Glomerulonephritis (GN) represents a collection of kidney diseases characterized by inflammation within the renal glomeruli and small blood vessels. The lesions that occur in other nephron structures mainly result from the harmful effects of proteinuria. In recent years, an emphasis has been placed on gaining a better insight into the pathogenesis and pathophysiology of GN in order to facilitate diagnoses and provide efficient and targeted treatments of the disease. Owing to the advanced molecular and genetic diagnostic techniques available today, researchers have been able to elucidate that most cases of GN are determined by genetic risk factors and are associated with the abnormal functioning of the immune system . MicroRNAs (miRNAs) are a group of single-stranded, non-coding molecules, approximately 20 nucleotides in length, that act as regulatory factors in the post-transcriptional processes capable of regulating the expression of multiple genes. In this paper we present the available research aiming to determine effects of miRNAs on the development and progression of GN and discuss the potential role of miRNAs as new diagnostic markers and therapeutic targets. Glomerulonephritis (GN) is a non-homogeneous group of diseases characterized by inflammatory lesions within the renal glomeruli and small blood vessels. The lesions observed in other nephron structures are of a secondary nature and mainly result from the harmful effects of proteinuria. GN is caused by the improper functioning of immunological processes, although the detailed pathogenesis of most GN cases is yet to be understood. The large number of different forms of GN can be divided into two major subgroups: primary and secondary. Primary forms of GN are distinguished by pathological lesions that are initially only found in the glomeruli, where the clinical symptoms and laboratory test deviations are caused by disorders to their structure and function. Secondary forms of GN, however, originate from non-glomerular diseases that are rather systemic. Another way to classify GN is based on histopathological criteria distinguishing between proliferative and non-proliferative instances of GN. In most of the cases, histopathological lesions ensure the emergence of a typical set of clinical symptoms. Proliferative glomerulopathies are accompanied by the nephritic syndrome that is comprised of arterial hypertension, active urine sediment, oliguria, and proteinuria of <3.5 g/d, while non-proliferative glomerulopathies are accompanied by nephrotic syndrome, which is characterized by proteinuria >3.5 g/d, hypoalbuminemia, lipid disorders, and edemas .In recent years, emphasis has been placed on gaining a better insight into the pathogenesis and pathophysiology of GN that could facilitate quicker diagnosis and a more efficient targeted treatment. Owing to the advanced molecular and genetic diagnostic techniques available today, researchers have discovered that most cases of GN are determined by genetic risk factors and the immunologically mediated forms of GN are associated with the abnormal functioning of the immune system. In genetically predisposed persons, environmental factors may cause overexpression of certain immunological factors which, when deposited in the glomeruli, lead to damage .MicroRNAs (miRNAs) are a group of single-stranded, non-coding molecules containing approximately 20 nucleotides that act as regulatory factors of post-transcriptional processes. These small molecules regulate the expression of multiple genes. Up to 30% of the genes encoding human proteins are regulated by miRNAs . miRNAs In this paper we review available reports on the effects of miRNAs on the development and progression of GN and discuss their new roles as diagnostic markers and potential therapeutic targets.As mentioned above, miRNAs are single-stranded non-coding regulatory molecules responsible for modifying the expression of multiple genes, mainly in the post-transcriptional stage, although they have also shown to affect the process of transcription itself in certain cases. Most miRNAs are transcribed from DNA to pri- and then pre-miRNA, and finally cleaved to form mature miRNA. In a majority of cases, miRNAs interact with the three prime untranslated region (3’-UTR) of the target mRNA and cause translational inhibition or degradation of mRNA .Caenorhabditis elegans. Researchers isolated the lin-4 molecule, responsible for the transition through larval stages [MicroRNAs were first identified in 1993 when Ambros and Ruvkun extracted the molecules from the model nematode, l stages ,7. Sincel stages .MicroRNAs form in several stages. A typical miRNA synthesis pathway begins in the nucleus, where the original transcript, or so-called primary miRNA (pri-miRNA), is transcribed. As a result of these hairpin-shaped structures processing, a precursor miRNA (pre-miRNA) is formed. The main functional role of this process is facilitated by the RNA-binding protein complex, DGCR8 , and Drosha ribonuclease, an RNase III enzyme. DGCR8 binds to the single-stranded ends of the pri-miRNA and makes it possible for the RNase catalytic domain to specifically cleave transcripts to release pre-miRNA molecules approximately 60–100 nucleotides in length. The pre-miRNA is then transported by Exportin 5 to the cytoplasm, where as a result of the activity of the Dicer enzyme (another RNase III enzyme), it is cleaved into a double-stranded miRNA-miRNA* molecule (approx. 20 nucleotides in length). The miRNA-miRNA* complex is composed of the guide strand and the passenger strand; the latter being marked with an asterisk. By way of numerous transformations, the passenger strand is degraded and the guide strand is incorporated into the RISC complex containing argonaute (AGO) proteins, and as a mature single-stranded miRNA molecule, it is finally able to bind to the 3′ untranslated regions (3’-UTR) of mRNAs ,5.The Ago family proteins, mainly Ago2, are one of the most important components of the mRNA-induced silencing complex (mRISC). Ago proteins facilitate both mRNA degradation and translation silencing possible through facilitating interactions between microRNA molecules and their target mRNAs. Full complementarity of the mRNA:miRNA system results in mRNA separation that promotes transcript degradation, whereas partial complementarity results in translation inhibition .miRNA molecules are of key importance to the functioning of the human system and are responsible for the normal course of many biological processes. Disturbance to miRNA expression promotes development of numerous diseases. For example, such a relationship has been observed with regard to chronic kidney disease. Further, miRNA molecules have been identified as components of extracellular fluids, including as components of urine and blood serum, and as a result they have the potential to be used as easily available, diagnostic biomarkers ,5.An association has been shown between individual miRNAs and their location in given organs. In 2004, miRNA molecules specific for the kidney were identified, namely miR-192, miR-194, miR-204, miR-215, and miR-216 . In anotIgA nephropathy (IgAN) is the most prevalent type of primary GN worldwide . In IgANNumerous studies have shown that the expression of various miRNAs is altered in IgAN patients. A study published in 2018 identified 48 miRNAs from IgAN patients in which expression levels differed from healthy individuals of the control group . Of thosIn the study carried out by Wang et al. researchers showed that the expression of selected miRNAs in the urine of IgAN patients depended on the histopathological advancement of their lesions (Lee’s classification) as compared to patients with other GN forms, including minimal change disease and membranous GN . Four miAn increasing number of studies have pointed to miRNAs having roles in the pathogenesis of IgA nephropathy . The sigmiRNAs are also believed to play an essential role in the pathogenesis of IgAN by regulating the release of proinflammatory cytokines . It has Perhaps in the future, apart from their role in the diagnosis of renal diseases, miRNAs will become therapeutic targets for IgAN treatment. Inhibiting the activity of selected miRNAs could lead to blocking the first stage of pathogenesis of IgAN, thus reducing the levels of poorly galactosylated IgA1, or curbing renal tissue fibrosis, thus slowing down the progression of disease. Nonetheless, it should be remembered that each of the miRNAs is responsible for multiple transcriptional processes, which is why miRNA inhibitors need to be used cautiously in order to minimize the risk of inducing adverse effects .Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown etiology, which is characterized by inflammation that can be found in a large set of tissues and organs. One of the most frequent organ manifestations of SLE is LN, which in many cases leads to end-stage renal disease. In the progression of LN, immune complexes are deposited or formed in situ in the kidney, which invokes cytokine responses and leads to damage to the endothelium of the glomerular capillary loops and small renal vessels, podocytes, tubules, and renal interstitium. Lesions in the kidneys are observed in approximately 50% of patients diagnosed with SLE. The degree of renal lesions is assessed on the basis of biopsies and the histopathological classification of the lesions is done according to ISN/RPS , which distinguishes VI classes .The first association between miRNAs and LN was shown in 2009 by Dai et al. who identified 66 miRNAs in renal biopsy specimens from LN patients in which the expression of miRNAs was modified as compared to healthy individuals of the control group . In anotRecently, studies have also confirmed the important role of miRNAs in LN. In a 2018 study, elevated plasma concentrations of miR-125a, miR146a, and miR155 were shown in LN patients compared to the control group comprising healthy individuals . The plaIn a study by Navarro-Quiroz et al. not only differences in abundance of plasma circulating microRNAs in LN have been measured, but also particular microRNAs have been assessed as potential contributors to LN pathogenesis . In thisFSGS, or focal segmental glomerulosclerosis, is comprised of group of forms of GN that are caused by podocyte injury resulting in subsequent glomerulosclerosis and mesangial matrix expansion . FSGS isIn 2013, a study was published which assessed the association of miR-192 and miR-205 with the occurrence and course of FSGS and MCD . The FSGThe microRNAs play also an important role in the pathogenesis of FSGS . As statDespite the advancements in medicine, the diagnosis and monitoring of the disease course and response to treatment of different forms of GN remain a challenge to clinicians. Currently, a histopathological assessment of renal biopsy specimens is the only commonly practiced diagnostic mean to evaluate GN. Renal biopsy is an invasive method, which is why it is important for new, non-invasive diagnostic markers to identify GN. Presently, microRNAs, single-stranded non-coding nucleotide molecules responsible for regulating the expression of multiple genes, are attractive potential markers of GN. Some evidence also suggests the role of miRNA in the pathogenesis or as potential therapeutic agents, especially in IgA nephropathy. Nonetheless, further multicenter studies are required in order to assess the exact utility of microRNAs in the general diagnosis and monitoring of the course of selected forms of GN. The exact role of microRNAs as therapeutic strategies in GN is also yet to be determined.


The enormous advancements in the medical imaging methods witnessed in the past decades have allowed clinical researchers to study the function of the human brain in vivo, both in health and disease. In addition, a better understanding of brain responses to different modalities of stimuli such as pain, reward, or the administration of active or placebo interventions has been achieved through neuroimaging methods. Although magnetic resonance imaging has provided important information regarding structural, hemodynamic, and metabolic changes in the central nervous system related to pain, magnetic resonance imaging does not address modulatory pain systems at the molecular level . Such important information has been obtained through positron emission tomography, bringing insights into the neuroplastic changes that occur in the context of the pain experience. Positron emission tomography studies have not only confirmed the brain structures involved in pain processing and modulation but also have helped elucidate the neural mechanisms that underlie healthy and pathological pain regulation. These data have shown some of the biological basis of the interindividual variability in pain perception and regulation. In addition, they provide crucial information to the mechanisms that drive placebo and nocebo effects, as well as represent an important source of variability in clinical trials. Positron emission tomography studies have also permitted exploration of the dynamic interaction between behavior and genetic factors and between different pain modulatory systems. This narrative review will present a summary of the main findings of the positron emission tomography studies that evaluated the functioning of the opioidergic system in the context of pain. The changes observed in brain areas related not only to the sensory but also to the emotional and cognitive aspects of pain and also corroborated the concept of pain as a multidimensional experience, introduced many years before.41 Those findings were further expanded to other chronic pain conditions, such as trigeminal neuropathic pain19 and migraine headache.45 Although those studies used the selective mu-opioid receptor radioligand [11C] carfentanil, similar findings have been obtained using the nontype selective opioid receptor radiotracer [11C] diprenorphine, which has also been largely used to study the role of the whole opioid system in chronic pain syndromes. For instance, increased availability of opioid receptors in the frontal, temporal, and cingulate cortices was associated with decreases in the inflammatory pain of patients with rheumatoid arthritis in a past study.28 Another study with [11C] diprenorphine described the presence of reduced opioid receptor availability in both cerebral hemispheres of patients diagnosed with peripheral neuropathic pain.68 Of note, in patients with neuropathic pain, the decreased availability of opioid receptors was predominantly located in the hemisphere contralateral to the reported pain.37 Such findings could indicate a higher release of endogenous opioid peptides  driven by pain, resulting in higher occupancy and lower availability of opioid receptors  combines radioactive tracers and mathematical models to determine the kinetics of bioactive tracers involved in certain physiological phenomena. With this method, the measurement of a certain radiotracer concentration in the nervous tissue is represented through a 3-dimensional image, which ultimately represents the anatomical distribution of the biological process under evaluation. Positron emission tomography has allowed researchers to quantify the availability of several receptors in vivo. The receptors studied include serotonergic, dopaminergic, and opioid, among various others, which can be evaluated either in baseline conditions  or during specific challenges, such as cognitive tasks, experimental pain, or even the administration of a placebo.43 a definition that derives from the concept the multidimensionality of pain. This fundamental concept, initially introduced in the 1960s42 is still extremely useful to understand the complexity of the pain experience, which goes far beyond nociception (sensory dimension), with its important affective  and cognitive  dimensions. However, with the enhancement of neuroimaging techniques, it has been possible to corroborate and consolidate this model. Magnetic resonance imaging (MRI) studies have provided valuable information regarding functional changes  as well as structural changes  that occur in the brain driven by pain. In addition, some previous PET studies showed increases in the regional cerebral blood flow related to noxious thermal stimuli of different intensities when compared with innocuous thermal stimulation. The results of such studies suggest that changes in the regional cerebral blood flow associated with noxious but not to non-noxious thermal stimulation could represent neuronal changes associated with nociceptive processing driven by pain.11 Other PET studies have confirmed the presence of changes in cerebral blood flow induced by noxious stimuli and have even revealed the brain regions that contribute to each aspect of the pain experience. For instance, 1 PET study that evaluated the effects of noxious heat stimulation on the regional cerebral blood flow confirmed the major role of the thalamus in the processing of pain intensity. However, according to the results of that study, pain unpleasantness may be more associated with the activity of the posterior part of the anterior cingulate cortex (ACC).63 Nonetheless, these findings added limited, although extremely relevant, data regarding the activity of specific neurotransmitters and modulatory systems related to pain processing and modulation. With this respect, PET studies that applied selective or nonselective radiotracers to investigate the functioning of particular modulatory systems related to noxious stimulation have contributed significantly to the study of pain, providing important insights into the molecular mechanisms underlying the pain experience as well as the placebo analgesia and the nocebo effect.Pain has been described as “an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage,”8 In a later study, Zubieta et al.72 investigated the activity of mu-opioid receptor-mediated neurotransmission in vivo during a sustained pain challenge, obtained through a controlled injection of 5% hypertonic saline or a placebo into the masseter muscle of healthy volunteers. The results demonstrated the activation of the mu-opioid system associated with an ongoing pain experience. Such activation was represented by a lower availability of mu-opioid receptors during the pain challenge when compared with the placebo phase. Strikingly, those changes were found in a wide variety of pain-related brain structures, such as the amygdala, thalamus, hypothalamus, insula, prefrontal cortex (PFC), and dorsal ACC. These findings served as a basis to further PET studies, and consequent investigation ultimately confirmed the same pattern of mu-opioid activation in patients with different pain syndromes.45 Interestingly, a recent PET/MRI study demonstrated that the mu-opioid system is not only involved in self-experienced pain but also regulates the neural mechanisms that trigger the unpleasant sensation of seeing others in pain, namely vicarious pain . More precisely, a negative correlation between hemodynamic changes  and mu-opioid receptor availability was detected in the insula (anterior and posterior), thalamus, primary motor cortex (M1), primary and secondary somatosensory cortex (S2), paracentral lobule, supplementary motor area, and PFC. Given these findings, these brain regions appear to process the negative features of pain as well as play a role in the sensory-discriminative mirroring of others' pain. On the other hand, the availability of mu-opioid receptors in the orbitofrontal cortex (OFC) was positively correlated to its BOLD activation induced by vicarious pain, which could possibly be linked to a role of this brain region in socioemotional functions and mentalizing. Importantly, the involvement of dopamine D2 receptors could not be demonstrated in that study.29In one the first PET studies that used the selective mu-opioid receptor radiotracer [11C] carfentanil, Bencherif et al. demonstrated a decreased mu-opioid receptor availability in the contralateral thalamus related to a noxious stimulus . Interestingly, this decrease in mu-opioid receptor availability in the thalamus directly correlated with the subjective pain ratings. These results suggested a release of endogenous opioids driven by acute pain. They were also important to demonstrate, in vivo, the involvement of the mu-opioid neurotransmission in the supraspinal regulation of pain perception.72 also demonstrated a substantial interindividual variability in both receptor availability and in the activation of the mu-opioid system in healthy controls experiencing same level of pain intensity. In these, and subsequent studies, the pain intensity was controlled  . Indeed, genetic variants may not only interfere in the protein coded by a gene but also in the physiological activity of several neural systems, including the opioidergic system. Thus, the role of genetic polymorphisms on the antinociceptive activity of the opioid system has also been examined through PET.Zubieta et al.158metval), on the mu-opioid activation induced by a sustained pain challenge74 in healthy subjects. Since COMT is one of the enzymes that metabolizes catecholamines, it is involved in the regulation of noradrenergic and dopaminergic systems and may indirectly affect opioid neurotransmission. This study reported that the mu-opioid activation induced by the sustained pain challenge was significant lower in met/met healthy subjects, which have a lower COMT activity, when compared with val/met subjects, which present an intermediate function of the same enzyme. Those differences were found in the striatopallidal pathway nuclei and amygdala74  were also accompanied by higher sensory and affective ratings of pain in healthy subjects. A more recent study has confirmed that in chronic temporomandibular disorder pain, COMT polymorphisms  have induced higher pain sensitivity, but with changes in mu-opioid activity in the limbic system. Hence, further studies should be performed to better understand the effect of COMT polymorphism and mu-opioid activity in multiple pain conditions and disorders.44Early evidence regarding the genetic component of pain processing comes from a PET study that used a selective opioid mu-opioid receptor radiotracer to investigate the effects of the abundant polymorphism of the catechol-O-methyltransferase (COMT) gene, which codes the replacement of valine  by methionine (met) at the codon 158  to guanine (G) at codon 118 (A118G) in the human OPRM1. This is related to a lower expression of the OPRM1 gene.69 Hence, it interferes with pain sensitivity as well as the outcomes of pain treatments, with important effects on the modulatory activity of the opioid system. This polymorphism has been recently investigated in clinical pain conditions.66 For example, patients with primary dysmenorrhea were evaluated through functional MRI (fMRI). The results showed that the individuals who carried the G allele had functional hypoconnectivity between the ACC and periaqueductal gray matter (PAG) when compared with AA homozygotes, which also correlated with the spontaneous menstrual pain. Patients with primary dysmenorrhea carrying the G allele exhibited no correlation between PAG-seeded FC and their menstrual pain. These findings indicated a decreased opioid modulation of pain in G allele carriers. The results of that study also brought important insights into the mechanisms of pain in dysmenorrhea, and such data help to explain the great individual variability in the pain experience reported by those patients. A later study reported that other genetic variations in the mu-opioidergic system, more specifically, the presence of the T-allele of the rs563649 polymorphism on the mu-opioid receptor, may influence the prediction of the development of pain complaints at the ages of 16 to 17 years, from behavioral and functional indicators  obtained at earlier ages (14–15 years).46Other polymorphisms affecting the activity of the mu-opioid system have also been studied. For instance, the 73 The presence of the same correlation in the PAG, although not reaching the statistical level of significance, assigns an antinociceptive function to the amygdala, possibly through its connections to the PAG, a region with a dense concentration of mu-opioid receptors and an essential element of the descending pain modulatory system.40 The correlation between the NAc mu-opioid activation, a key component of the reward circuitry21 with the sensory pain ratings found in the same study, indicates the contribution of this structure to the antinociceptive effects produced by mu-opioid receptor activation. In addition, significant negative correlations were found between MPQ affective scores and mu-opioid receptor activation in the NAc as well as in the ACC and thalamus. The changes in thalamic mu-opioid activity are also relevant. A recent fMRI-PET study demonstrated a colocalization of changes in opioid receptor availability measured with [11C] diprenorphine and the hemodynamic changes in the brain obtained through changes in the BOLD signal within the thalamus, mediated by noxious pressure stimulus in healthy subjects.67 These findings were interpreted as a possible inhibition of thalamic neurons by a release of endogenous opioids induced by the painful stimulus.A negative correlation has also been demonstrated between the sensory features of pain, evaluated through the sensory score of the McGill Pain Questionnaire (MPQ) and the degree of mu-opioid activation in the thalamus, NAc, and amygdala using the model of experimentally induced sustained pain previously described.Taken together, these results suggest that the antinociceptive effects of mu-opioid receptor activation by endogenous opioids are not only important to reduce the sensory aspects of pain but also to relieve its affective features. Such results also opened possibilities to investigate the relationship between mu-opioid receptors and their relationships with other systems important to pain and inflammation, which was explored in a following study.52 On the other hand, during a pain challenge (sustained pain applied to the masseter muscle), the activation of mu-opioid receptors was positively correlated with the plasma levels of Il-1ra (antinociceptive cytokine), but not with the plasma levels of IL-1β in the dorsomedial NAc. Nevertheless, the analgesic effects produced by the mu-opioid system activation during the pain challenge were counterbalanced by changes in IL-1β. For instance, subjects that displayed higher pain-induced release of IL-1β experienced less opioid-induced analgesia. The effects of IL-1β in the functioning of the opioidergic system have also been demonstrated in in vitro and in animal model studies. For instance, in a past study, IL-1β promoted an increase in the expression of proenkephalin and opioid receptors mRNA in primary astrocyte-enriched cultures.54 Furthermore, the release of IL-1β in the peripheral ganglia has been proposed as a possible mechanism to the attenuation of the analgesic effects related to morphine, a process that possibly involves the activation of satellite glial cells in the peripheral ganglia.9 In fact, bidirectional interactions between peripheral inflammation and central mechanisms as well as their contributions to the development of pathological conditions had been largely reported.4 Corroborating this concept, changes in the functioning of the opioid system have been found in patients with inflammatory pain.28 In addition to mechanistic studies, the contribution of the opioid system to pain has been extensively investigated through PET in cohorts of patients with episodic/chronic pain, and the role of mu-opioid activation in the pathophysiological mechanisms of different painful syndromes has just started to be unveiled.Using the model of sustained pain previously described, a PET study revealed a significant negative correlation between the baseline plasma levels of the proinflammatory cytokine interleukin 1β (IL-1β) and the availability of mu-opioid receptors in the amygdala. In that study, subjects with higher levels of IL-1β showed lower mu-opioid receptor availability in the amygdala along with higher pain sensitivity.24 or changes in the cortical volume, eg, decrease cortical volume found in a cohort of patients with back pain.5 More importantly, those changes have been proven to affect multiple cortical structures involved not only in pain perception but also in the modulation of nociceptive stimuli. In addition, fMRI studies suggest the occurrence of colocalized functional and structural changes in the brain of patients with chronic pain.14 Nevertheless, beyond the presence of structural and metabolic changes in the brain induced by a persistent pain experience, PET studies have brought insights into the molecular neuromechanisms underlying the maladaptive neuroplasticity related to chronic pain. The use of PET in pain research has also helped researchers understand the individual viability related to pain, as well as long-lasting resistance to treatment, including to opioids found in refractory patients. Recent studies with (PET) using nonselective  radiotracers or [11C] carfentanil, a selective mu-opioid receptor radiotracer, have shown reduced mu-opioid receptor availability in some chronic pain disorders, including rheumatoid arthritis,28 neuropathic pain,37 and fibromyalgia.25The results of MRI studies indicate that chronic pain is associated with structural changes in the central nervous system. Such changes include altered cortical thickness, eg, cortical thickening or cortical thinning, previously demonstrated in patients with migraine28 Similar results were obtained in a sample of 5 patients with central poststroke pain. Those patients presented lower opioid receptor availability in several pain-related brain structures, such as the thalamus, anterior and posterior cingulate cortex, midbrain, insula, PFC, S2, and parietal cortex, when compared with a control group of healthy subjects.68 Although still preliminary, those studies evidenced the presence of an altered opioid neurotransmission in chronic pain and highly suggested that a decreased availability of opioid receptors could be a common finding in subjects with chronic pain. In another PET study conducted in patients with arthritis  using [11C] diprenorphine, higher chronic pain levels were associated with increased availability of opioid receptors in the insula, PAG, basal ganglia, NAc, and subcallosal area in the group of patients, which could suggest upregulation of opioid receptor sites in patients with chronic pain who present higher levels of pain.10 The availability of opioid receptors in the caudate nucleus was also positively correlated to thermal pain thresholds in both patients and healthy subjects, supporting the role of the upregulation of opioid receptors within the striatum as an adaptive response to inhibit pain. Finally, the same study showed reduced levels of opioid receptor availability in the caudate nucleus of patients with chronic pain compared with controls, supporting the findings of the other studies. Nevertheless, a common limitation of those studies was the use of a nonselective radiotracer, which did not permit the identification of the specific opioid receptor affected by chronic pain.The first evidence of changes in the opioid system in patients with chronic pain dates back to the 1990s. A preliminary PET study performed in 4 patients with rheumatoid arthritis and using [11C] diprenorphine showed increased binding related to pain relief in several brain regions. However, more significant changes were reported in the temporal, frontal, and cingulate cortices.25 used the selective mu-opioid receptor radioligand [11C] carfentanil to study the functioning of the mu-opioid system in fibromyalgia syndrome, a disease with unclear etiology and pathophysiology. The results indicated a reduced availability of mu-opioid receptors in the amygdala, NAc, and dorsal ACC of 17 patients with fibromyalgia when compared age- and sex-matched healthy controls. When analyzing the group of patients with fibromyalgia separately, the availability of mu-opioid receptors in the striatum and cingulate cortex was negatively correlated with the relative amount of affective pain, defined by the affective/sensory scores of the MPQ. Furthermore, the availability of mu-opioid receptors in the NAc was negatively correlated to the affective pain ratings. When expanding the findings obtained with fibromyalgia to other chronic pain diseases, reduced availability of mu-opioid receptors in the NAc was also found in a cohort of patients with trigeminal neuropathic pain.19 Those changes were also correlated with the clinical pain symptoms. Nonetheless, in the cohort of patients with trigeminal neuropathic pain, the availability of mu-opioid receptors in the NAc was negatively associated with the sensory and total pain ratings of the MPQ. The NAc is the main structure in the ventral striatum. Although very small, this brain region plays a crucial role in the reward circuitry and modulation of the nociceptive information. It receives afferent nociceptive inputs through connections with the amygdala, thalamus, cingulate cortex, and parabrachial nucleus. Moreover, the axis formed by the PAG, rostralventromedial medulla, and NAc exerts a crucial role in the descending pain modulatory system.40 The analgesic effects of several analgesic drugs, including opioids, highly depend on the proper functioning of those structures.2 Remarkably, changes in the BOLD signal of the NAc has been related to the onset (aversive) and offset (reward) of painful heat stimuli.7 However, even more important seems to be the changes in activity of the NAc in response to the offset phase of painful heat stimuli, which permitted the differentiation between patients with chronic low back pain and healthy subjects in a previous study.6In another work, Harris et al.56 Based on such findings, the authors hypothesized that in patients with fibromyalgia, tonic higher levels of endogenous opioids would promote a lower affinity or a downregulation of mu-opioid receptors on GABAergic interneurons of brain regions involved in nociception. Hence, an inhibition of GABAergic interneurons related to a phasic release of endogenous opioid triggered by noxious stimulation, an important mechanism for the descending pain modulation, could be altered in fibromyalgia. More specifically, although in healthy individuals, opioid release results in the inhibition of GABAergic interneurons that tonically inhibit the neurons located in several brain regions such as the PAG, PFC, and ACC, thus causing disinhibition and excitation of antinocicpetive neurons, in patients with fibromyalgia, such phasic endogenous opioids release in response to nociceptive stimuli fails in producing adequate inhibition of GABAergic interneurons and a faulty antinociceptive mechanism occurs.56A PET/fMRI study performed in patients with fibromyalgia revealed a positive relationship between pain-evoked neural activity and mu-opioid receptor availability in the rostral ACC (rACC) and in the dorsolateral PFC (DLPFC). On the other hand, fMRI BOLD signals in several brain regions such as the medial frontal gyrus, posterior cingulate cortex, and ACC were negatively correlated with the affective/sensory pain ratio. Furthermore, higher mu-opioid receptor availability was negatively correlated with the affective/sensory pain ratio in the medial frontal gyrus, posterior cingulate cortex, ACC, and precentral gyrus.16 Indeed, PFC activation had been previously shown in both triggered and spontaneous migraine headache attacks.18 Moreover, it seems that an increased mu-opioid activity in the PFC enhances the connectivity with the PAG, which is important to produce analgesia.65 In addition, the mu-opioid activity during the ictal and interictal phases of patients with migraine was positively correlated, corroborating the interindividual variability in the mu-opioid system largely reported and already discussed in this article.73 In a following PET study also performed in patients with episodic migraine, the activity of the mu-opioid neurotransmission in the red nucleus and PAG was positively correlated to the development thermal allodynia in an experimental paradigm using a heat stimulus applied to the ophthalmic (V1) region of the trigeminal nerve.45 The results of that study substantiate the concept of a dysfunctional response of midbrain structures to the nociceptive inputs received from activated afferent trigeminal projections as a mechanism of migraine headaches.61 Conversely, a recent PET study did not find differences in the availability of opioid receptors when comparing patients with episodic migraine and healthy subjects.34 However, the lower sensibility of diprenorphine,53 used to evaluate the functioning of the opioidergic system in that study along with the comparison at baseline conditions , might explain their distinct results when compared with the previous studies.45The activity of mu-opioid receptors has also been studied in patients with episodic migraine during both its ictal (painful) and interictal (pain-free) phases. Increased activity of mu-opioid receptors in the PFC was reported during the ictal spontaneous migraine phase compared with interictal periods.62 The authors scanned Sprague-Dawley rats with [18F] FDPN, a compound analog of diphrenorphine. Animals were scanned 3 months after the spared nerve injury or sham surgery was performed. Animals that underwent the spared nerve injury exhibited lower availability of opioid receptors in the motor cortex and basal ganglia when compared with sham. Although a baseline scan was not performed, the observed changes were attributed to the presence of neuropathic pain, since all animals were exposed to the same experimental conditions. More importantly, the same study found a decreased expression of mu-opioid receptors in the basal ganglia and insula of the animals that developed a peripheral neuropathy, which was not followed by changes in enkephalin or in the neuronal marker NeuN, as demonstrated by immunohistochemistry. Considering those findings, the decreased availability of opioid receptors observed in the brain of patients with chronic pain might be interpreted as variations in the expression of opioid receptors triggered by pain rather than loss of opioidergic neurons (no changes in NeuN-immunoreactivity cell count between nerve-injured and control rats) or higher release of endogenous opioids .35 Nevertheless, the lack of a longitudinal evaluation  is an important limitation that should be addressed in future studies. Moreover, to which extent those findings can be translated to other (non-neuropathic) chronic pain disorders, as well as to humans, are important aspects that must considered when analyzing such results. The use of selective radiotracers by future studies will also be important confirm the specific opioid receptor affected in this whole process. In addition, another important feature that has been scarcely explored and should be investigated in depth by future studies is the difference in opioid activation between somatic and visceral pain. For instance, at least 1 study reported that in contrast to somatic pain, sustained visceral pain is not related to an increased release of mu-opioid receptors, despite similar intensities, thus highly suggesting that endogenous opioids could play distinct roles in each type of pain .36As previously mentioned, the findings of these studies could indicate a higher release of endogenous opioids related to pain or downregulation of opioid receptors, particularly mu-opioid receptors, or even both. Those hypotheses have only been recently tested by a PET study performed in an experimental animal model of neuropathic pain.33 Further studies demonstrated the central pathways involved in the placebo effect. This includes cortical areas, such as the ACC and DLPFC, the descending modulatory system components , as well as the dorsal horn of the spinal cord.64 In addition, the neurobiology of placebo and nocebo effects has been investigated through PET studies. Overall, these works have confirmed, in humans, the fundamental role of the dopaminergic and opioidergic system on placebo/nocebo as well as the specific areas engaged in the processing of both phenomena, mainly the reward circuitry with its functioning centered in the activity of the NAc. This progress in the understanding of the precise neuromechanisms underlying placebo and nocebo effects can potentially impact the evaluation of clinical trial results, development of therapeutic strategies, and even from a broader perspective, the comprehension of the interindividual variability related to pain experience. The activation of the mu-opioid system in the placebo response was demonstrated in an early PET study. Significant activation of the mu-opioid system was found in the insula, rACC, DLPFC, and NAc.70 More importantly, the mu-opioid system activity was correlated with the pain experience. For instance, the DLPFC mu-opioid activation found in that study was related to the degree of the expected analgesia induced by placebo. Interestingly, the same study applied a system for pain delivery that allowed the measurement of variations in the requirements of the algesic substance used before and after the placebo administration. Therefore, it provided an objective evaluation of changes in pain sensitivity induced by placebo, or in other words, the activation of antinociceptive mechanisms. Mu-opioid activation in the rACC was highly correlated with the requirements of the algesic infusion to keep the pain during the study.70 In fact, the role of rACC in the placebo mechanisms has been shown by other studies.22 It has also been demonstrated that the coupling between the ACC and PAG induced by placebo can be blocked by naloxone.22The opioid system has been widely associated with the development of placebo effects since the discovery that that the placebo effect can be prevented by the administration of naloxone, an opioid receptor antagonist.50 In the same study, individuals with low expectations and higher effectiveness  exhibited the strongest placebo responses, whereas subjects with higher expectations and lower effectiveness  displayed nocebo responses.The investigation of the role of expectations and learning processes to the activation of the mu-opioid system during placebo has revealed that the prediction error signal  is related to the analgesic responses produced by placebo and to the mu-opioid activation in the ACC, insula, amygdala, thalamus, and OFC.59 Remarkably, nocebo and placebo produced opposite effects in those systems. Activations of both dopaminergic and opioidergic systems were found with placebo while deactivations occurred with nocebo. Placebo-induced activation in the dopaminergic system was found in the basal ganglia, including the NAc. Indeed, the dopaminergic release in the NAc was responsible for 25% of the variance in the analgesic effects produced by placebo. In the opioidergic systems, those effects were also detected in the NAc but extended to other brain regions such as the PAG, amygdala, OFC, insula (anterior and posterior), and ACC . On the other hand, 25% of the sample experienced hyperalgesia with the administration of the placebo, which represented the nocebo effect. Nocebo correlated to deactivation of the dopaminergic and opioidergic systems with plentiful overlap of the brain regions affected by both effects (placebo and nocebo). When comparing placebo-induced changes in the activity of mu-opioid and dopamine neurotransmissions between high placebo and nocebo responders, again, significant deactivations were found in nocebo responders opposing to the activations found in the group of placebo responders. Regarding the mu-opioid neurotransmission, the changes were found in the subgenual ACC, anterior insula, OFC, and thalamus, while in the dopaminergic system, similar findings were detected in the NAc and putamen. Another important finding of that study was that the degree of placebo-induced dopaminergic and opioidergic activations in the NAc predicted high and low placebo responsiveness. Furthermore, the activation of dopamine neurotransmission in the NAc correlated with the degree of mu-opioid activation in regions that responded to placebo. Such findings corroborate the importance of dopamine receptors to the activity of mu-opioid neurotransmission12 as well as the participation of mesolimbic dopaminergic projections and its interactions with the opioidergic system to the placebo analgesia.58 In fact, it has been described that placebo characterizes a type of reward expectation; therefore, both share the same pathways.17A further study investigated compared the roles of the opioidergic and dopaminergic systems with the development of placebo and nocebo effects in healthy volunteers.34 As expected, few subjects from each group reported higher pain levels with the placebo treatment. Those individuals were classified as nocebo responders instead of placebo responders. Another study investigated the neuromechanisms related to a method of noninvasive modality of neuromodulation, namely transcranial direct cranial stimulation (tDCS) on healthy subjects through PET.20 The results demonstrated that sham and real stimulation (2 mA for 20 minutes) induced mu-opioid activation in shared (precuneus and PAG) as well as in specific brain regions . These data support the presence of a placebo effect contributing to the clinical effects of tDCS. Moreover, those findings suggest that both shared and specific mechanisms underlie the effects of such method. Similar findings have also been demonstrated with other neuromodulatory techniques, such as transcranial magnetic stimulation (TMS), confirming the activation of the mu-opioid system driven by noninvasive methods of neuromodulation.31 More specifically, in a study published by Lamusuo et al.,31 the availability of mu-opioid receptors was lower after real TMS applied to S1/M1 when compared with sham stimulation in the right ventral striatum, medial orbitofrontal, prefrontal and anterior cingulate cortices, left insula, superior temporal gyrus, dorsolateral PFC, and precentral gyrus. These results might represent a higher release of endogenous opioids driven by TMS. Another study assessed the availability of opioid receptors, measured through the nonselective radiotracer [11C] diprenorphine, preoperatively and postoperatively, in patients with neuropathic pain treated with motor cortex stimulation (MCS). The results indicated that the levels of opioid receptors assessed through preoperative PET scans may be valuable predictors of MCS outcomes when used to treat neuropathic pain. For instance, in that particular study, patients exhibiting values of opioid receptor density below the lower limits in age-matched controls in the contralateral insula, PAG, and thalamus were presented the lowest probabilities to benefit from MCS treatment.38 An additional PET study also reported a focal release of endogenous opioids driven by deep brain stimulation (DBS) of the PAG. However, those effects were neither affected by the systemic administration of the opioid antagonist naloxone nor correlated to the analgesic effects produced by DBS. Therefore, such findings were not able to determine whether the analgesic effects produced by DBS are indeed driven by a DBS-related opioid release.60 Another study that explored the effects of traditional and sham (placebo) Chinese acupuncture in patients with fibromyalgia26 also suggested differential mechanisms, both involving the mu-opioid neurotransmission, underlying each one.The activation of the mu-opioid system induced by placebo has also been investigated from the perspective of specific clinical conditions and alternative pain treatment modalities. One of those studies investigated the effects of a placebo treatment in patients with episodic migraine during their interictal phase using PET with [11C] diprenorphine. Although the placebo treatment resulted in decreased pain scores, the magnitude of the placebo effect was not significantly different between placebo and controls. Moreover, patients with migraine and healthy controls did not differ regarding activity of opioid receptors induced by placebo.48 and influenced by genetic factors.49 Such information could be extremely useful when designing clinical trials, especially in cases of therapies previously expected to produce large placebo effects. The use of such information would permit the subdivision of clinical pain study samples based on the potential development placebo/nocebo effects and the presence of genetic polymorphisms or personality traits that exert direct influence over the placebo response. Another fact still scarcely explored in the literature is how the degree of placebo responses can predict the effectiveness of nonpharmacological therapies such as cognitive-behavioral therapy or neuromodulatory therapies.55Interestingly, some studies have indicated that placebo effects and the related mu-opioid activation may be predicted by some personality traitsThe possibility of studying the human opioid system in vivo has contributed important information to the understanding of the antinociceptive mechanism related to acute and chronic pain, as well as the neurochemical basis of both placebo and nocebo effects. The altered functioning of the opioid system in the brain of patients with chronic pain represents one of the most important findings obtained with clear therapeutic implications. The interindividual variability in the mu-opioid system activation is another prominent result provided by PET studies. Future studies are expected to expand the overall knowledge regarding the contribution of genetic polymorphisms to the differential responses of the mu-opioid system to pain and to the individual resiliency or susceptibility to chronic pain development. The use of that information, together with the investigation of genetic, potentially inflammatory markers, or even associated personality traits, will be extremely useful in the development of clinical trials with better stratification of the individuals, analyzed based on their expected activation of the mu-opioid system as well as on their predisposition to experience placebo or nocebo effects.The authors have no conflict of interest to declare.A.F. DaSilva is funded by the National Institute of Health–National Institute of Neurological Disorders and Stroke—R01 NS094413, and National Institute of Dental and Craniofacial Research—U01 DE025633. M.F. DosSantos is funded by Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Jovem Cientista do Nosso Estado (JCNE), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ).


Although the efficacy of herbal medicines (HMs) and traditional Chinese medicines (TCMs) in human diseases has long been recognized, their development has been hindered in part by a lack of a comprehensive understanding of their mechanisms of action. Indeed, most of the compounds extracted from HMs can be metabolized into specific molecules by host microbiota and affect pharmacokinetics and toxicity. Moreover, HMs modulate the constitution of host intestinal microbiota to maintain a healthy gut ecology. Dietary interventions also show great efficacy in treating some refractory diseases, and the commensal microbiota potentially has significant implications for the high inter-individual differences observed in such responses. Herein, we mainly discuss the contribution of the intestinal microbiota to high inter-individual differences in response to HMs and TCMs, and especially the already known metabolites of the HMs produced by the intestinal microbiota. The contribution of commensal microbiota to the inter-individual differences in response to dietary therapy is also briefly discussed. This review highlights the significance of intestinal microbiota-associated metabolites to the efficiency of HMs and dietary interventions. Our review may help further identify the mechanisms leading to the inter-individual differences in the effectiveness of HM and dietary intervention from the perspective of their interactions with the intestinal microbiota. The function of herbal medicines (HMs) and traditional Chinese medicines (TCMs) in the remedial and prophylactic management of human diseases has been recognized for a long time , while tin vivo drug activity, including pharmacokinetics and toxicity, is closely associated with the gut microbiota  may ameliorate T2D with hyperlipidemia by enriching beneficial bacteria, including Blautia and Faecalibacterium spp.  . The majrove T2D ; Table 3ium spp. . In addit of GQD ; Table 3t of GQD  and joint of GQD . Furthert of GQD , which iAkkermansia and Parabacteroides confer seizure protection to mice fed a controlled diet by reducing the level of gamma-glutamyl amino acids and increasing the GABA and glutamate content in the brain (Lactobacillus in the mammary gland microbiota and subsequently elevates the levels of bile acid and bacterial-modified metabolites in breast cyst fluid (Bifidobacterium increases the abundance of Akkermansia muciniphila, with both microbes being able to generate SCFAs, thereby improving insulin resistance and limiting inflammation and consequently improving the symptoms of obesity (Faecalibacterium, provide full protection against acute arsenic toxicity in a mouse model (Dietary interventions have become an effective method for treating some refractory diseases, with the effects being associated with the commensal microbiota of the host he brain . In addihe brain . The Medst fluid . Howeverst fluid . Furtherst fluid ; this re obesity . Further obesity . A promise model .Lactobacillus reuteri restores oxytocin levels, synaptic plasticity, and healthy social behaviors in mice (Bacteroides and Prevotella and not in those whose gut microbiota exhibited a higher proportion of Bifidobacterium (Bacteroides and Prevotella in elevating insulin levels. Indeed, convincing epidemiological studies have indicated that specific dietary components may be crucial for the pathogenesis of some diseases such as asthma and allergies (N-oxide by hepatic flavin monooxygenases, thereby promoting the development of atherosclerosis. However, the specific microbial taxa contributing to this process require further investigation.However, some of the observed dietary effects have not yet been associated with specific intestinal microbes or with specific metabolites. For instance, a maternal high-fat diet negatively impacts the social behavior of offspring, resulting in a deficiency in synaptic plasticity in the ventral tegmental area and in oxytocin production, but the administration of  in mice . It has  in mice . Notably in mice . Such re in mice , suggestacterium . This sullergies . For exallergies . SpecifiSalmonella (in vivo, it will also be valuable to further examine these associations in order to assist in developing novel approaches to reducing the toxicity of HMs and TCMs.The beneficial effect of HMs and dietary therapy in several refractory diseases is generally appreciated, but the underlying mechanisms involved remain obscure. However, their interaction with the host microbiota seems to be a critical factor in such processes. Indeed, a growing number of studies indicate that the commensal microbiota plays a crucial role in maintaining host health and that the constitution of the intestinal microbiota exhibits large inter-individual differences. Moreover, most components in HMs and dietary interventions can modulate the constitution of the microbiota, which may disrupt or maintain homeostasis in the host. Collectively, it is not surprising that the gut microbiota, and especially microbiota-associated metabolites, may be a crucial mediator linking HMs or dietary therapy and the physiological status of the host. Therefore, it is important to consider the effects of biotransformation by commensal microbiota when designing herbal formula dietary therapy to achieve optimal success in treating diseases, particularly in the case of precision medicine. It is also essential to determine the optimal timing of administrating HMs and specific diets, in particular given that the composition of the gut microbiota exhibits diurnal variation. Indeed, microbiota-associated metabolites have several attractive properties, including known chemical structures and long shelf lives . In partlmonella . HoweverYlW contributed to the conception, design, collection and assembly of references, discussion, interpretation, and writing of the manuscript. SQ contributed to the collection and assembly of references, interpretation of the article, and writing of the manuscript. JJ, LH, and FL contributed to the collection and assembly of references. FJ, ZR, and YfW contributed to the conception, design, interpretation of the article, and the final article approval.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.


Infectious laryngotracheitis (ILT) of chickens is a substantial issue to be studied in Iraq because this disease is one of the most highly contagious respiratory diseases in the world caused by a herpesvirus. However, in Iraq, the ILT virus (ILTV) infection and disease have yet not been confirmed in layers, so farm owners do not vaccinate these layers. The current study aimed to document the detection and characterization of ILTV in layer hens from Al-Diwaniyah city, for the first time in Iraq, using molecular techniques like polymerase chain reaction (PCR) and sequencing.Four layer farms  in Al-Diwaniyah province, Iraq, suffered a severe ILT outbreak, was diagnosed and reported by clinical and PCR tests. This disease has been reported in Iraq, and more recently, it began to show outbreaks in Al-Diwaniyah city. The current work opted to investigate the ILTV using PCR and DNA sequencing techniques. The study targeted the p32 gene of ILTV using pooled tracheal swabs and organs including the trachea, lung, and kidneys which were collected from dead and clinically infected chickens.The analyses revealed that four of six suspected field samples showed positive results by PCR. The DNA sequencing results showed the homology of the amplified fragments with the studied gene.This study confirmed the presence of ILTV in hens with respiratory signs during the outbreak. Infectious laryngotracheitis (ILT) has gained accelerated attention worldwide because this disease can cause fundamental economic losses including decline egg production, low growth rates, and mortality .ILT virus (ILTV) is susceptible to disinfectants, but it also can survive for weeks to months if protected by organic materials such as manure, deep litter, biofilms, or respiratory secretions . AlthougStudies have found that herpes simplex virus-1 gene has a 63 homology open reading frame sites out of 77 of the whole genome. ILTV detection by isolation techniques is time-consuming, while other methods such as serological tests such as fluorescent antibody technique, enzyme-linked immunosorbent assay, serum neutralization, and agar gel immunodiffusion are generally of low sensitivity and laborious. Nowadays, identification of the viral DNA by conventional polymerase chain reaction (PCR) such as using infected cell polypeptide 4 (ICP4) gene  or real-st time in Iraq, using molecular techniques like PCR and sequencing.The current study aimed to document the detection and characterization of ILTV in layer hens from Al-Diwaniyah city, for the 1The samples were collected from the outbreak area. The research was done according to global and national ethical criteria for animal care and use.In this study, the isolation of ILT virus was conducted by collecting the samples pooled tracheal swabs and organs such as the tracheas, lungs, and kidneys, from four layer farms  that suffered respiratory problems and 17-30% mortality rate. The study was conducted during January 2018-May 2018 in Al-Diwaniyah province after severe outbreak. After doing the postmortem examination on infected layers, the suspected diagnosis was ILT. Six positive samples were used to perform additional analyses to confirm the ILTV.DNA Extraction Kit  was used for the extraction of DNA from pooled samples of layer hens suspect of ILTV infection.et al. [Primers, designed previously by Vögtlin et al. , were uswww.ncbi.nlm.nih.gov/BLAST).To confirm the specificity of the amplicons, two samples of the PCR products were sent for DNA sequencing, using the same primers mentioned above to laboratory in Korea . BLASTn in NCBI website was used to evaluate the sequence homology of ILTV with sequences from GenBank (The fragment of 588bp of the p32 gene of ILTV is shown in four samples . The posPhylogenetic analysis and divergence relationship of the two selected Iraqi isolates that are illustrated in Avian ILT was classified as a specific viral disease of chickens in the United States in 1926 . It was The current work confirms the diagnosis of ILTV in layer hens from farms that experienced respiratory disease outbreak in the region of Al-Diwaniyah city, Iraq. Other extensive studies are needed to conduct epidemiological surveillance in this region to determine the incidence, prevalence, and economic impact of the disease. Furthermore, it is of importance to detect the sources of the viruses to be field or vaccine strains to gain valuable information about the origin of the disease and routes of transmission. The phylogenetic results of the studied samples of this outbreak suggested that ILTV was brought by carrier birds and spread rapidly to neighboring flocks due to the stressed situation of non-vaccinated layers. In conclusion and according to the study, it is highly recommended to improve the biosecurity measures in Iraq to avoid ILT infection.FA and HH collected the samples, recorded the case history, monitored the cases, making an experimental design, performed the PCR, and drafted the manuscript. AAA and YIK prepared the phylogenetic tree and searched the literature for citation purposes. All authors read and approved the final manuscript.


Arabidopsis thaliana indicate that the mechanisms of development of the inner and outer integuments depend on largely different sets of molecular players. However, it was not known, in most cases, whether these differences were already present in early flowering plants, or arose later in the Arabidopsis lineage. Here, we analyze the expression patterns of integument regulators in Amborella trichopoda, the likely sister to all other living angiosperms. The data obtained indicate that regulators of the YABBY, KANADI, and homeodomain-leucine zipper class III transcription factor families have largely conserved their integument-specific expression profiles in the Amborella and Arabidopsis lineages since the most recent common ancestor (MRCA) of living angiosperms. We identified only one case, involving the paralogous genes ETTIN and AUXIN RESPONSE FACTOR4, in which integument-specific expression patterns had clearly diverged between Amborella and Arabidopsis. We use the data obtained to partially reconstruct molecular mechanisms of integument development in the MRCA of living angiosperms and discuss our findings in the context of alternative hypotheses for the origin of the angiosperm outer integument.The ovules and seeds of most angiosperm groups are enclosed by two integuments, whose evolutionary origins are considerably separated in time, as the inner integument arose over 300 million years ago (MYA) in an ancestor of all living seed plants, while the outer integument arose, perhaps as recently as 164 MYA, in an ancestor of all living angiosperms. Studies of the model angiosperm  The ovules and seeds of most seed plants are covered by one or two integuments. These maternal tissues function to (1) protect the internal tissues of the ovule and later the seed, (2) define a route, via the micropyle, for pollen or pollen-tube entry, and (3) contribute in many cases to the regulation of seed hydration and dormancy . GymnospArabidopsis thaliana (hereafter referred to as Arabidopsis), which possesses anatropous, bitegmic ovules. Ovule initiation in Arabidopsis begins with the expression of the homeobox transcription factor WUSCHEL (WUS) (see Much of what is known of the molecular mechanisms of integument development comes from the study of the model angiosperm WUS) see , which iWUS) see .WUS at the apex of the Arabidopsis ovule primordium defines the presumptive nucellar tissue. The outgrowth of the integuments from the chalazal region, immediately below the nucellus, depends on the expression of three homeodomain-leucine zipper class III (HD-ZIP III) transcription factors, PHABULOSA (PHB), PHAVOLUTA (PHV), and CORONA (CNA) (WUS expression (ETTIN/AUXIN RESPONSE FACTOR3 (ETT/ARF3) and ABERRANT TESTA SHAPE/KANADI4 (ATS/KAN4), both of which are expressed in the abaxial (away from the growth axis) domain of the inner integument, act to remove auxin from the zone between the two integument primordia  , which apression . Expressrimordia . InteresArabidopsis, there is a clear division of labor between the inner and outer integuments, as ATS is the principal KANADI family member expressed in the inner integument , is expressed specifically in the abaxial zone of the Arabidopsis outer integument, and an orthologue of this gene shows a largely similar expression pattern in the basally diverging angiosperm Nymphaea   , 2011, sINO, mentioned above), the mechanisms of integument development have been investigated to date uniquely in Arabidopsis, and it is therefore not known, in most cases, which of these were already present in early flowering plants and which evolved later in the Arabidopsis lineage. In the present work, we investigate the conservation of mechanisms of integument development from very early stages of angiosperm evolution by analyzing the expression patterns of the orthologs of Arabidopsis integument regulators in the basally diverging angiosperm Amborella trichopoda (hereafter referred to as Amborella). Most recent molecular phylogenetic studies place Amborella, the only known representative of Amborellales, as sister to all other living angiosperms  toluidine Blue-0 in 0.1 M sodium phosphate buffer (pH 6.8).Amborella ortholog of the Arabidopsis YABBY-family integument regulator INO has previously been reported (ETT and AUXIN RESPONSE FACTOR4 (ARF4)  . HoweverSupplementary Figure S1), generated from the protein alignment shown in Supplementary Figure S2, succeeded in identifying clear Amborella orthologs of the Arabidopsis genes REV and CNA, and a clear pro-ortholog of the Arabidopsis genes PHB and PHV, which accordingly appear to be derived from a duplication within the crown group of living angiosperms. In all these cases, the Amborella genes identified as orthologs or pro-orthologs of Arabidopsis integument regulators occupied basal positions within their respective clades, in agreement with the likely phylogenetic placement of Amborella.Phylogenetic reconstructions of the HD-ZIP III family in the present study , generated from the protein alignment shown in Supplementary Figure S4, succeeded in identifying a clear Amborella ortholog of the Arabidopsis inner integument regulator ATS (KAN4), which grouped in a small basal clade of ATS-like sequences. Relationships of gene orthology between Amborella sequences and the Arabidopsis outer integument regulators KAN1 and KAN2 were less clear in phylogenetic reconstructions.Phylogenetic reconstruction of the KANADI family , Atr.ATS, (XM_006856161.3), AtrPHB/PHV (XM_006845952.3), Atr.REV (XM_006846714.3), Atr.ARF4 (XM_020671056), and Atr.ETT (XM_011627988.2). The longer alternative splicing variant of Atr.ARF4 , spreading towards the micropyle-end with the growth of the outer integument . This expression pattern closely resembles that of INO in Arabidopsis. Atr.INO expression was stronger in early stages of Amborella ovule development, becoming absent in the mature ovule . These data reinforce observations in Nymphaea , are summarized in Figure 2.Nymphaea  that alrAtr.ATS was found to be expressed specifically in the abaxial domain of the inner integument , as is ATS in Arabidopsis. This expression persisted into the final stages of ovule development in almost mature flowers . No expression of Atr.ATS was apparent in any female reproductive tissue other than the inner integument. These data strongly suggest ATS orthologs to have conserved their function in inner integument development in both the Amborella and Arabidopsis lineages since the MRCA of living flowering plants.Atr.PHB/PHV, the Amborella pro-ortholog of Arabidopsis PHB and PHV, to be strongly expressed at early stages of development in the chalazal region of the ovule, which gives rise to the integuments, and very strongly expressed in the adaxial tissues of the ovary wall . At later developmental stages, Atr.PHB/PHV appeared weakly expressed in the adaxial zone of the inner integument . At early stages of ovule development, Atr.REV showed a similar expression pattern to Atr.PHB/PHV in the chalaza and ovary wall, though arguably with a slightly wider expression in the chalaza . At later stages of ovule development, reduced levels of Atr.REV expression were visible in the adaxial regions of both integuments . The association of Atr.PHB/PHV expression with the inner integument, and that of Atr.REV with both integuments , suggests the paralog-specific conservation of function between the Amborella and Arabidopsis lineages since their separation at the base of the living flowering plant clade. Atr.PHB/PHV and Atr.REV expression patterns are nonetheless very similar, and these genes are clearly expressed together in parts of the chalaza, the inner integument and the abaxial region of the ovary wall.We found ETT and ARF4 act redundantly in the definition of abaxial tissue identity in the Arabidopsis leaf , with Atr.ARF4 giving the stronger hybridization signal. Expression levels of both genes diminished in mature integuments . A switch may therefore have occurred in the major paralog active in the inner integument, with ETT playing the principal role in Arabidopsis, while Atr.ARF4 is more highly expressed in Amborella. In addition, whereas the role of ETT is limited to the inner integument in Arabidopsis, Atr.ARF4 and Atr.ETT are expressed in the abaxial domains of both integuments in Amborella.sis leaf , though mborella  suggesteArabidopsis ETT has been found to interact physically with ATS, both of which are expressed specifically in the inner integument . Of course, Atr.ETT and Atr.ARF4 might interact in the outer integument with other Amborella KANADI proteins  whose expression patterns were not investigated in the present study.It is interesting to note that tegument . Atr.ETTFigure 1N) and a mature  female flower are provided to help interpret anatomical details of in situ hybridization images shown in Figures M.Toluidine-blue stained sections of the carpel wall and ovule in a Stage 7   , as it i 105 MYA .Figure 3). Predicted intermolecular interactions shown in this model are based on functional data from Arabidopsis alone and the conservation of tissue-colocalization of the molecules concerned in Amborella.We have used the data presented here, together with information from published studies, to construct a partial molecular model for the control of integument development in the MRCA of living angiosperms (Like the carpel, the outer integument is a pleisiomorphic feature of angiosperms. Two contrasting hypotheses have been proposed to account for the origin of this structure, prior to the radiation of living angiosperms. One of these hypotheses (reviewed by WUS leads, in transgenic Arabidopsis, to the production of supernumerary integuments (The main alternative hypothesis for the origin of the outer integument, as discussed by eguments , demonstThe data presented here strongly suggest that the precise mechanisms of integument development were already substantially distinct in the inner and outer integuments some 164 MYA in the MRCA of living flowering plants. This conclusion clearly favors the hypothesis of a distant or indirect homology between the inner and outer integuments, such as might be explained by the more direct origin of the outer integument from an ovule-containing seed–fern cupule. It does not provide support for the possible origin of the outer integument by a modular reiteration of the inner integument, as this mechanism would be expected to yield two integuments whose development initially depended, in early flowering plants, on near-identical sets of molecular regulators.GA performed the experiments and phylogenetic analyses. AV supervised the experimental work and phylogenetic analyses. AA-R supervised the experimental work. BF and GG provided the plant material and contributed to writing the paper. CS planned and supervised the research and wrote the paper.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.


Whole-genome sequencing (WGS) will soon replace traditional phenotypic methods for routine testing of foodborne antimicrobial resistance (AMR). WGS is expected to improve AMR surveillance by providing a greater understanding of the transmission of resistant bacteria and AMR genes throughout the food chain, and therefore support risk assessment activities. At this stage, it is unclear how WGS data can be integrated into quantitative microbial risk assessment (QMRA) models and whether their integration will impact final risk estimates or the assessment of risk mitigation measures. This review explores opportunities and challenges of integrating WGS data into QMRA models that follow the Codex Alimentarius Guidelines for Risk Analysis of Foodborne AMR. We describe how WGS offers an opportunity to enhance the next-generation of foodborne AMR QMRA modeling. Instead of considering all hazard strains as equally likely to cause disease, WGS data can improve hazard identification by focusing on those strains of highest public health relevance. WGS results can be used to stratify hazards into strains with similar genetic profiles that are expected to behave similarly, e.g., in terms of growth, survival, virulence or response to antimicrobial treatment. The QMRA input distributions can be tailored to each strain accordingly, making it possible to capture the variability in the strains of interest while decreasing the uncertainty in the model. WGS also allows for a more meaningful approach to explore genetic similarity among bacterial populations found at successive stages of the food chain, improving the estimation of the probability and magnitude of exposure to AMR hazards at point of consumption. WGS therefore has the potential to substantially improve the utility of foodborne AMR QMRA models. However, some degree of uncertainty remains in relation to the thresholds of genetic similarity to be used, as well as the degree of correlation between genotypic and phenotypic profiles. The latter could be improved using a functional approach based on prediction of microbial behavior from a combination of ‘omics’ techniques . We strongly recommend that methodologies to incorporate WGS data in risk assessment be included in any future revision of the Codex Alimentarius Guidelines for Risk Analysis of Foodborne AMR. Antimicrobial resistance (AMR) represents a major threat to public health, with an estimated 700,000 deaths attributable to AMR every year in the world, and a projected 10 million deaths per year by 2050 in the absence of additional control measures . AMR is Salmonella carrying plasmid borne extended-spectrum beta-lactamase (ESBL) resistance  and person-to-person contact . The presistance . The relsistance . Howeverlobacter . AMR haslobacter . Until rlobacter .Salmonella and Campylobacter, in addition to some sequencing of resistant strains of Escherichia coli and Enterococcus collected from food-producing animals, retail meats and humans  and E. coli (since 2018) collected from agri-food samples and human clinical isolates  by public health and food safety laboratories has ramped up in recent years, and these new methods are set to replace traditional phenotypic methods for routine surveillance of AMR and other food safety hazards in the near future . In typid humans . Similarisolates .in silico speciation and sub-species level differentiation of isolates , as well as a description of the molecular mechanisms underlying observed resistance phenotypes, the use of WGS is expected to assist AMR surveillance by providing a greater understanding of the transmission of AMR bacteria and genes throughout the food chain, and therefore support risk assessment of foodborne AMR . This is typically done using homology-based algorithms such as BLAST  . Some prIn general, AMR genes can be located either on bacterial chromosomes or on MGEs, allowing AMR gene transmission via clonal spread or horizontal transfer, respectively. MGEs include factors which enable AMR genes to move within or between DNA molecules within the same cell , as well as factors which enable AMR genes to be transferred between bacterial cells  . PlasmidE. coli from humans, chicken meat, pigs and poultry farms, E. coli strains from poultry to humans, as had been suggested based on traditional, low-resolution typing methods , also known as Whole Genome Association Studies (WGAS), make it possible to identify genetic markers or genetic risk factors associated with increased pathogenicity or virulence. Briefly, GWAS identify molecular markers such as genes, k-mers, insertions/deletions or single base-pair changes  in the DNA sequence of an organism that are significantly associated with a phenotypic trait of interest  or virulence  of a given hazard. Prediction tools can be used to detect the presence of known pathogenicity or virulence genes, such as those determining the capacity of attachment, adhesion, invasion or replication of an isolate. As an example, the Center for Genomic Epidemiology (CGE) has developed web-based tools to identify acquired virulence genes among several bacterial species  among 38 Shiga toxin-producing E. coli (STEC) O157 isolates of human and animal origin . Using methods akin to the prediction of AMR and pathogenicity or virulence genes, bioinformatics pipelines can be used to identify the presence of genes known to provide higher ability to grow or survive under stress conditions. As an example, iccation . In the biofilms , the abin cattle  or the aeratures .2 to 103 genes are compared between genomes). The latter is an expansion of the traditional multilocus sequence typing (MLST) approach  and includes whole-, core- and accessory-genome MLST   . The str agMLST) , and it eritidis , L. monoytogenes  and quin. jejuni  showed h. jejuni . It requ. jejuni . As two C. jejuni isolates from poultry in 12 European countries and a set of 536 previously published C. jejuni genomes retrieved from the European Nucleotide Archive, C. jejuni. In other words, they explored whether the emergence of fluoroquinolone-resistant C. jejuni was related to the transmission among countries or to the selection through fluoroquinolone use in individual countries  and New York (NY) in the United States, highlighted several geographic location-specific clones , as well as broadly distributed clonal groups with similar AMR profiles that likely emerged a long time ago and successfully disseminated to wider populations , S. Heidelberg infections in Canada . WGS was successfully used for source attribution of L. monocytogenes in the European Union  or comparative exposure or risk assessments  to point-of-exposure . This can be done using comparative genomics of isolates collected at successive points or stages of the farm-to-fork continuum . Such anCampylobacter, E. coli and Salmonella populations circulating on-farm, at abattoir and at retail for major livestock species or their meat products  clustering with those found at step n. The sensitivity of this probability estimate as a function of the clustering threshold could also be explored. The uncertainty around the probability estimate could be assessed using bootstrapping as proposed by Second, a quantitative outcome describing the probability of a hazard to be transmitted from one step of the food chain to the next is needed. L. monocytogenes in a meat establishment has already been described . Just like any other molecular comparative analyses, direct interpretation of the observed relationships may be hampered by the complexity of the food chain, with food products being increasingly produced, processed, transformed and consumed in different regions or countries and within different timeframes (as influenced by the product shelf life) .As a simple, deterministic example, if WGS analyses of surveillance data revealed that only half of direct hazards in retail beef are genetically related to isolates collected in on-farm cattle, then the probability of the hazard to be transmitted from the farm to retail product would be 50%. This would have profound implications of the evaluation of interventions in the exposure assessment, as interventions modeled on-farm would only have an impact on 50% of the population of the hazard that humans are exposed to in retail beef, in this hypothetical example (assuming that the other 50% of the hazard population sampled in retail beef have a different origin). Therefore, in a purely linear model, any intervention that reduces prevalence of the hazard by 90% on-farm would only reduce overall exposure to humans from beef consumption by 45%.E. coli and from E. coli to environmental bacteria in the food-processing chain) has also been described  to a vancomycin-susceptible E. faecium of human origin in the intestines of three out of six human volunteers (In those cases where risk assessors decide to focus on the AMR gene(s) as the hazard of interest, and if AMR profiling via WGS shows that the AMR gene(s) are located on MGEs , then horizontal transfer of AMR genes should preferably be included in the exposure assessment step of a QMRA. This will contribute to providing a comprehensive depiction of the exposure pathways . Horizonescribed . Plasmid minutes . Followi minutes . For exalunteers .E. coli in the large intestine of cattle) that include horizontal transfer of plasmids between bacteria in the presence or absence of antimicrobial treatment have been developed  which is a critical piece of information, but also help to better characterize those plasmids. For example, WGS can be used to identify the incompatibility group or replicon type of a plasmid, which usually correlates with the dynamics and efficiency of horizontal transfer, as well as the plasmid host-spectrum . WGS canAs part of the hazard characterization step of a foodborne AMR QMRA, risk assessors aim to translate levels of exposure to the hazard into a probability of one or more adverse health outcomes in humans . Wherevein vitro model of the human gastrointestinal tract, S. Heidelberg and S. Typhimurium in the gastrointestinal tract and ability to cause infection was highly variable  before reaching a suitable site for colonization, attachment and invasion that will eventually lead to infection and subsequently to disease . Dose–revariable . WGS provariable .Once the dose is adjusted, the probability of infection (or disease) could further be refined using WGS profiling of the pathogenicity of the hazard, with highly pathogenic strains requiring lower doses to cause infection or disease . HoweverS. Heidelberg in Canada attributable to prior antimicrobial consumption in humans  prior to ingestion of a contaminated product. This refers to the notion of ‘etiologic fraction’ for prior antimicrobial use, defined by the Institute of Medicine as the proportion of human cases that would not occur but for the resistance of the infecting bacterial strain to the antimicrobial(s) being administered to the patient prior to infection . Human un humans . Howevern humans .TEM) gene copies present in food, but there are currently no data to convert a number of gene copies into a probability of infection or illness , and better characterize the expected response (based on pathogenicity and AMR profiles), WGS focuses on the characterization of individual isolates and therefore cannot provide any quantitative estimates of the dose being ingested initially . Other genomic approaches such as metagenomics , is likely to help in this regard. For example, bioinformatic analytical pipelines, such as Resfinder, SEAR or AMRplusplus can be used to quantify the abundance of AMR genes in metagenomes of diverse origin, e.g., animal feces or farm effluents . However illness .Salmonella infections between 1996 and 2006 in the United States revealed significant differences among serovars in terms of case-fatality rates, hospitalization rates and ability to cause invasive disease  to a cluster-based  or a strain-based approach  . Insteadl origin . Expresspic data .E. coli  via WGS makes it possible to predict the phenotypic behavior of a hazard, and therefore to adjust the model parameters accordingly. For example, predicting the hazard’s ability to grow during processing, transport and storage will provide a more accurate estimate of the number of cells ingested at the point of consumption. Consequently, incorporation of WGS data into QMRA models will help reduce the uncertainty in the final risk estimates. Yet, there is some uncertainty in the degree of correlation between genotypic and phenotypic profiles. The detection of genes or genetic markers does not mean this gene or genetic marker will be functional or expressed. Previous literature has shown very high correlation  between AMR WGS and phenotypic profiles among E. coli , Staphyls aureus , Campyloter spp.  and non-enterica . WGS evelmonella . Howevera toxins .S. Enteritidis and S. Typhimurium, both being highly clonal pathogens  following a voluntary withdrawal of ceftiofur use, and an increase in resistance (from 7 to 20%) after reintroduction of ceftiofur use  are unlikely to provide additional information. Comparison of WGS profiles of S. Heidelberg populations over time, however, would enable the assessment of the effect of withdrawing ceftiofur use on the prevalence of ceftiofur-resistant S. Heidelberg in broiler chicken meat in Canada to be refined. For example, WGS could help to describe whether a single bacterial population underwent resurgence after reintroduction of ceftiofur use, or whether the original ceftiofur-resistant S. Heidelberg population died out and a second population took over after ceftiofur use reintroduction.As an example, CIPARS reported that changes in the frequency of isolates resistant to ceftiofur  among chicken ofur use . These oThe integration of WGS data into QMRA modeling represents a critical step toward the development of next generation QMRA of foodborne AMR. Yet, there are a number of challenges that need to be addressed before the transition can be effective. There is still a lack of knowledge translation between bioinformaticians and risk assessors, with bioinformaticians having a limited understanding of the type of information required for QMRA modeling, and risk assessors having poor comprehension of how WGS data are analyzed, including what type of information can be generated and any limitations on these analyses. Risk assessors and epidemiologists need to be better trained in next generation sequencing techniques and molecular epidemiology . Risk as1 combines several free tools for rapidly performing simultaneous analyses of draft Salmonella genome assemblies, including serovar prediction and sequence-based typing analyses , as well as metadata driven visualization to examine the phylogenetic, geospatial and temporal distribution of genome-sequenced isolates in comparison with a database comprising over 4,000 publicly available genomes  are influenced by the microbiota present in food, food environments or gastrointestinal tracts of humans and animals. Yet, metagenomics approaches also come with new challenges. While WGS rely on ‘clean’ genome sequence analyses, metagenomics require ‘noisy’ sequencing efforts where pathogenic bacteria only represent a minute amount of the bacterial community in a sample, so enrichment or removal of DNA from other sources are needed to improve data quality; these procedures introduce significant bias in the interpretation of metagenomics analyses . Additiomcr-1 gene , which codes for plasmid-based resistance to polymyxin in Gram-negative bacteria (polymyxin is one of the few available drugs for treating infections caused by carbapenem-resistant Enterobacteriaceae)  . A novelin China . Whole pa (mcr-1 . The rapntinents , and subxpansion . This exThe integration of WGS data into foodborne AMR QMRA modeling offers the opportunity to move toward the next-generation of AMR risk assessment. Instead of considering all hazard strains as equally able to cause disease, WGS can substantially improve the hazard identification by focusing on those strains of highest public health relevance and/or stratifying the hazards into subsets of similar genetic profiles that are expected to behave similarly. The distribution of QMRA model parameters, including growth/survival and pathogenicity/virulence can be adjusted accordingly, making it possible to capture the variability in the strains of interest while decreasing the uncertainty in some model input parameters. The high discriminatory power of WGS also offers an opportunity to improve the exposure assessment by analyzing the degree of relatedness between bacterial populations and AMR profiles found at successive stages of the food chain. Overall, WGS can contribute to substantial improvements to the accuracy of QMRA models, and should be considered in any future revision of the Codex Alimentarius Guidelines for Risk Analysis of Foodborne AMR. WGS data, however, also introduce new sources of uncertainty, especially related to the thresholds of relatedness to be used and the degree of correlation between genotypic and phenotypic profiles. The latter could be improved using a functional approach of microbial behavior based on a combination of omics techniques.LC wrote the initial draft of the manuscript. PB, CC, BC, AF, BH, SM, EP, RR-S, ET, and BS provided substantial comments and edits to improve the text and figures and prepare the final version of the 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.