Sinomenium acutum, sinomenine is traditionally used in China and Japan for treating rheumatism and arthritis. Previously, we have demonstrated that sinomenine possessed a broad analgesic spectrum in various chronic pain animal models and repeated administration of sinomenine did not generate tolerance. In this review article, we discussed sinomenine's analgesic mechanism with focus on its role on immune regulation and neuroimmune interaction. Sinomenine has distinct immunoregulative properties, in which glutamate, adenosine triphosphate, nitric oxide, and proinflammatory cytokines are thought to be involved. Sinomenine may alter the unbalanced neuroimmune interaction and inhibit neuroinflammation, oxidative stress, and central sensitization in chronic pain states. In conclusion, sinomenine has promising potential for chronic pain management in different clinical settings.Purified from the roots of the plant  Sinomenium Acutum, sinomenine was originally found to be effective in treating rheumatism in Japan since the early 1930s  naloxone in Chinese hamster ovary cells transfected with the opioid μ-receptor [μ-receptors may be relevant to its ability to prevent the development of morphine tolerance [A) receptor [There is evidence suggests sinomenine is an opioid receptor . Sinomenolerance . Howeverolerance , 8, sincreceptor  or blockreceptor , could aHistamine has been known to act on sensory neurons to produce itch, and histamine-induced itch converts to pain in states of neuropathic hyperalgesia . In rodeSinomenine has a potent histamine-releasing ability associated with degranulation of mast cells in mammalian tissues . In ordeNitric oxide (NO) is a messenger molecule synthesized by three isoforms of nitric oxide synthases (NOSs), which diffuses from the site of production across cellular membranes. All three isoforms of NOS are present in the nervous system, namely, the constitutive neuronal (nNOS), endothelial (eNOS), and the inducible (iNOS) isoforms. Under normal conditions, iNOS is mainly absent in neural tissues but is upregulated during inflammation, while eNOS is present in the brain vasculature and nNOS is present in the spinal cord .There is an essential role played by the NO system in the development of chronic pain. Clinically, a significant increase in the plasma levels of nitrate/nitrite concentrations (used as an indicator of NO synthesis) has been noted in chronic pain patients in comparison with healthy individuals. In animal studies, upregulation of nNOS at messenger RNA or protein levels has been detected in large neurons of laminae III and IV after sciatic nerve transection (SNT), while the selective inhibitor of nNOS blocked hyperalgesia and allodynia following SNT . NO can Notably, beneficial effects of nonselective NOS inhibitors were limited, as they also suppress eNOS expression and therefore antagonize its protective effects. In certain circumstances, the nonselective NOS inhibitor even aggravates ischemic pain . SinomenOnce a nerve is damaged, Wallerian degeneration occurs that subsequently results in activation of Schwann cells and an influx of inflammatory cells into injured nerve tissue. These infiltrated cells produce a wide array of inflammatory mediators. Cyclooxygenase 2-dependant prostaglandin E2 (COX2/PGE2) is one of the most important mediators, which abundantly produced in the nerve injury site and involved in the genesis of neuropathic pain. COX2/PGE2 can promote neuropathic pain by upregulating the iNOS in macrophages, which could be reversed by nonselective COX inhibitors . BesidesSinomenine can potently suppress the pronociceptive PGE2 synthesis through COX2 inhibition in lipopolysaccharide-treated macrophages  and in e+ ion conductance in a non-voltage-gated fashion leading to overall neuronal hyperexcitability and hence facilitates neuropathic pain [The tumor necrosis factor (TNF) is an inflammatory cytokine that involved in the development of neuropathic pain. Using the standard model of chronic constriction injury (CCI) of the sciatic nerve in rats, TNF has been detected at the injury site and shows temporal upregulation . Intereshic pain .α2 receptor. During chronic pain, inhibition of norepinephrine release in the hippocampus by TNF is significantly enhanced, which could be reversed by the TNF blockade. Interestingly, the norepinephrine inhibitory function of TNF transforms to facilitation following chronic administration of antidepressant drugs or during the natural dissipation of hyperalgesia [Besides, multiple studies have demonstrated that clinically administration of agents that antagonize TNF-attenuated chronic pain , 47. Othralgesia .A clinical study also indicated that nerve biopsies of human patients with painful neuropathy show higher levels of TNF expression, especially in Schwann Cells . In addiγ) is an inflammatory cytokine that associated with a number of autoimmune diseases. The immunemodulatory role of INF-γ involves activation of macrophages to produce NO/TNF and upregulates the major histocompatibility complex (MHC) antigens [γ can facilitate the nociceptive flexor reflex in rats [γ can induce thermal hyperalgesia [γ has participated in the establishment of chronic pain. Spinal microglia cells express the receptor for INF-γ (INF-γR), which converts microglia into activated cells when being stimulated and produces a long-lasting pain hypersensitivity [γR severely impairs periphery nerve injury-evoked microglia activation and tactile allodynia without affecting microglia in the contralateral dorsal horn or basal pain sensitivity [γ in patients with mesangial proliferative nephritis [γ and antibody production in spleen cells of CIA rats [γ level.Interferon gamma  in the infraorbital nerve of rats, levels of IL-6 and IL-1β in the ventromedial medulla (RVM) were increased [β into RVM increased NR1 phosphorylation of the NMDA receptor and subsequently generated hyperalgesia, which could be reversed by an NMDA antagonist [β than healthy subjects [β and IL-6 in macrophages and decrease the serum concentrations of IL1-β and IL-6 in CIA rats [β, thereby suppressing the activation of microglia cells.In response to never injury, microglia transforms into macrophage-like cells that express MHC antigens to secrete proinflammatory cytokines including IL-1ncreased . Injectitagonist . In additagonist . Furthersubjects . SinomenCIA rats . It is pIL-4 is a peptide that has both proinflammatory and antiallodynic properties. Inoculation of vector S4IL4 to express IL-4 in DRG neurons 1 week before sciatic nerve ligation (SNL) delayed the onset of thermal hyperalgesia and tactile allodynia in rats but did not prevent the ultimate development of chronic pain manifestations . Treatmeβ or substance P and prevents the development of neuropathic pain [P38 mitogen-activated protein kinases (p38-MAPKs) are the mammalian orthologue of the yeast high-osmolarity glycerol response kinase which participates in a signaling cascade controlling cellular responses to cytokines and stress. Stimulation of microglia by proinflammatory cytokines and spinal astrocytes activation is mediate via the p38-MAPK system and directly contributed to central sensitization . For inshic pain , 57, 58.Following treatment with sinomenine, a significant decrease of the p38 MAPK activity has been seen in activated RBL-2H3 cells . After nMatrix metalloproteinases (MMPs) are zinc-dependent endopeptidases which degrade various kinds of extracellular matrix proteins. They are known to be involved in the synthesis of apoptotic ligands, chemokines, and cytokines . Recent κB) is a key regulator of inflammatory responses. Inhibiting NF-κB activity is beneficial in controlling both pain and inflammation. For instance, transgenic inhibition of NF-κB in glial fibrillary acidic protein (GFAP) expressing glial cells attenuated pain and neuronal inflammation after peripheral nerve injury [κB [β by inhibiting the NF-κB binding activity, through upregulation of the inhibitor of kappa B (IκB-α) expression in peritoneal macrophages and synoviocytes [κB activity in nerve injury sites and improve neuropathic pain conditions.The transcription factor nuclear factor kappa B  are chemically-reactive molecules containing oxygen. ROS are generated as a natural byproduct of the normal metabolism of oxygen and have important roles in cell signaling. However, during environmental stress , ROS levels can increase dramatically and result in significant damage to cell structures. This phenomenon is known as oxidative stress.Recent experimental data suggest that ROS are involved in the maintenance of chronic pain. In rats with neuropathic pain, the mechanical allodynia developed after nerve ligation was significantly reversed after treatment with ROS scavengers. In addition, the superoxide dismutase (SOD) mimetic which converts free-radical superoxide to hydrogen peroxide was very effective in reducing inflammation and hyperalgesia after carrageenan injection into the rat paw . MoreoveCentral sensitization is defined by increased responsiveness of dorsal horn neurons to nociceptive peripheral stimulation . BehavioκB activation and proinflammatory cytokine production [Sinomenine exhibited anti-inflammatory properties by reducing superoxide ions, inhibiting the microglial nicotinamide adenine dinucleotide phosphate (NADPH) oxidase  and upreoduction .Protein kinase B (PKB) is a serine protein kinase which can be stimulated by ROS to protect cells against oxidative stress. Studies have shown that PKB is activated in response to neuropathic pain, which subsequently acts on sensory neurons to stimulate the expression of nociception-related genes . SinomenPain signal is generated in the peripheral nerve terminals, through the afferent sensory nerve fibers, transmitted into the DRG , and then transduced by the nerve fibers to the dorsal horn of the spinal cord. In the dorsal horn of the spinal cord, pain signals usually require pain-relay neurons to enter the higher levels of the nervous system  to complete the process of pain perception . Here, wIn DRG (as shown in a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors on the postsynaptic membrane, generating sustained activation in pain-relaying neurons, as well as producing cytotoxicity at the same time. In the dorsal horn, sinomenine can reduce the excessive ATP and glutamate release while enhancing GABAergic neurotransmission. These effects will subsequently help to suppress the production of inflammatory cytokines from microglial cells and astrocytes and decrease their active states . In turn, the dorsal horn neurons will be protected from harmful levels of glutamate and proinflammatory cytokines, and the ongoing sensitization in the same neurons will be suppressed.In the spinal dorsal horn . Although the application criteria of sinomenine need to be further verified before its widely usage, we conclude that sinomenine has promising potential for chronic pain management in different clinical settings.To summarize, sinomenine is a rare anti-inflammatory and antihyperalgesic substance that can act on both peripheral and central nervous systems. Although it possessed different types pharmacological properties, no clear drug target has been found. So, sinomenine is likely to be a typical multitarget active substance (purified from TCM). To further describe the state of the art on the pro's and con's of potential sinomenine use in chronic pain therapy, we introduced a notion of “pharmacological cloud” (as shown in


Early stages of the novel coronavirus disease (COVID-19) have been associated with ‘silent hypoxia’ and poor oxygenation despite relatively small fractions of afflicted lung. Although it has been speculated that such paradoxical findings may be explained by impairment of hypoxic pulmonary vasoconstriction in infected lungs regions, no studies have confirmed this hypothesis nor determined whether such extreme degrees of perfusion redistribution are physiologically plausible. Here, we present a mathematical model which provides evidence that the extreme amount of pulmonary shunt observed in patients with early COVID-19 is not plausible without hyperperfusion of the relatively small fraction of injured lung, with three-fold increases in regional perfusion to afflicted regions. Although underlying perfusion heterogeneity  exacerbated existing shunt in the model, the reported severity of hypoxia in early COVID-19 patients could not be replicated without considerable reduction of vascular resistance in nonoxygenated regions. Patients initially present with a “Type L” phenotype characterized by “Low” lung stiffness  despite poor oxygenation. Type L may transition into a frequently fatal “Type H” phenotype characterized by “High” lung stiffness (reduced compliance) accompanied by features reminiscent of conventional acute respiratory distress syndrome (ARDS) 1. Although a strict dichotomy of phenotypes based on respiratory compliance is controversial 2, there are nonetheless frequent reports of severe hypoxia in patients with only a small fraction of nonaerated lung 5, including so-called ‘silent hypoxia’. Thus the early stages of COVID-19 appear to be unique and poorly understood, manifesting in the lung as peripheral lesions characterized by ground-glass opacification on computed tomography (CT) 7. Curiously, the fraction of lung affected in this way is often surprisingly low given the severity of the associated hypoxia and estimated shunt fractions (average 50%) 8. If one assumes that ground-glass opacification represents lung that is nonventilated, these CT studies imply abnormally high ratios of shunt fraction to nonaerated lung fraction of 3.0 for COVID-19 compared to 1.3 for ARDS 8.Gattinoni et al. described disease due to the novel coronavirus (COVID-19) as following two stages, or phenotypes 8. Accordingly, Gattinoni et al. hypothesized that their seemingly paradoxical observations of hypoxia and high compliance were related to an underlying impairment of hypoxic pulmonary vasoconstriction (HPV) 8. Normally, HPV is a feedback mechanism whereby pulmonary arterioles constrict in lung regions with poor oxygenation 9. This response results in increased regional vascular resistance, reduced regional perfusion, and thereby reduced overall shunt fraction. It seems reasonable, therefore, that an impairment in HPV might upset the balance between ventilation and perfusion enough to explain the intriguing clinical findings that have been reported in COVID-19 patients.A possible interpretation of the Type L phenotype is that a disproportionately large fraction of the pulmonary circulation is being directed through nonaerated lung inj) with impaired oxygen transport, what increase in regional blood flow and hence vasodilation would be necessary to manifest a ratio of shunt fraction (Fshu) to Finj above 3? What are the limitations on oxygen diffusion in injured lung that would be compatible with clinical findings? What role might gravitational gradients play? In the present study we used a mathematical model of perfusion and oxygen transport to address these questions with the goal of determining if the altered HPV hypothesis can potentially explain the Type L phenotype of COVID-19, or whether we need to look for an alternative explanation.Whether the above explanation is actually plausible, however, requires a quantitative analysis of the factors responsible for determining shunt fraction, ventilation maldistribution, and their relationship to each other. For example, given a certain fraction of injured lung  impaired HPV function produced no change in resistance; and 3) “reversed” HPV reduced resistance regardless of oxygenation. Measured outcomes included Fshu, ratio Fshu:Finj, and ratio PaO2:FiO2.The lung model was partitioned into 6 compartments , represeshu:Finj with respect to injury location and HPV alterations is shown in aO2:FiO2 < 300 mmHg %−1 until Finj > 30%. Given a relatively small fraction of injured lung, with Finj ranging from 0 to 30%, both a complete shunt  and “reversal” of HPV  are necessary conditions for Fshu:Finj > 2 and PaO2:FiO2 < 300 mmHg %−1. By contrast, impairment of HPV alone is not sufficient to produce the same level of severe hypoxia at low values of Finj as found by Gattinoni et al. 8. With HPV impairment, Fshu more closely follows Finj such that the ratio of Fshu to Finj lies between 0.7 and 1.3. For all considered alterations to HPV, focusing the injury in the lower zone  results in higher Fshu and worse hypoxia. Interestingly, as Finj decreases in the reversed HPV model, the Fshu:Finj ratio increases, indicating that Fshu decreases more slowly than Finj. Note that the impaired HPV model represents unaltered vascular resistances from baseline values, and therefore corresponds to a model with relatively uniform perfusion distribution.The variability in Fshu:Finj = 3 is represented in each panel by contours of Fshu at 30, 60, and 90% for Finj at 10, 20, and 30%, respectively. Note that baseline perfusion gradient does not explicitly require the definition of upright vs. supine vs. prone positioning, but instead simply reflects discrepant perfusion in 3 arbitrary lung compartments.The interplay between baseline perfusion gradients and vasodilation in the HPV reversal model is shown in Our analysis based on a simple mathematical model of perfusion in normal and shunted compartments suggests that the extreme amount of pulmonary shunt observed in patients with early stage severe COVID-19 is not plausible without hyperperfusion of the relatively small fraction of injured lung, with up to 3-fold increases in regional perfusion to the afflicted regions. Furthermore, the obstruction to oxygen diffusion in the injured regions must be nearly complete such that there is less than 5% equilibration between alveolar gas and end-capillary blood.8, characterized by severe hypoxia but relatively normal lung compliance, cannot be recapitulated in this model without dramatic reductions to vascular resistance in the injured regions. To replicate the reported values for Fshu of 50% and Fshu:Finj of 3 (implying Finj of 17%), the model requires reductions in injured resistance of 60 to 70%, depending on the baseline perfusion gradient  14, it is possible that earlier stages instead promote local vasodilation or impairment of HPV 15. Other evidence of vasodilation due to COVID-19 includes recent discovery of cardiovascular complications reminiscent of vasodilatory shock and Kawasaki disease 16, which is associated with weakened walls of the coronary artery.The Type L early stage of COVID-19 described by Gattinoni et al. gradient . Approxishu:Finj > 2 in our model. Instead, the Fshu:Finj ratio in the impaired HPV model is limited by the magnitude of the baseline perfusion gradient. With zero baseline perfusion gradient and impaired HPV, vascular resistance is uniform across all compartments, and thus the fraction of shunted blood flow is equal to the fraction of lung with impaired oxygen transport (Fshu:Finj = 1). Heterogeneous perfusion may increase the risk for larger Fshu, especially when the injured region also receives more baseline perfusion , Fshu:Finj in the model is still limited to 2 at most. For example, in shu:Finj does not exceed 3 even at 100% baseline perfusion gradient until the resistance reduction is 40% for Finj = 10%, or 55% for Finj = 20%. Therefore it appears unlikely that the degree of pulmonary shunt reported in COVID-19 patients (Fshu = 50% and Fshu:Finj = 3) could occur without a substantial degree of vasodilation and hyperperfusion in the small fraction of injured lung, even when considering the possibility that one-third of the lung is physiologic dead space.In COVID-19, the lower left and lower right lobes are most commonly afflicted according to radiographic abnormalities 19. Pulmonary embolism may reduce or eliminate perfusion to well-aerated or ventilated lung regions, resulting in physiologic dead space and redistribution of perfusion to other lung regions. This alone does not necessarily produce hypoxia, but can exacerbate hypoxia if perfusion is redistributed to regions of pulmonary shunt. In our simple model, a baseline perfusion gradient of 100% corresponds to zero perfusion in one-third of the lung, which may be interpreted similarly to the result of severe thrombotic pulmonary emboli. Even in this case, Fshu:Finj is still limited to at most 2 with only impaired HPV, and vasodilation in injured regions remains necessary to explain Fshu:Finj ≥ 3. A case report using dual-energy CT demonstrated no indications of pulmonary emboli in the well-aerated lung, but rather vasodilation of pulmonary arteries and hyperperfusion adjacent to infected regions 5.It should be noted that the model assumptions do not require gravity to explain the presence of a baseline perfusion gradient. Given arbitrarily defined compartments, differences in regional perfusion could also represent conditions manifesting abnormal perfusion defects such as pulmonary emboli. Coagulation and thrombosis have been identified as symptoms of COVID-19, and in many cases are associated with mortality due to stroke, myocardial infarction, or pulmonary embolism shu:Finj may be obtained at a lower level of vasodilation in the injured region , compared to the 60 to 70% reduction required without considering any physiologic dead space. Another factor that may contribute to systemic hypoxia is increased oxygen uptake by lung tissues, which may account for up to 20% of total oxygen metabolism in patients with lung injury compared to only 5% at baseline 21.Compounding the effects of large perfusion defects with “reversed” HPV, a given ratio of Fgion see . A recen3 may reflect the Type L phenotype. It is intriguing that hypoxia in these patients is not associated with hypercapnia. One might assume that severe pulmonary shunt would produce deficiencies in both oxygen and carbon dioxide exchange. Hypercapnia is less likely to be “silent”, considering that even small increases in arterial carbon dioxide tension elicit feedback response from pH-sensitive central chemoreceptors to increase respiratory drive. Although our model suggests that oxygen equilibration must be less than 5% to produce high ratios of Fshu:Finj, it is possible that elimination of carbon dioxide is less impaired given that diffusion of carbon dioxide across the alveolar-capillary membrane is roughly 20-fold faster than that of oxygen 3.Reports of “silent hypoxia” in early stages of COVID-19 ClinicalTrials.gov identifiers NCT04290871, NCT04304313, NCT04332666). Notwithstanding other systemic effects of pharmacological interventions, our model suggests that vasodilation throughout the noninjured lung may counterbalance disease-induced vasodilation in the injured regions, restoring a more uniform baseline perfusion distribution similar to the impaired HPV model. Vasodilation may also reduce the severity of thrombotic pulmonary emboli, thereby reducing physiologic dead space and perfusion heterogeneity. Prone positioning is another intervention which may reduce baseline perfusion gradients compared to supine positioning 22, and is also reportedly beneficial for COVID-19 patients 24. Therefore, our model may explain a mechanism by which pulmonary vasodilators and prone positioning may improve ventilation-to-perfusion matching and reduce hypoxia, perhaps providing palliative care for COVID-19 patients.Pulmonary vasodilators such as inhaled nitric oxide, sildenafil, and angiotensin- are currently involved in clinical trials for treatment of COVID-19  where there is only a small fraction of injured lung and before the initiation of mechanical ventilation or other respiratory support. The purpose of the model was to quantitatively assess the plausibility of the hypothesis that severe hypoxia in early COVID-19 is the result of hyperperfusion within a small amount of injured lung. The model demonstrates that vasodilation of injured, unoxygenated regions appears to be a necessary feature of early COVID-19 to explain the reported severity of hypoxia, with or without shunt amplification by thrombotic pulmonary emboli.It should be noted that no consensus has yet been established for these interventions, current evidence is largely anecdotal, and the theories proposed herein based on our simple model are speculative. The direct implications of this study are furthermore limited to palliative care, and cannot be applied to identify or verify underlying viral mechanisms that initiate or progress the disease, or to explain why asymptomatic patients exhibit radiographic indicators of viral pneumonia 25. Each compartment was perfused, receiving deoxygenated mixed venous blood and returning end-capillary blood with oxygen content determined by injury severity. The model described time-averaged gas exchange, i.e., neglecting within-breath and within-beat fluctuations. Normal lung compartments had normal oxygen diffusion such that end-capillary oxygen tension (PcO2) equilibrated with alveolar oxygen tension (PAO2). Injured lung compartments had limited or zero oxygen diffusion such that PcO2 was either equal to mixed venous oxygen tension (PvO2) or a weighted average of PvO2 and PAO2:The lung model was partitioned into 6 compartments , represevO2 = 40 mmHg and PAO2 = 100 mmHg, representing patients upon admission without supplemental oxygen. These values result from the alveolar gas equation, with 21% inspired oxygen, 47 mmHg water vapor pressure at 37 C, 40 mmHg arterial carbon dioxide tension, and 0.8 respiratory quotient:Note that in the normal lung compartments, B was assumed to have a value of 1. A value of 0 for B corresponds to a complete shunt with no oxygen diffusion. Oxygen tensions were assumed to be Pbas) were determined to establish a baseline perfusion gradient in 3 equal-sized normal compartments . Baseline perfusion gradient was defined as half the range of perfusion across all height levels divided by the average. Baseline vascular resistance (Rbas) at each height level (h), relative to baseline total pulmonary vascular resistance (PVRbas), was determined as follows:tot is total pulmonary perfusion , and Qbas is baseline perfusion at each height level. Vascular resistance was then adjusted in injured regions to reflect possible abnormalities arising in COVID-19. Three types of modification were examined: 1) normal HPV function increased resistance exponentially in regions with low PcO2; 2) impaired HPV function produced no change in resistance; and 3) “reversed” HPV reduced resistance by a factor 0 < K < 1 regardless of oxygenation. The following equations were used:inj is resistance of the injured compartment. Note that resistances were defined in a volumetric manner, such that the effective compartmental resistance was inversely proportional to the fraction of lung it represented. Following these HPV modifications and now accounting for an injured compartment at each height level with altered vascular resistance, total pulmonary vascular resistance (PVR) was computed by the parallel combination of compartmental resistances.Perfusion distribution in the model reflected the relative vascular resistance in each compartment. First, baseline resistances  was computed based on PcO2 for each compartment:cO2 is oxygen saturation of hemoglobin determined by the Severinghaus equation fit to the oxygen-hemoglobin dissociation curve 26:Perfusion to each n’th compartment was then allocated in inverse proportion to compartmental resistance.vO2) was calculated in the same manner. Mixed arterial oxygen content (CaO2) was then determined as a perfusion-weighted average of compartmental end-capillary oxygen contents.Mixed venous oxygen content (Cshu) was defined as the ratio of unoxygenated to total blood flow:c*O2 represents a normal(noninjured) compartment.Shunt fraction (Fshu, ratio Fshu:Finj, and ratio PaO2:FiO2. Simulations were conducted over a range of Finj from 0 to 30%. For example, 15% of the lower lung zone injured reflects an overall Finj of 5%, because the lower zone represents 1/3 of the total lung. For simplicity, the extent of injury in each simulation was restricted to only 1 height zone: lower, middle, or upper. Baseline perfusion gradient varied between 0 and 100% representing a range of perfusion heterogeneity from uniform with ⅓-⅓-⅓ distribution at 0% gradient to 0-⅓-⅔ distribution at 100% gradient. The degree of limited oxygen diffusion in the injured compartment varied between 0 and 20%, where 0% represents complete shunt and 100% represents complete equilibration with alveolar gas.Measured outcomes included FA Matlab script for evaluating the mathematical model described herein is available as Supplement


Trichechus manatus latirostris) mortality event following a red tide bloom in Southwest Florida, an RNA sequencing experiment was conducted. Gene expression changes in white blood cells were assessed in manatees rescued from a red tide affected area (n = 4) and a control group (n = 7) using RNA sequencing. The genes with the largest fold changes were compared between the two groups to identify molecular pathways related to cellular and disease processes. In total, 591 genes  were differentially expressed in the red tide group. Of these, 158 were upregulated and 433 were downregulated. This suggests major changes in white blood cell composition following an exposure to red tide. The most highly upregulated gene, Osteoclast associated 2C immunoglobulin-like receptor (OSCAR), was upregulated 12-fold. This gene is involved in initiating the immune response and maintaining a role in adaptive and innate immunity. The most highly downregulated gene, Piccolo presynaptic cytomatrix protein (PCLO), was downregulated by a factor of 977-fold. This gene is associated with cognitive functioning and neurotransmitter release. Downregulation of this gene in other studies was associated with neuronal loss and neuron synapse dysfunction. Among the cellular pathways that were most affected, immune response, including inflammation, wounds and injuries, cell proliferation, and apoptosis were the most predominant. The pathway with the most differentially expressed genes was the immune response pathway with 98 genes involved, many of them downregulated. Assessing the changes in gene expression associated with red tide exposure enhances our understanding of manatee immune response to the red tide toxins and will aid in the development of red tide biomarkers.To investigate a Florida manatee ( Trichechus manatus latirostris) mortalities (n = 830) were reported, with one-third of those (n = 276) attributed to prior exposure to an expansive and prolonged red tide bloom. Historically, the majority of red tide blooms in Florida occurred in the southwest part of the state with Sarasota serving as the epicenter [Karenia brevis (K. brevis), ‘blooms’ and emits algal neurotoxins, which have been responsible for numerous mortality episodes of manatees and other wildlife in the past. In free-ranging manatees rescued from red tide conditions, decreased lymphocyte proliferation responses have been observed [In 2013, a record number of Florida manatee  and the Florida subspecies (Trichechus manatus latirostris) are listed as threatened by the Endangered Species Act and the state of Florida [K. brevis, undergoes a rapid population growth under favorable environmental conditions, red tides can form quickly and begin releasing toxins termed “brevetoxins” into the environment in higher concentrations. The brevetoxins released during red tide blooms can have large consequences to fish, seabirds, marine mammals, and humans [The West Indian manatee  experiment was conducted. Differential gene expression of message RNA (mRNA) was compared in manatees rescued from red tide exposure and a non-exposed control group from geographically separated areas, not impacted by red tide . The mRNA transcripts in an organism comprise the transcriptome or the collection of all transcribed RNAs and these can be used to determine an organism’s health status at any given time . Using tlater  immediately in the field, to preserve the buffy coat samples in a ratio of 1.2 mL RNAlater to <0.5 mL buffy coat according to the manufacturer’s protocol (Life Technologies™) and samples were refrigerated at minus 20°C, as recommended by the manufacturer.Manatee white blood cell ‘buffy coat’ samples were opportunistically obtained from two groups (red tide and control) and analyzed using RNA-Seq to detect changes in gene expression . The redPrescreening was done to ensure control manatees did not have any indication of a preexisting illness or trauma, such as cold stress or boat-related injuries. Clinical data from the manatee health assessments were obtained and reviewed to prescreen manatees for inclusion in this study based on blood chemistries and acute phase protein serum amyloid A (SAA) values. SAA measurements were performed on manatee serum by the University of Miami Avian & Wildlife Laboratory  following their published protocol . ResearcPlasma was separated from whole blood via centrifugation at 500 x g, 15 min. Brevetoxin was measured in plasma using modifications of a competitive enzyme linked immunosorbent assay (ELISA)  previous™ 1000  to assess 260/280 and 260/230 ratios. To remove DNA contamination, the samples were DNase treated using the TURBO™ DNA-free™ kit according to the manufacturer’s protocol (Life Technologies™). After DNase treatment, Qiagen RNeasy columns were used to further purify the samples . The Agilent 2100 Bioanalyzer  was used to obtain RNA integrity (RIN) values. Most RIN values ranged from 7.3–9.4, with the exception of one that had a RIN value of 5.9 . The purity of the samples was determined using a NanoDrope of 5.9 . RNA-Seq® EpiCentre ScriptSeq™ Complete Gold (Blood)-Low Input Kit  was used along with the Illumina® FailSafe™ PCR Enzyme Mix and ScriptSeq™ Index PCR primers (set 1) was used for RNA processing and sequencing library preparation. This kit’s ‘Globin and Ribo-Zero’ reagents were designed to remove globin and rRNA from RNA samples . The Zymo Research RNA Clean and Concentrator-5  was used to purify the RNA (>200 nt) and concentrate the depleted samples. Individual libraries were made for each of the samples which were then pooled at equimolar concentrations. The library pool was prepared for sequencing following the manufacturer’s protocol. The Illumina® NextSeq® 500  sequencing platform was used to create paired-end reads using 2 X 150 cycles.The Illumina™ instrument and a paired-end whole-genome shotgun approach with partial annotation of the manatee genome [Several quality measures were performed using the FastQC  on Galaxe genome , 20. Fore genome , 20. Thee genome  was usede genome  in DEB [e genome  to genere genome . Digitale genome . For alle genome . Signifie genome  with the™ V9 (operating with the ResNet 9.0 database) was used to identify molecular pathways associated with exposure to red tide. The differentially expressed genes were further analyzed by subnetwork enrichment analysis (SNEA) to determine the cell processes involved [PathwayStudioinvolved –28. The The RNA-seq samples included in the downstream analysis (n = 11) had a mean number of 57 million reads per sample with a range of 44–70 million reads. The average assembled sequences across samples were 21,420 . The range in GC content was 40–44% with a mean GC content of 41%. After removal of redundant identifications, a total of 15,301 unique genes were identified by RNA-Seq. Of these, 15,260 mapped to human homologs with E score values <E-10, indicating good confidence in the annotation. A total of 636 genes had 100% match with human homologs and 14,575 had more than 50% identity with human homologs.In The RNA-seq data identified 2,149 genes that were differentially expressed with a p-value <0.05. Considering only adjusted p-values (FDR<0.05), there were 595 differentially expressed genes. The top 50 upregulated and downregulated genes with adjusted p-values are reported in The 10 upregulated genes with the highest fold change were Osteoclast associated 2C immunoglobulin-like receptor (OSCAR), myotubularin related protein 2 (MTMR2), Transmembrane protein 56 (TMEM56), thymocyte selection associated family member 2 (THEMIS2), matrix metallopeptidase 9 (MMP9), Haptoglobin (HP), Chemokine (C-X-C motif) receptor 2 (CXCR2), Chemokine (C-X-C motif) receptor 1 (CXCR1), monoacylglycerol 0-acyltransferase 2 (MOGAT2) and basic leucine zipper transcription factor 2C ATF-like (BATF) . Eight o™. This program identifies relationships between genes and disease based on knowledge harvested from millions of published articles in PubMed and compares the genes that are differentially expressed in an experiment to the knowledgebase using Fisher’s exact test to derive a statistical inference. The cellular pathway with the most differentially expressed genes (DEGs) was the immune response pathway with 98 genes involved .After comparing the red tide to the control group, the top 595 differentially expressed genes (both up and downregulated) were grouped into pathways involved in cellular and disease processes using PathwayStudioBrevetoxins are neurotoxins that primarily affect voltage-gated sodium channels in nerve cells . While tWhile the manatee genome sequence is not completely annotated, we used it as a scaffold for Illumina reads and then annotated the genes by BLAST against the human genome. The analysis provided excellent annotation by comparing sequences with human homologs. This great similarity between manatee and human transcriptomes allowed us to use pathway analysis to identify pathways most likely affected by the exposures. Because manatees are mammals, it is reasonable to assume that many of the basic pathways described for other mammals likely operate in the same way in manatees; although manatees probably utilize species-specific pathways as well. Transcriptomics analysis of white blood cells in the current study provided molecular data to support previous gross pathology of red tide-affected manatees. Bossart et al  found th−17 . PCLO is associated with neuronal loss and synapse, neurotransmitter release, and cognitive dysfunction [−2), is associated with both immunity and leukocyte activity. Lastly the MMP9 gene, which is associated with leukocyte migration, was upregulated . Functional information about each of the ten most up- or downregulated genes in mammals appears in the At the level of the individual gene changes in manatees, we observed changes in gene functions related to immune response , leukocyte production and differentiation , and neuronal activity and synapse . The most differentially expressed gene, piccolo presynaptic cytomatrix protein (PCLO), was downregulated  were collected from manatees within 24 hours of their arrival at ZooTampa for rehabilitation and two (#13 and #14) were collected from manatees after they received a one-week course of treatment for red tide-related illnesses at the Zoo. As expected, the two groups showed minimal separation from each other in the heat map , with thGene expression changes related to immune response, neurodegenerative processes, and inflammation were the most prominent observations in our study in response to brevetoxin exposure. This aligns with the research findings of other studies which showed that the health effects of red tide exposures are not only gastrointestinal and respiratory, but neurological as well, which is emerging as an increasing concern , 37. AddS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.


Background: The prognosis of patients with advanced gastric cancer remains overall poor despite some recent innovations and the development of new therapeutic approaches. Current European guidelines do not recommend any specific treatment for patients with advanced gastric cancer refractory to two or more previous chemotherapy regimens, making this setting “orphan.” Immunotherapy is quickly evolving also for this malignancy even if with controversial results and the correct patient selection is still debated, especially for Western patients. The phase III ONO-4538-12 “ATTRACTION-2” represents the current landmark trial for the development of immunotherapy for pretreated Asian patients and led to the approval of Nivolumab in some Asian countries, while only previous phase trials are available for Caucasians. Complete radiological response is anecdotic and has never been described both in the pivotal trial both in the others with Western patients enrolled.Case presentation: We report two cases of heavily pretreated Western elderly patients with metastatic gastric cancer who experienced durable complete radiological response to Nivolumab “off label” (more than 20 months to date) in a clinical practice context. Molecular analysis of potential predictive factors has been performed  on primary tumor sample.Conclusions: Despite the lack of evidence for Western patients and the controversial outcome with the use of checkpoint inhibitors in previous settings, immunotherapy may dramatically change the prognosis and the natural history of pretreated Western metastatic gastric cancer, in a correctly selected population. Microsatellite instability and tumor mutational burden may be reliable predictive factors also for Caucasians. There is an urgent need for a change in clinical practice also for this “orphan” patients and more efforts are needed in order to clarify the role of predictive factors for a correct patient selection and better chances of survival for this awful malignancy. The prognosis of patients with advanced gastric cancer (GC) remains poor and the median overall survival is still around 1 year despite some recent innovations and the development of new therapeutic approaches .Current European guidelines do not recommend any specific treatment for patients with disease refractory to two or more previous regimens. Anyway, the amount of patients who reach this setting is increasing and nowadays only some Asian countries and the US registered immunotherapy for advanced GC in this specific population –5.Immunotherapy is quickly evolving also for GC even if it is not yet a standard of care worldwide and the results are controversial. Furthermore, the correct patient selection remains a target not fully achieved, especially for Western patients.For Asian patients the phase III trial ONO-4538-12 “ATTRACTION-2”  represenMoreover, in first and second line setting immunotherapy did not significantly improve survival compared to chemotherapy both in Asian and Western patients in recent phase III randomized trials KEYNOTE-062  and KEYNWith regards to biomarkers selection, a recent prospective phase II trial with Pembrolizumab (anti-PD-1 antibody)  clarifieThe same molecular features (Mismatch repair and EBV status), also with PD-L1 expression and tumor mutational burden (TMB), have been recently analyzed in a single institution Japanese study with 80 patients treated with Nivolumab after two or more chemotherapy regimens . These mHowever, there are no results regarding the subgroup analysis according to MSI and programmed death-ligand 1 (PD-L1) status in the ATTRACTION-2 population.Genetic, epidemiologic, and molecular differences between Asian and western patients are well-known. Even if data from clinical trials are lacking for non-Asians, we should consider that there is some evidence that Western patients seem to have a more favorable “immune-environment” and a stronger immune-signature. In fact, a large global retrospective study with 1,600 GC samples from both Asian and non-Asian cohorts showed different tumor immune-signatures between Asian and non-Asian patients, particularly with infiltrating T-cells and T-cell gene expression patterns in Caucasians that might condition a possible more frequent and better response to immunotherapy , 14.off label” Nivolumab in a clinical practice context.Here we report two cases of elderly pretreated Western patients with metastatic GC who have experienced extraordinary and dramatic durable response with “In particular, we report the first radiologic complete responses to Nivolumab in Western patients with advanced pretreated GC at our knowledge, highlighting that immunotherapy is not a standard of care for GC and complete responses are anecdotal at current time and still not described even in the landmark phase III trial such as in the other mentioned studies with Western patients enrolled .Radiological evaluation for advanced disease has been performed every 3 or 4 months approximately using total body computed tomography (CT) with contrast with consecutive comparisons and response assessment performed according to response evaluation criteria in solid tumors (RECIST) v1.1 . A 18- fTreatment toxicity has been evaluated according to Common Terminology Criteria for Adverse Events (CTCAE) v5.0 .Histological and molecular analysis have been performed on the surgical sample of primary tumor in local laboratory according to current guidelines, with the exception of “FoundationOne CDx” test (a next generation sequencing based comprehensive genomic profiling offered to patients free of charge at our institution as companion diagnostic) .in situ hybridization for EBV, in addition to human epidermal growth factor receptor 2 (HER2) status evaluation, were performed in local laboratory according to the European Society of Medical Oncology recommendations  proteins status  and PD-L1 expression (tumor proportion score), Epstein-Barr early RNA (EBER) ndations .Tumors lacking either MLH1, MSH2, PMS2, or MSH6 expression were considered “MMR-deficient,” while MSI and TMB have been assessed only by FoundationOne CDx.At the moment of disease progression after the second or third line of treatment, patients signed the informed consent and an off label use request for Nivolumab was submitted for each patient in light of the results of phase III Asian trial “ATTRACTION-2” presented at ASCO GI 2017 and ESMO congress 2017. It is noteworthy that current guidelines did not recommend any specific treatment for this specific setting. Nivolumab has been administered 3 mg/Kg iv in a 14 days cycle until progression of disease or unacceptable toxicity. Every biweekly administration has been considered a cycle of treatment.Local ethical committee has been regularly updated about this off label program and a specific informed consent was obtained from both patients for the publication of this report.The survival and safety follow up cut-off date for this report was June 2019, 20th.Patient 1, a Caucasian 78 year-old female, has been diagnosed on February 2015 with a well-differentiated adenocarcinoma of the body of the stomach, intestinal type according to Lauren classification, with staging CT scan evidence of liver metastasis (28 mm at IV segment) . She wasIn March 2015, she underwent a palliative total gastrectomy with roux-en-y esophagojejunostomy and D2 lymphadenectomy due to symptomatic luminal obstruction. The stage according to TNM was pT4 G1 pN0 (0/26) cM1 HER2 negative.From April 2015 to September 2015 she received 8 cycles of mFOLFOX-6 as first line chemotherapy, achieving a stable disease as best objective response, and subsequently continued maintenance treatment with capecitabine single agent in light of the side effects and the age until December 2015, when she was referred for follow-up due to worsening skin and GI toxicity.On September 2016 (PFS = 16 months), a liver progression occurred  and patient started the second line treatment with Ramucirumab 8 mg/kg  plus Paclitaxel 80 mg/mq  administered in a 28 days cycle .After 6 cycles, she obtained a partial response and continued Ramucirumab monotherapy until May 2017 due to grade 3 neurotoxicity.In May 2017, patient underwent a hip bone and femurs MRI with contrast in order to evaluate a new onset skeletal pain. MRI showed a secondary osteolytic lesion at right femur  and the Therefore, in June 2017 patient was referred for antalgic radiotherapy (8 Gray single fraction) and antiresorptive therapy with zoledronic acid iv. Moreover, she refused radiation therapy for liver lesion and further chemotherapy also considering age and the toxicity of previous chemotherapy regimens.After 3 months of follow up, the off label immunotherapy with Nivolumab was available and patient started treatment on 25 September 2017.In January 2018, the first CT and PET evaluation demonstrated a stable disease according to RECIST criteria. No weight loss, skeletal pain, or other symptoms occurred. Meanwhile, patient experienced a global clinical condition improvement and a significant recovery from previous chemotherapy toxicity. No immune-related adverse events were recorded.In June 2018, she experienced a slight deterioration of renal function (likely related to zoledronic acid use) with a serum creatinine of 2 mg/dl and ultrasound evidence of monolateral grade 1 hydroureteronephrosis and concurrent lithiasis. A CT scan without contrast confirmed the nephrological findings and no more evidence of liver disease  as well as no potential signs or peritoneal involvement.Zoledronic acid was discontinued and immunotherapy temporarily interrupted due to the persistence of creatinine increase. In July 2018, patient underwent ureteral stent placement (subsequently removed in October 2018 due to recurrent urinary tract infections) and creatinine returned to baseline levels during the next weeks.After about 2 months of interruption, patient resumed Nivolumab in August 2018 and performed subsequently a new PET and CT scan (with contrast) in October 2018, which confirmed the complete radiological response . MeanwhiIt is noteworthy that MLH1 promoter methylation analysis could be performed in order to explain the not mutation-caused changes in the protein functions .Patient continued treatment and a further PET and CT evaluation confirmed no evidence of disease in April 2019 with no new onset relevant symptoms or toxicity after more than a total of 25 cycles of immunotherapy.An 82 year-old Caucasian male presented with dyspepsia and was found to have a poorly differentiated adenocarcinoma of the gastroesophageal junction (GEJ) (Lauren type unknown) causing 70% luminal obstruction. He was in good clinical condition (ECOG performance status 0); relevant comorbidities were blood hypertension, allergic rhinitis and cataract.Staging CT scan confirmed a large mass of the GEJ with no evidence of distant disease .He subsequently underwent total gastrectomy with roux-en-y esophagojejunostomy and D2 lymphadenectomy in October 2015. The stage according to TNM was pT4a G3 pN3a (9+/16) cM0 HER2 negative (0 IHC score).From December 2015 to May 2016, he received adjuvant chemotherapy with mFOLFOX-6 with modified dose due age and passed to De Gramont regimen at seventh cycle because of hematological toxicity.In July 2016, PET and CT scan demonstrated a liver (single 37 mm lesion at VIII segment) and abdominal nodes recurrence for which patient started the second line treatment with paclitaxel 80 mg/mq  plus ramucirumab 8 mg/kg  administered in a 28 days cycle.He was still in good clinical condition (ECOG performance status 0) with the exception of a slight weight loss and sporadic episodes of abdominal pain. He received 6 cycles with no grade 3–4 toxicity and achieved after the third cycle a partial response.In March 2017 (PFS = 7 months), treatment was interrupted due to further liver disease progression with volumetric increase (35 × 32 mm) of the known lesion as confirmed by abdominal MRI with contrast (with no evidence of new lesions).Because of the age and the unavailability of standard additional therapeutic options, patient was referred for stereotactic radiotherapy  and subsequently continued with radiological and clinical follow up until further liver (same VIII segment lesion) and nodal progression diagnosed at the CT scan performed 4 months later in September 2017 (with concomitant high blood levels of carcinoembryonic antigen CEA: 230 ng/mL).On October 2017, 16th, patient started treatment with Nivolumab off label. At baseline the CT scan demonstrated a 39 mm liver lesion and several abdominal lymph nodes.In December 2017, he performed, due to abdominal pain, a first CT scan evaluation, which showed stable disease according to RECIST criteria; he was still in good clinical condition with no immune-related toxicities.After 4 months of treatment, he experienced episodes of pruritus without skin rash treated with oral cetirizine.In April 2018, CT scan revealed a minor response both on liver and lymph nodes , while subsequently in September 2018 no evidence of disease was demonstrated with a completely negative evaluation .Patient refused to undergo Foundation One CDx test, but local laboratory analysis on primary tumor sample revealed a so-called “patchy” expression pattern of MLH1 and PMS2  and PD-L1 negative status . MoreoveIn January 2019 and May 2019, further CT scans confirmed complete radiological response and patient has been continuing immunotherapy with no new immune-related adverse events and good clinical condition after more than 30 cycles.Precision medicine has largely remained elusive in GC to date. Immunotherapy represents a promising weapon although it is not yet a standard of care and the majority of evidence is limited to Asian population. The effectiveness of checkpoint inhibitors remains controversial in different settings of treatment.Furthermore, the correct molecular patient selection is still debated and few data are available for Western patients although Pembrolizumab received US Food and Drug Administration approval across gastric tumors with PD-L1 positivity [as well as for MSI-H solid tumors in 2017 .Nevertheless, our report presents one of the first complete radiological responses to immunotherapy ever described to our knowledge in Western GC patients and the first ever to Nivolumab, considering the results of trials available to date.Although only two cases are reported (with indolent course and low disease burden), we suggest that immunotherapy is promising also for non-Asian patients, and MSI testing and TMB are reliable and important also in this population despite the lack of evidence at current time.Immunotherapy led to a real “disruption” of the prognosis and the natural history of these patients.More data are expected for this “orphan” population and dedicated phase III trials, with a correct molecular patient selection, are urgently needed for this specific setting also in Western patients, despite the failure of previous line immunotherapy studies.continuum of care strategy.A further reflection regards the great benefit and good tolerability that also elderly patient (over 75 years old) can experience with immunotherapy and the results achievable thanks to a correct The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The studies involving human participants were reviewed and approved by Comitato Etico AOU Università degli Studi della Campania Luigi Vanvitelli—AORN Ospedali dei Colli, Via Santa Maria di Costantinopoli no 104, 80138 Naples. The patients/participants provided their written informed consent to participate in this study.FD and GT designed the general outline of the paper supervising the writing of the paper. GT and APe wrote the main part and contributed to the clinical documentation collection and FC declares the following interests: Advisory Boards  and Institutional research grants . FD declares the following interests: Advisory Boards . GT declares the following interests: Travel grant , Advisory (Lilly). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Type-1 Gaucher disease (GD1) is considered to be non- neuronopathic however recent evidence of neurological involvement continues to accumulate. There is limited evidence of retinal abnormalities in GD1. The purpose of this study was to evaluate the retinal findings of patients with GD1. Thirty GD1 individuals and 30 healthy volunteers between the ages 40–75 years were prospectively enrolled. Macular and optic nerve optical coherence tomography (OCT) scans of both eyes of each patient were performed and thickness maps were compared between groups. Patients with a known neurodegenerative disease, glaucoma, high myopia and previous intraocular surgeries were excluded. It was shown that patients with GD1 presented with higher incidence of abnormal pRNFL OCT scan and showed significantly thinner areas of pRNFL and macular ganglion cell complex (GCC) when compared to a healthy control population. Changes in retinal thickness were not associated with GD1 genotype, treatment status, disease monitoring biomarker (lyso-Gb1) and severity score index (Zimran SSI). Further investigations are needed to determine whether these findings possess functional visual implications and if retinal thinning may serve as biomarker for the development of future neurodegenerative disease in this population. Gaucher disease (GD), although rare, is one of the most common inherited lysosomal storage disorders ,2. It reThe human retina, an extension of the central nervous system, is a light-sensitive neuronal tissue that consists of nine layers of cells that together convert light rays into a neuronal impulse. The visual information is produced by the photoreceptors and spread to the retinal ganglion cells (RGC) via bipolar cells. The RGC somas reside in the ganglion cell layer (GCL) in the inner retina and it is their axons that form the retinal nerve fiber layer (RNFL) which comprise the optic nerve (ON). From the ON the visual information is transmitted via axons leading to the lateral geniculate body . Damage While ocular symptoms may present as manifestations of GD, they are primarily observed in type-3 Gaucher disease (GD3), a neuronopathic form of GD . PreviouP < 0.01). Significant pRNFL and macular ganglion cell complex (GCC) thinning was observed in different ON quadrants  and ETDRS sectors , respectively . Informed consent was obtained from all participants.This prospective study included a cohort of 30 consecutive patients with GD1 followed at the SZMC Gaucher unit and 30 healthy controls. Participants between the ages of 40–75 years were included. Patients with a known ND and movement disorder, high myopia (−6.0 D>), previous intraocular surgeries apart from cataract extraction, glaucoma and poor-quality scans  were excluded from the study.®, AG, Rostock, Germany) . Th. Th30]. For optic disc OCT assessment, the 3D optic disc scan was utilized ). Peripapillary RNFL is defined by the device as the RNFL at the 3.4 mm circle automatically centered on the optic disc. The pRNFL map is automatically presented as a circle, which is divided to four, 12 and 36 sectors for analysis . For thep value of ≤0.01 was considered statistically significant. Analyses were performed using the SPSS-software .In order to test the association between two categorical variables, the chi-squared test was performed. Independent sample Mann–Whitney U Test was used to compare OCT data between patients with GD1 and controls and within patients with GD1 using the following variables: GD genotype and treatment status. The Pearson’s correlation coefficient was applied to observe the correlations between OCT data to GD disease monitoring marker (lyso-Gb1) and disease severity score (Zimran SSI). Due to multicomparison, a


The simultaneous analysis of different regulatory levels of biological phenomena by means of multi-omics data integration has proven an invaluable tool in modern precision medicine, yet many processes ultimately paving the way towards disease manifestation remain elusive and have not been studied in this regard. Here we investigated the early molecular events following repetitive UV irradiation of in vivo healthy human skin in depth on transcriptomic and epigenetic level. Our results provide first hints towards an immediate acquisition of epigenetic memories related to aging and cancer and demonstrate significantly correlated epigenetic and transcriptomic responses to irradiation stress. The data allowed the precise prediction of inter-individual UV sensitivity, and molecular subtyping on the integrated post-irradiation multi-omics data established the existence of three latent molecular phototypes. Importantly, further analysis suggested a form of melanin-independent DNA damage protection in subjects with higher innate UV resilience. This work establishes a high-resolution molecular landscape of the acute epidermal UV response and demonstrates the potential of integrative analyses to untangle complex and heterogeneous biological responses. In contrast to this however, solar UV irradiation is also the most abundant risk factor for skin cancer and other extrinsically influenced skin disorders3. It is well established that UV irradiation both directly and indirectly induces DNA damage. Direct damage is mainly a result of UVB and to lesser extent UVA irradiation, causing dimerization of adjacent pyrimidine bases, a frequent cause of mutations during replication4. Indirect DNA damage results mainly from oxidative stress, caused by free radicals and cellular reactive oxygen species, which increase after UV irradiation5. Damaged DNA, if not properly repaired, interferes with many cellular mechanisms such as transcription, the cell cycle and replication and can give rise to mutations and epigenetic alterations, driving genomic instability and ultimately carcinogenesis.Solar UV irradiation has complex and ambivalent effects on the human organism. Beneficial effects of sun exposure are thought to be mainly mediated by vitamin D, which is synthesized in the skin through a photosynthetic reaction triggered by exposure to UVB. Vitamin D was primarily acknowledged for its importance in bone formation, increasing evidence however points to its influence on the proper functioning of nearly every tissue in our bodies6. The stratification of individual UV response is thus highly important for risk assessment in cancer prevention (UV-protection), therapeutic dose determination (PUVA therapy) and in the understanding of the biological processes leading to malignancies (e.g. squamous skin cancers). Fitzpatrick skin type categories7 have been widely used as an indicator and predictor of sun sensitivity in epidemiology and experimental photobiology. However, this categorization is hampered by subjectivity and is prone to recall error8. In a study assessing the reliability of Fitzpatrick skin type classifications for instance, only ~ 60% of all study participants self‐identified as the same skin type after repeated questioning a few months later9. Several authors have investigated the relationship between Fitzpatrick skin type and minimal erythema dose (MED)13, a more objective measure of UV sensitivity frequently used in clinical or research settings, showing increasing MED with higher Fitzpatrick classification in general, but with considerable intergroup variation.Human skin has developed several defense systems to guard against the damaging effects of UV: Prominently these include structural changes to the tissue such as epidermal thickening and the synthesis of melanin, but they also comprise quick molecular adaptations like the suspension of cell cycle and gene transcription, as well as the activation of DNA repair pathways. The extent of protection afforded by these mechanisms however is characterized by high inter-individual variation15. Although methylations of adenine have been reported as well, these have so far received considerably less attention. Methylation of CpG sites in the human genome is an important regulatory mechanism that can lead to the activation or repression of gene transcription. Modifications are established and maintained by a set of specific enzymes called DNA methyltransferases. DNA methylation is generally considered to represent a regulatory interface between environmental cues and the genome and might cause or allow long-lasting changes in gene transcriptional activity16. Our current knowledge about epigenetic changes associated with acute UV irradiation, its contribution to transcriptomic alterations and implication in skin photobiology, remains very limited. Previous studies have shown however, that chronic solar UV gives rise to large hypomethylated blocks of DNA in the healthy epidermis and that these blocks are conserved in cutaneous squamous skin carcinomas17, underlining the importance of studying DNA methylation in the context of solar irradiation. In addition, a multi-omics analysis of UV irradiated keratinocytes recently identified several new UV target genes including CYP24A1, GJA5, SLAMF7 and ETV118, demonstrating the value of multi-layered omics analyses in unraveling biological phenomena and enabling more reliable biomarker detection, as it has similarly been shown in cancer research, allowing molecular diagnosis and prognosis, often utilizing DNA methylation markers20.DNA methylation is a covalent epigenetic modification of cytosine to 5-methylcytosine, occurring within CpG dinucleotidesHere we hypothesized that integrative analysis of UV induced epigenetic and transcriptomic alterations in vivo might help to decipher inter-individual responses to environmental challenges and give hints towards early pathogenesis. For this reason, we generated high-resolution multi-omics molecular profiles of the in vivo irradiated epidermis. Our results provide evidence that a UV induced epigenetic memory might be established already after short term repetitive UV irradiation. Integrative analyses of methylation and expression data reveal previously unnoted pathways involved in the acute epidermal UV response and allow the precise inter-individual prediction of MED without the need for prior UV irradiation. Finally, analysis of these molecular phototypes indicates the existence of a melanin-independent form of damage protection in individuals with higher innate resilience to UV irradiation.Elucidating the complex molecular mechanisms underlying UV-gene interaction might offer new insights into how UV modulates skin homeostasis and disease pathogenesis to help improve the prevention of UV-induced skin aging and related pathologies. In order to obtain a comprehensive picture of the molecular events regulating acute epidermal photobiology, 32 female Caucasian volunteers (Fitzpatrick phototypes 1–4) where irradiated with individually calibrated doses of 0.9 MED using a full spectrum solar simulator on three subsequent days on a sun-protected area on their lower backs. 24 h after the last irradiation, suction blister roofs were extracted from irradiated and control sites of each subject and gene expression profiling (Illumina RNA seq) and concomitant DNA methylation profiling (Illumina EPIC Arrays) were performed. Paired differential expression and methylation analyses between irradiated and control areas revealed that in total 20.5% (FDR < 0.05) of all interrogated CpGs and 32.4% (FDR < 0.05) of all detected gene transcripts were significantly altered in response to irradiation. These considerable changes were spread over the whole genome, with notable exceptions only occurring in the regions around the centromeres and in some constitutively heterochromatic regions e.g. on chromosome 13  the observed methylation changes after acute irradiation correlated very well with the reported patterns  harbored at least three differentially methylated CpGs (FDR < 0.05 and log2FCabs > 0.2), whereas for downregulated genes this number increased significantly further to 34.3%  revealed 2,267 significant associations after multiple testing correction. Again, most of these associations were found with alterations in methylation patterns in enhancer regions. Among these highly correlated differentially expressed and methylated genes we identified several known and previously described actors in the UV response, such as CYP24A1, BRCA2, NOTCH2, FOXO3 and GATA3. Examples also included the observed hypomethylation and upregulation of CSNK2A2, a catalytic subunit of Casein kinase II, a ubiquitous serine/threonine protein kinase involved with a manifold of cellular processes, such as cell cycle control and apoptosis and the immune-modulatory keratin KRT17 , and acidic/neutral lipases38. When the permeability barrier is perturbed, both the secretion and synthesis of lamellar bodies is stimulated, which allows for the rapid repair and normalization of permeability barrier function39. So far only few studies have evaluated the effect of UV on the stratum corneum. They provide evidence for an increased epidermal lipid synthesis in response to UV radiation and alterations of lipid profiles42, however these studies gave no functional correlation to genes or mechanisms involved. In addition, atopic dermatitis and psoriasis patients display modified lipid profiles and both groups are known to benefit from UV therapy. Induced DEMGs related to lipid biosynthetic processes might therefore provide evidence of an understudied UV response mechanism and potentially aid in identifying novel targets to help the regeneration of diseased skin. Differentially upregulated genes involved with other notably positively enriched pathways, such as TYMS and DHFR  and 5.08 mJ/cm2 (methylation-based). Models built using DNA methylation features in particular were able to predict individual UV sensitivity to a remarkable degree, indicating a strong epigenetic component associated with UV tolerance. The combination of both expression and methylation data yielded the most accurate prediction model with a median absolute error of 4.6 mJ/cm2, suggesting further complementarity in the two data levels , a frequent cause of mutation in the skin after UV irradiation, that directly links UV damage to carcinogenesis51. Analysis of the extent of CPDs detectable in the samples revealed lower abundances of CPD-alterations in the DNA of MP 3 subjects compared to the other molecular phototypes  during childhood and adolescence in the late epidermal cancer pathogenesis44. In support of this, the molecular data and methylation-based features in particular allowed the highly precise prediction of individual UV sensitivity without any prior irradiation, delivering new and strong evidence of an epigenetic component to individual UV tolerance. Further molecular evidence of the heterogeneity in response to irradiation was delivered by the molecular subtyping analysis, where clustering on the integrated multi-omics data revealed three different molecular phototypes (MPs) among irradiated samples with distinct biological signatures. The MPs differed most prominently in their association with pathways regarding cellular stress response, apoptosis/autophagy and DNA damage sensing and repair . In an attempt to validate the predicted improved damage sensing and DNA repair of the most UV resilient MP 3, we analyzed the extent of cyclobutane pyrimidine dimers (CPDs) at 24 h after the last irradiation, as readout of UV-induced DNA damage and mutagenic potential. Significantly, this data indeed revealed a decreased amount of CPDs after UV irradiation in MP 3 compared to lower MPs. This is in line with some previous studies which showed lower CPD counts in irradiated samples derived from higher phototypes63, although no molecular mechanisms could so far be elucidated. Interestingly, studies of this type often suffered from substantial biological variation within each phototype as well, once more highlighting the need for better stratification and potentially explaining why the mechanisms leading to these observations could so far not be elucidated in ex vivo tissue. Notably, our study setup differed slightly from most of the previous by making use of a repetitive irradiation scheme, potentially widening the window for detecting inter-individual differences in response mechanisms.In general, substantial inter-individual variation in UV tolerability and cancer risk can be observed among Caucasian subjects. Genetic factors like polymorphisms of the melanocortin 1 receptor (MC1R) gene correlate with fairness of skin, UV sensitivity and enhanced cancer risk, however do not fully explain the diversity of UV responses, suggesting the possibility of epigenetic involvement in UV sensitivity and pathogenesis64. The efficacy of melanin as a sunscreen in darker skin is two- to four-fold higher compared to Caucasians65. However individuals with highly pigmented skin have been found 16–500 times less likely to present with skin cancer compared to individuals with fair skin68. The type of melanin produced also plays an important role in skin cancer risk determination. The photoprotective effects of melanin are mainly attributed to eumelanin. Pheomelanin on the other hand has only weak photoprotective properties and has even been found to contribute to carcinogenesis by a mechanism of oxidative damage69. Still, even less deeply pigmented ethnicities such as Asians present far lower skin cancer rates compared to Caucasians53, hinting towards the existence of additional cancer protective mechanisms apart from melanin. One possible explanation involves MC1R variants, which have been shown to confer an increased risk of melanoma and non-melanoma skin cancers, independently of skin pigment (including red hair phenotype)70. The increased expression of transcripts which are associated with nucleotide metabolism and DNA repair in our dataset might present another previously uncharacterized mechanism leading to higher cancer protection afforded by skin with high UV tolerance. The detailed characterization of these biological pathways and the analysis of their clinical significance will be important aspects for future studies.The most intensely explored UV-protection mechanism of the human skin is melanin pigmentation. Melanin serves as a physical barrier that scatters UVR and as an absorbent filter that reduces the penetration of UV through the epidermisTaken together, our analyses demonstrate the benefit of using multi-omics integration for elucidating complex and diverse responses by disentangling inter-individual variation caused by insufficiently precise subject groupings, such as Fitzpatrick phototypes. The presented data illuminates the diverse and interconnected impacts of repetitive UV irradiation on both transcriptomic and epigenetic patterning in the human skin and provides new insights on protective mechanisms of subjects with high innate UV resilience, that might have further-reaching implications for UV-induced carcinogenesis.71, exclusion criteria included tattoos or scars in the test area, pigmentation disorders, pregnancy and medication such as anti-histamines or anti-inflammatory drugs within two weeks prior to study start. A detailed listing of exclusion criteria can be found in the 32 healthy female Caucasian subjects belonging to Fitzpatrick phototypes 1–4 were recruited, with twelve subjects belonging to phototype 1 + 2, ten to phototype 3 and ten to phototype 4. Subjects were aged between 30 and 65 years, with homogenous age distributions in each phototype group. Similar to previous studies13.Minimal erythema dose (MED) estimation is a quantitative method to report the amount of UV (particularly UVB) needed to induce sunburn in the skin 24–48 h after exposure, by determining erythema (redness) and edema (swelling) as endpoints. Individual MED was determined for every subject on the first day of the study following the protocols described in DIN EN ISO 2444472. For each subject, two suction blister roofs of 7 mm diameter were extracted from both control and test sites, one of each to be used to extract RNA for sequencing, the other to extract DNA for the DNA methylation analysis. This amounted to four suction blister roofs extracted per subject and a total of 128 samples.The study sites were located in a sun-protected area on the subjects’ lower backs and were randomly split into control and test areas. On the second day of the study, the first irradiation of the test sites was performed using a SOL 500 full spectrum solar simulator (Hönle UV Technology). Intensities were chosen individually to reach 0.9 MED for all subjects or in other words 90% of the required minimal dose causing erythema in a given test subject. Irradiation to 0.9 MED was repeated in the same manner on the third day and once again on the fourth day of the study, leading to a cumulative irradiation of all test sites three times. On the fifth day of the study and 24 h after the last irradiation session of each subject, epidermal samples were taken using the suction blister method, as previously described71. Tissue samples were suspended in the respective lysis buffers for DNA or RNA extraction and homogenized using an MM 301 bead mill (Retsch). DNA was then extracted using the QIAamp DNA Investigator Kit (Qiagen) according to manufacturer’s instructions. RNA was extracted using the RNeasy Fibrous Tissue Mini Kit (Qiagen) according to manufacturer’s instructions.Nucleic acid extraction was performed as previously described73 for quality control, Trimmomatic v0.3674 for quality based read trimming and Salmon v0.8.175 for read mapping and quantification of transcript expression in the form of read counts and transcripts per million (TPM).Transcriptome libraries were prepared using TruSeq Library Prep Kit (Illumina) and sequencing was performed at 1 × 50 bp on Illumina’s HiSeq system to a final sequencing depth of approximately 100 million reads per sample. Sequencing data was processed using a pipeline including Fastqc v0.11.776 using DESeq277. Linear models were fitted using a paired design matrix to account for inter-individual variation unrelated to the irradiation treatment. Genes were considered significantly differentially regulated with FDR < 0.05 after multiple testing adjustment by the Benjamini–Hochberg procedure.Differential gene expression analysis was performed based on the quantified read counts in R v3.6.176. Methylation data was processed using the minfi package78 in R. Normalization was carried out using the functional normalization method79, which makes use of internal control probes present on the array to infer and correct for technical variation between arrays. Subsequent analyses used M values to describe CpG methylation levels, as their approximate homoscedasticity renders them superior for statistical testing compared to Beta values80.Methylation profiling was performed using Infinium MethylationEPIC arrays (Illumina)81. Linear models were fitted using a paired design matrix to account for inter-individual variation unrelated to the irradiation treatment. CpGs were considered significantly differentially methylated with FDR < 0.05 after multiple testing adjustment by the Benjamini–Hochberg procedure. To compare DNA methylation patterns with those previously described in chronically sun-exposed skin and cancer, we used lists of the respective genomic regions and their methylation status in photoaged skin17 and different types of cancer22, which were available from the Differential CpG methylation analysis was performed in R using limmaFor the calculation of overlap between genes and CpGs, only differentially expressed genes with absolute log2 fold-changes above 0.5 with at least three differentially methylated CpGs with absolute log2 fold-changes above 0.2 were considered, in order to uncover the most reliably differentially expressed and methylated genes. Pearson’s correlation coefficients of gene-CpG pairs were calculated as the sum of all gene transcripts for a given gene correlated with the mean of all CpGs belonging to a functionally annotated group  annotated to a given gene. Annotations such enhancer status, location in transcription start sites or within exons were extracted from the official manifest files for the Infinium MethylationEPIC array provided by Illumina via their website. Significance was assessed using linear models in R, with p-values being adjusted for multiple testing using the Benjamini–Hochberg procedure.25 database, accessed through the STRINGdb package25 in R. PPI information was retrieved for all differentially expressed genes (FDR < 0.05) with absolute log2 fold-changes above 0.7 with at least three differentially methylated CpGs (FDR < 0.05) with absolute log2 fold-changes above 0.5. The interaction query was performed using the standard combined interaction score threshold of 400. The resulting network was refined using in R using igraph82 by retaining only the top 20% of the most reliable edges based on the combined interaction score, with nodes disconnected from the core network being trimmed in the process. The resulting PPI-network was then visualized using igraph82.Information on protein–protein-interactions (PPI) retrieved from the STRING83 as implemented in the GSVA R package84, applied to the log2 transformed TPMs. GO term gene sets85 were downloaded originated from the Molecular Signatures Database v6.286 and included all three sub-ontologies: biological processes (BP), molecular functions (MF) and cellular compartments (CC).Enrichment analyses were performed using the z-score method87 for the prediction of MED were built in R using the implementation provided in the glmnet package88 and interfaced using the machine learning framework mlr89. As lasso regression models perform automatic feature weighting by regularizing the absolute magnitude of coefficients, the models were trained on the full datasets, forgoing the necessity of prior feature selection. Furthermore, data from both control and irradiated samples was included in the training process, in order to allow accurate predictions irrespective of previous UV or sun exposure of a given sample. Model predictions and accuracy scores were extracted from tenfold cross-validation to avoid overfitting and derive unbiased predictions and estimates for the quality of model fit. Metrics used for judging model performance were median absolute error (MAE), as well as the Pearson correlation coefficient (r) and the coefficient of determination (R2).Lasso regression models45 using parameter settings of 45. Measures used for the selection of cluster numbers were the eigen-gap statistic and rotation cost as proposed in the original method description45.After a filtering step, removing features which showed little correlation to MED and reducing feature matrices to 10% of their original size, gene expression (log2 transformed TPMs) and CpG methylation data  from irradiated samples were integrated via similarity network fusion as previously described85 downloaded from the Molecular Signatures Database v6.286. The pathway models were based on the support vector machine (SVM) implementation from the e1071 R package90, interfaced via the mlr89 machine learning framework. The models were trained by restricting the expression data (log2 transformed TPMs) to that of genes annotated within a given pathway and trained to predict sample irradiation status (control or irradiated to 0.9 MED) stratified by molecular phototype. The SVMs used the radial basis function kernel with hyperparameters set to Pathway predictivity analysis was performed using GO term gene setsCPD concentrations were determined using the OxiSelect UV-Induced DNA Damage ELISA Kit (Cell Biolabs) according to the manufacturer’s instructions.91 and dplyr92 for diverse data handling tasks, as well as the packages ggplot293, ggpubr94, circlize95, and pheatmap96 for visualization. Mapping and annotation of gene identifiers was performed using the biomaRt97 and org.Hs.eg.db98 packages, utilizing the GRCh37 (hg19) human genome build.Data analysis in R further included the usage of the package data.tableThe study was performed in agreement with the recommendations of the Declaration of Helsinki and all test subjects provided written, informed consent. Approval of the study protocol was granted by the Ethics Committee of the University of Freiburg (study code 016/1672).Supplementary Figure S1.Supplementary Figure S2.Supplementary Figure S3.Supplementary Figure S4.Supplementary Figure S5.Supplementary information.