopia poorly protect the breastfeeding right of most new mothers. Ethiopian revised Labor Proclamation (No.1156/2019) incorporates most of the International Labour Organization maternity protection recommendations. However, it poorly safeguards breastfeeding rights and abilities of employed women. The provided maternity leave period is also shorter than the recommended exclusive breastfeeding duration. The revised Federal Civil Servant Proclamation of Ethiopia (NO.1064/2017) mandates the establishment of a nursery in government institutions where female civil servants could breastfeed and take care of their babies in a private room. Though, it protects only a small proportion of working mothers in Ethiopia, as majority women employed in the agriculture and informal economy sectors. So far, there are no notable workplace breastfeeding arrangements and support for employed mothers by employers and other initiatives. The ILO recommendation and experience of other middle income and low-income countries can be legal and practical grounds for establishment of breastfeeding-friendly workplace in Ethiopia.The lack of workplace breastfeeding laws, arrangements, and supports in Ethiopia limits mothers’ right to practice optimal breastfeeding. Policymakers, the government, and all concerned bodies should give due attention to enacting and enforcing sound laws and arrangements that will enable employed mothers to practice optimal breastfeeding upon return to work. Optimal breastfeeding is the single most cost-effective intervention to reduce child mortality in developed and developing countries . The impWomen’s employment is one of the means for achieving the global effort of gender equality. Women empowerment interventions have positive impacts on child and maternal nutritional and health status . DespiteSuboptimal breastfeeding is among the main factors contributing to the high burden of child mortality, morbidity, and malnutrition in Ethiopia , 10. ConA woman shall be provided with the right to one or more daily breaks or a daily reduction of hours of work to breastfeed her child. . The period during which nursing breaks or the reduction of daily hours of work are allowed, their number, the duration of nursing breaks, and the procedures for the reduction of daily hours of work shall be determined by national law and practice. These breaks or the reduction of daily hours of work shall be counted as working time and remunerated accordingly.  [.Different international, regional, and national laws protect motherhood and childhood worldwide. Enabling mothers to continue breastfeeding upon return to work is one of the essential elements of maternity protection. As early as 1919, the ILO acknowledged that breastfeeding was an integral part of motherhood and reproduction and deserved protection at the workplace . The ILOBreastfeeding law, policies, and arrangements at the workplace should ensure optimal breastfeeding practice, work productivity, and women’s right to equal work opportunity. Evidence shows that workplace breastfeeding arrangement and support for working mothers increase women’s work motivation, attendance, satisfaction, and productivity and is essential to meet Infant and young child feeding (IYCF) standards of breastfeeding , 19.The breastfeeding right of most new mothers in Ethiopia is unprotected by public legislation and policies. The revised Labor Proclamation No.1156/2019) incorporates most of the International Labour Organization maternity protection recommendation. However, it does not have specific legislation on breastfeeding protection and support in the workplace 19 incorp. The CivMaternity leave is one of the opportunities that will promote breastfeeding for working mothers . The durThe ILO recommendation and experience of other middle- and low-income countries can be legal and practical grounds for establishment of breastfeeding-friendly workplace in Ethiopia . For insAll employed women in Ethiopia have a right to practice optimal breastfeeding and deserve laws, arrangements, and support in the workplace. The law and policy options could include; adopting ILO breastfeeding conventions, introducing inclusive and explicit laws that protect all employed mothers’ breastfeeding rights, and ensure implementation of the Federal Civil Servant Proclamation act. Besides, the involvement, mutual understanding, and coordination of all stakeholders are essential for establishing organizational, structural, and social arrangements of breastfeeding in the workplace. These emotional and physical arrangements could be made through initiatives like workplace awareness creation and promotion of breastfeeding, employer and colleague support of breastfeeding, the establishment of breastfeeding facilities and childcare centers, and flexible work arrangement etc. . AlthougThe absence of breastfeeding laws, arrangements, and supports for employed mothers limits their ability and right to practice optimal breastfeeding. Policymakers, governments, and all concerned bodies should give due attention to enacting and enforcing sound laws and establishing arrangements and supports that will enable employed mothers to practice optimal breastfeeding upon return to work. Future researchers could focus on assessing the consequences of the problem and contextualizing and piloting different breastfeeding-friendly workplace interventions.


Autophagy is a key but undervalued process in root ontogeny, ensuring both the proper development of root tissues as well as the senescence of the entire organ.Autophagy is a process which occurs during plant adaptation to changing environmental conditions as well as during plant ontogeny. Autophagy is also engaged in plant root development, however, the limitations of belowground studies make it challenging to understand the entirety of the developmental processes. We summarize and discuss the current data pertaining to autophagy in the roots of higher plants during their formation and degradation, from the beginning of root tissue differentiation and maturation; all the way to the aging of the entire organ. During root growth, autophagy participates in the processes of central vacuole formation in cortical tissue development, as well as vascular tissue differentiation and root senescence. At present, several key issues are still not entirely understood and remain to be addressed in future studies. The major challenge lies in the portrayal of the mechanisms of autophagy on subcellular events in belowground plant organs during the programmed control of cellular degradation pathways in roots. Given the wide range of technical areas of inquiry where root-related research can be applied, including cutting-edge cell biological methods to track, sort and screen cells from different root tissues and zones of growth, the identification of several lines of evidence pertaining to autophagy during root developmental processes is the most urgent challenge. Consequently, a substantial effort must be made to ensure whether the analyzed process is autophagy-dependent or not.The online version contains supplementary material available at 10.1007/s00425-021-03668-3. ATG) and/or the proteins they encode  and consist of 18 genes  and in the central vacuole , all types of autophagy have been documented to occur  has been documented in root segments with differentiating primary and secondary xylem; while high expression of ATG11 was exclusively noticed in roots showing secondary growth. Importantly, concurrent detection of ATG8 protein was characterized immunohistochemically during the early development of TEs and xylary fibers , which is a typical example of vacuolar cell death in dPCD, has been suggested to be autophagic . Unfortunately, as compared to xylogenesis, there is a lack of comprehensive literature data pertaining to the involvement and mechanisms of autophagy in the process of phloemogenesis. At their maturity, SEs do not contain many organelles and are they devoid of a nucleus, dictyosomes of the Golgi apparatus and vacuoles. The only visible intracellular components are a few plastids, mitochondria, endoplasmic reticulum, specific vesicles and phloem-specific P proteins  and salicylic acid (SA); and a decrease in cytokinins (CKs). Interestingly, the upregulation of ATG genes, which may confirm the role of autophagy in this process, has not been detected. However, the authors emphasized that seminal roots of H. vulgare do not contain the same abundance of proteins in comparison to leaves. Additionally, the plants were exposed to a continuous supply of nutrients which may have suppressed nutrient remobilization; and thus, the overall process of autophagy  was observed, in addition to an overall increase in the number of autophagosomes as well  has been detected during the senescence of fine, absorptive roots  the mechanism regulating the degradation of cytoplasmic structures in differentiating root cells has not yet fully elucidated, however, it is of particular interest; (ii) determining if selective autophagy may be responsible for the process of phloemogenesis in roots; (iii) characterization of a signal which may determine the abrupt cessation of degradative processes when the cell matures . Scientific advancement in each of these areas will undoubtedly provide an opportunity to reconsider how roots regulate and use the autophagic machinery to ensure proper growth and functioning at the whole plant level. The research that will be necessary to address these questions can be facilitated by novel methods; a basis of which can be provided through methodological progress for immunolocalization techniques such as the use of specific antibodies, in addition to fluorescent fusion proteins (FFP) that can be implemented to visualize the subcellular distribution of autophagy-related proteins. Taken together, these approaches can ultimately provide a powerful tool and possess capacities for creating generic knowledge that can be of wider relevance; which may ultimately prove to be critical for understanding the role of autophagy in developmental processes in plants. Therefore, it is necessary to perform future investigations, using cutting-edge cell biological methods to track, sort and screen cells from different root tissues. These approaches can be used in addition to tailored strategies at the molecular level which will increase our understanding of autophagy function within particular zones of roots.AB-Z, NW: conceptualization and original draft preparation; KM, NW: experimental data; KM: figures; AB-Z, KM, NW: review and editing.Representative images of negative control reactions which omitted the primary antibody anti-ATG8. LM light microscopy image of the same pioneer root section (TIF 13048 KB)a, b Aerenchyma formation in Zea mays roots, c, d Populus trichocarpa fine root senescence (TIF 7865 KB)Representative images of negative control reactions by omitting primary antibody anti-ATG8. LM light microscopy image of the same root section. Below is the link to the electronic supplementary material.