Patients in the exploratory cohort had significantly decreased uVLL, lVLL, tVLL, and l/uVLL ratio compared with controls . Receiver operating characteristic curve analyses demonstrated that lVLL , tVLL , and l/uVLL ratio  had high diagnostic accuracies for IIPs. These results were reproduced in the validation cohort. IIP patients thus have decreased VLLs, and measurements of VLL may thus aid the accurate detection of IIPs.Detection of idiopathic interstitial pneumonias (IIPs) on chest X-ray is difficult for non-specialist physicians, especially in patients with mild IIPs. The current study aimed to evaluate the usefulness of a simple method for detecting IIPs by measuring vertical lung length (VLL) in chest X-rays to quantify decreased lung volume. A total of 280 consecutive patients with IIPs were randomly allocated to exploratory and validation cohorts, and 140 controls were selected for each cohort by propensity score-matching. Upper ( Although their precise prevalence is unclear, IIPs are not uncommon and idiopathic pulmonary fibrosis (IPF), as the main subtype of IIPs, was estimated to account for up to 65,000 s death in Europe and up to 17,000 in the United States in 20142, while an epidemiologic survey in Japan reported a prevalence of IPF of 10.0 per 100,000 population and a median survival time of 35 months3. Although IIPs have been poorly treated in the past, early intervention has become increasingly important over the last decade in line with the emergence of anti-fibrotic agents7, and routine screening in primary care practice is expected to improve the early detection of IIPs.Idiopathic interstitial pneumonias (IIPs) are progressive fibrotic diseases of the lungs with unknown etiology11. Pre-existing interstitial pneumonias, such as IIPs, are recognized as a major risk factor for drug-induced interstitial lung disease, and patients should thus be screened for interstitial pneumonia before administering these therapies13.IIPs have also become clinically important for non-pulmonologists in relation to the increasing use of biological, molecular targeted, and immune checkpoint-inhibitor agents as standard therapies in various fields of clinical medicine. Despite their efficacy, these novel treatments have raised concern about the occurrence of drug-induced interstitial lung diseases as a life-threatening toxicity17, and chest computed tomography (CT), especially high-resolution CT, is the gold standard for evaluating these features. However, the risk of radiation exposure and/or overuse of medical resources mean that routine chest CT screening of IIPs is not always practicable, especially in developing countries, where access to CT examinations is sometimes poor because of problems with medical resources and insurance systems.Radiologic imaging plays an important role in the screening and diagnosis of IIPs. Honeycombing, reticular shadows, and ground-grass opacity are typical radiologic features of IIPs18 and can be confirmed by reduced lung fields and an elevated diaphragm on chest X-ray. However, precise evaluation of IIP-related pulmonary abnormalities on chest X-ray is difficult for non-pulmonologists, such as primary physicians or oncologists, and even well-trained pulmonologists and radiologists may not find it easy to detect early-stage IIPs on chest X-ray19. In contrast, reduced lung fields and an elevated diaphragm are easily evaluable and quantifiable on chest X-rays, even by doctors without expertise in diagnostic radiology, by measuring the vertical length of the lung. However, whether or how decreased lung volume is quantified on chest X-rays in patients with IIPs remain unclear. In the present study, we aimed to develop a method to quantify decreased lung volume by measuring the vertical lung length (VLL) on chest X-ray, and to evaluate its usefulness for the detection of IIPs.Decreased lung volume, resulting from pulmonary fibrosis, is another feature of IIPsThe current study included an exploratory and validation cohort, each comprising 140 patients with IIPs and 140 propensity-score matched control subjects Fig. .Figure 1tVLL); (2) from the apex to the carina of the trachea ; and (3) from the carina to the costophrenic angle   from the apex to the costophrenic angle ;  from theuVLL, lVLL, tVLL, and l/uVLL ratio compared with the control subjects  . In addition, patients with mild IIPs also had significantly lower lVLL, tVLL, and l/uVLL ratio, but not uVLL, compared with the control subjects .In the exploratory cohort, patients with IIPs had significantly lower lVLL (area under the curve [AUC] 0.86), tVLL (AUC 0.83), and l/uVLL ratio (AUC 0.80), but not uVLL (AUC 0.60) for the detection of IIPs , 163 mm/m for tVLL , and 1.68 for l/uVLL ratio . When limited to patients with mild IIPs, lVLL, tVLL, and l/uVLL ratio, but not uVLL, also demonstrated high diagnostic accuracies  , 164 mm/m for tVLL , and 1.70 for l/uVLL ratio .Cut-off values were 98 mm/m for uVLL, lVLL, tVLL, and l/uVLL ratio compared with the control subjects  . Patients with mild IIPs had significantly lower lVLL, tVLL, and l/uVLL ratio, but not uVLL, compared with the control subjects .The differences in VLLs between patients with IIPs and the control subjects were reproduced in the validation cohort. Patients with IIPs had significantly lower uVLL, lVLL, tVLL, and l/uVLL ratio for the detection of IIPs were comparable to those in the exploratory cohort  .The AUCs of ly) Fig. E–H. The uVLL, lVLL, tVLL, and l/uVLL ratio were also comparable to those in the exploratory cohort  .Regarding patients with mild IIPs, the AUCs of lVLL , 163 mm/m for tVLL , and 1.68 for l/uVLL ratio  , tVLL (p = 0.030), and l/uVLL ratio (p < 0.001). There was no significant difference in AUCs between tVLL and l/uVLL ratio.The final cut-off values for the detection of IIPs in the total cohort were 98 mm/m for lVLL , 164 mm/m for tVLL , and 1.70 for l/uVLL ratio   and tVLL (p < 0.001). There was no significant difference in AUCs between lVLL and l/uVLL ratio, or between tVLL and l/uVLL ratio.When limited to patients with mild IIPs, the final cut-off values in the total cohort were 103 mm/m for When using the VLL cutoff values in round numbers in clinical practice, an lVLL of 100 mm/m had a sensitivity of 0.71 and specificity of 0.86 for all IIPs, and a sensitivity of 0.54 and specificity of 0.86 for mild IIPs. A tVLL of 160 mm/m had a sensitivity of 0.70 and specificity of 0.83 for all IIPs, and a sensitivity of 0.49 and specificity of 0.83 for mild IIPs. An l/uVLL ratio of 1.70 had a sensitivity of 0.76 and a specificity of 0.75 for all IIPs, and a sensitivity of 0.70 and specificity of 0.75 for mild IIPs.tVLL and lVLL showed moderate positive correlations with %predicted FVC  and %predicted forced expiratory volume in 1 s (FEV1) , and weak inverse correlations with body mass index (BMI)  and FEV1/FVC ratio   and %predicted FEV1 , and weak inverse correlations with FEV1/FVC ratio . The uVLL was negatively associated with the extent of the reticular pattern, but not with honeycombing or GGO  , honeycombing , and ground-glass opacity (GGO) on chest computed CT images  . When compared with the controls, both patients with and without honeycombing had significantly lower uVLL , lVLL (both p < 0.001), tVLL (both p < 0.001), and l/uVLL (both p < 0.001) values. Percentage low-attenuation area (%LAA), defined by the percentage area below − 950 HU in the total lung area on chest CT images had weak positive correlations with uVLL, lVLL, and tVLL  and a very weak correlation with the l/uVLL (r = 0.19) , tVLL (both p < 0.001), lVLL (both p < 0.001), and l/uVLL ratio (both p < 0.001) compared with the control subjects. Compared with the IPF group, non-IPF patients had significantly higher lVLL and l/uVLL ratio , but similar uVLL and tVLL . After adjusting for age, sex, BMI, %predicted FVC, %predicted FEV1, and %LAA in multivariate logistic regression analysis, there was no significant difference in uVLL, lVLL, tVLL, or l/uVLL ratio between the IPF and non-IPF groups , female-dominant (p < 0.001), and had lower BMI (p = 0.006), higher %predicted FVC, higher %predicted FEV1 (p < 0.001), and lower FEV1/FVC (p = 0.009) (Supplementary Table uVLL (p = 0.014) and higher lVLL, tVLL, and l/uVLL ratio , compared with those with IIPs . The secondary IPs comprised collagen vascular disease (CVD)-associated IPs (n = 116), sarcoidosis (n = 76), chronic hypersensitivity pneumonia (CHP) (n = 40), and pneumoconiosis (n = 8). The types of CVD were rheumatoid arthritis (n = 49), dermatomyositis (n = 26), Sjögren syndrome (n = 17), scleroderma (n = 8), systemic lupus erythematosus (n = 2), a combination of two or more CVDs (n = 5), and others (n = 14). Compared with the patients with IIPs, those with secondary IPs were significantly younger (uVLL (p = 0.002) and higher lVLL, tVLL, and l/uVLL ratio , compared with those with IIPs , tVLL (p = 0.001), and l/uVLL ratio (p < 0.001); those with sarcoidosis had significantly lower uVLL (p = 0.009) and higher lVLL (p < 0.001), tVLL (p < 0.001), and l/uVLL ratio (p < 0.001); those with CHP had significantly lower uVLL (p < 0.001) and higher lVLL (p < 0.001) and l/uVLL ratio (p < 0.001); and those with pneumoconiosis had significantly higher lVLL (p < 0.001), tVLL (p = 0.003), and l/uVLL ratio (p = 0.002).Compared with patients with IPFlVLL, tVLL, and l/uVLL ratio demonstrated high diagnostic accuracies for the detection of IIPs, with lVLL having the highest accuracy. Importantly, decreases in lVLL, tVLL, and l/uVLL ratio were also observed in patients with mild IIPs, with a %predicted FVC > 80%. Chest X-rays are a non-invasive, low-radiation exposure, and low-cost diagnostic method, and the measurement of VLLs is simple and easy, even for doctors with no expertise in diagnostic radiology, thus aiding the early detection of IIPs in clinical practice.In the present study, we evaluated reduced lung volume by measuring VLLs on chest X-rays, and found that VLLs were significantly decreased in patients with IIPs. Among the measured VLLs, lVLL was more prominent compared with uVLL, possibly because pulmonary fibrosis occurs predominantly in the lower lobe of the lungs. Although the uneven distribution of pulmonary fibrosis in the lower lungs is well known in IPF20, the underlying mechanisms are unknown. One hypothesis suggests that mechanical stress caused by repeated ventilation is stronger in the lower lobe because of the large motion of the chest wall and diaphragm22. In addition to IPF, most IIPs demonstrate predominantly lower lobe lesions23. Patients in the current study with non-IPF IIPs showed decreased lVLL and l/uVLL ratio, similar to patients with IPF.Among the decreases in VLLs observed in patients with IIPs, the decrease in 24. In the present study, VLLs were only moderately correlated with %predicted FVC, and the decreases in VLLs were independent of clinical factors, including %predicted FVC. In addition, differences in the cut-off values between all IIPs and mild IIPs were small. These characteristics of VLLs thus enable the detection of early-stage IIPs, but conversely, VLLs may be of little clinical use for estimating the deterioration of pulmonary function.Interestingly, VLLs were decreased in patients with IIP but with no decline in %predicted FVC. In addition to lung elasticity, pulmonary function also depends on thoracic elasticity and the respiratory muscles, and patients with IPF were reported to show only a weak or moderate correlation between pulmonary function and radiologic severity at baseline and changes during follow-uplVLL demonstrated the highest AUC for the detection of IIPs, the diagnostic accuracies of lVLL, tVLL, and l/uVLL ratio were similar from a clinical perspective. In contrast to lVLL and tVLL, which need to be adjusted for body height, l/uVLL ratio does not depend on height and can be calculated from chest X-ray images, and can thus be used as an alternative to lVLL.Among the measured VLLs, although uVLL and higher lVLL than those with IIPs. This may be because secondary IPs, including sarcoidosis, CHP, and pneumoconiosis, demonstrate upper-lobe-predominant fibrosis, whereas IPF, the most dominant phenotype among IIPs, demonstrates lower-lobe-predominant fibrosis27. Furthermore, CVD-IPs are a heterogeneous group of different disease phenotypes, each with distinct lung abnormalities varying according to the phenotype. The utility of VLLs in secondary IPs needs to be investigated further. Third, the present study was retrospective in nature, with limited clinical information. The existence of respiratory symptoms, dyspnea, or abnormal respiratory sounds on auscultation may increase the diagnostic ability in combination with VLLs. The clinical utility of VLLs thus needs to be validated in a prospective, observational study in a real-world setting.The current study had three main limitations. First, VLLs are only evaluable on chest X-rays performed in an upright position, in patients with no abnormal changes other than interstitial pneumonias. Chest CT should be used to screen for IIPs in patients in the decubitus position, or those with a history of chest surgery or other obvious chest abnormalities. Similarly, severely obese patients may have an elevated diaphragm due to visceral fat and may therefore be unsuitable for the evaluation of VLLs. Second, the differential distribution of pulmonary fibrosis affects VLLs. Patients with secondary IPs demonstrated lower lVLL, on chest X-ray. The measurements of lVLL, tVLL, and l/uVLL ratio demonstrated high diagnostic accuracies for the detection of IIPs. This simple method may aid the early detection of IIPs by non-respiratory practitioners and physicians in areas with poor access to CT examinations.Patients with IIPs, including those with mild IIPs, had decreased VLLs, especially This retrospective observational study was conducted in accordance with the ethical standards described in the Declaration of Helsinki. The study protocol was approved by the Institutional Review Board of Hamamatsu University School of Medicine (No. 20-015). Each patient provided written informed consent to be included in the study. The study was registered with the University Hospital Medical Information Network Clinical Trial Registry (identification code: 000040724).1. The inclusion criteria were as follows: chest X-ray and spirometry performed within 1 week of each other, and clinically stable disease with no worsening for at least 4 weeks before spirometry and chest X-ray. If multiple data were available for the same patient, the earliest data from the initial diagnosis of IIP were evaluated. The exclusion criteria were as follows: patients with pleuroparenchymal fibroelastosis, characterized by upper-lobe-dominant fibrosis28; patients with anatomical chest abnormalities, abnormal chest shadows without IIP , or a history of chest surgery; patients with missing data for either chest X-ray or spirometry within 1 week; and patients with clinically unstable disease within 4 weeks (e.g. pneumonia or acute exacerbation). Clinically stable patients without IIPs were evaluated as control subjects. Control subjects were required to have normal spirometry, no pulmonary disease, no anatomical chest abnormality, and no history of chest surgery.We retrospectively evaluated the medical records of patients with IIPs in our institute from January 2000 to December 2019. The diagnosis of IIP was made according to American Thoracic Society/European Respiratory Society Clinical Practice GuidelinesA total of 280 consecutive patients with IIPs and 400 control subjects were evaluated. The patients with IIPs were randomly allocated to the exploratory or validation cohort Fig. . ControlA total of 240 consecutive patients with secondary IPs, including connective tissue diseases, sarcoidosis, hypersensitivity pneumonia, and pneumoconiosis, were evaluated as the secondary IP cohort.uVLL, lVLL, and tVLL in the right lung on chest X-rays taken in the upright position and at maximum inspiration  lung was evaluated to eliminate the possible effect of cardiac shadow. VLLs were evaluated by two independent investigators and the results are expressed as a mean of the two estimates. The extent of the reticular pattern, honeycombing, and GGO on chest CT images were semi-quantitatively evaluated as follows: grade 0 (0%), grade 1 (< 25%), grade 2 (25–50%), grade 3 (50–75%), and grade 4 (> 75%)29. The definitions of reticular pattern, honeycombing, and GGO were according to the Fleishner Society criteria30. The %LAA on chest CT images was calculated using image-analyzing software .Lung volume loss was evaluated by measuring ion Fig. A–C. The p value < 0.05 (two-sided) was considered significant. All statistical analyses were carried out using JMP v13.0.0 , except The Jonckheere–Terpstra test was performed using EZR , a graphical user interface for R .The randomization of patients with IIPs was stratified by sex and %predicted FVC. One-to-one propensity score-matching was performed using age, sex, and BMI as co-variables. Continuous variables were compared by Wilcoxon’s rank sum test and categorical variables by Fisher’s exact test. The predictive values of VLLs for the diagnosis of IIPs were evaluated by multivariate logistic regression analysis. The cut-off values of VLLs for the diagnosis of IIPs were estimated by ROC analysis and determined using Youden’s index . The correlations between VLLs and clinical factors were evaluated by Pearson’s correlation analysis. The correlations of VLLs with the increased extent of lung abnormalities in the patients with IIPs were evaluated by the Jonckheere–Terpstra test. The inter-observer reproducibility was evaluated by intraclass correlation analyses. A Supplementary Information.


Dopamine-dependent long-term plasticity is believed to be a cellular mechanism underlying reinforcement learning. In response to reward and reward-predicting cues, phasic dopamine activity potentiates the efficacy of corticostriatal synapses on spiny projection neurons (SPNs). Since phasic dopamine activity also encodes other behavioural variables, it is unclear how postsynaptic neurons identify which dopamine event is to induce long-term plasticity. Additionally, it is unknown how phasic dopamine released from arborised axons can potentiate targeted striatal synapses through volume transmission. To examine these questions we manipulated striatal cholinergic interneurons (ChIs) and dopamine neurons independently in two distinct in vivo paradigms. We report that long-term potentiation (LTP) at corticostriatal synapses with SPNs is dependent on the coincidence of pauses in ChIs and phasic dopamine activation, critically accompanied by SPN depolarisation. Thus, the ChI pause defines the time window for phasic dopamine to induce plasticity, while depolarisation of SPNs constrains the synapses eligible for plasticity. It remains unclear how corticostriatal synapses utilize reward prediction error signaling in order to reinforce reward-related behaviors. Here, the authors show that potentiation of corticostriatal synapses requires phasic dopamine activation, pauses in striatal cholinergic interneuron firing, and depolarization of spiny projection neurons. In reinforcement learning, phasic dopamine activity is thought to reinforce the actions that led to a reward to increase the chances of earning the same reward in future3. The cellular mechanism of reinforcement learning is believed to involve dopamine-dependent long-term plasticity, where increased dopamine levels enhance the efficacy of glutamatergic synapses between cortex and striatum6. Thus, striatal spiny projection neurons (SPNs) involved in executing a rewarded action will respond more effectively to subsequent presentations of the same cortical input pattern, in order to maximise future reward delivery. However, dopamine neurons not only increase their firing rate after reward and reward-predicting cues, but also increase to other encoded variables such as distance to reward8, movement10, and behavioural choices11, although these variables may be encoded in a smaller discrete population of dopamine neurons12. Therefore, it is unclear how corticostriatal synapses might identify the appropriate dopamine signal from which to induce long-term plasticity during reinforcement learning.In the wild, animals need to take particular actions to maximise reward in a given situation. After obtaining a reward, or a sensory cue that predicts a reward, midbrain dopamine neurons briefly increase their firing rate in synchrony18 that the striatal tonically active neurons (TANs), likely to be ChIs, play a critical role in determining which dopamine signal is involved in modulating synaptic transmission. Although only 1% of striatal neurons are ChIs, they can regulate SPNs directly19 and indirectly21, and can modulate axonal dopamine release during coincident phasic dopamine activity25. ChIs exhibit excitation-pause-rebound multiphasic activity in response to reward and reward-indicating cues28. Critically, the pause phase coincides with phasic dopamine activity during learning15. Therefore, the ChI pause has been suggested to facilitate dopamine-dependent plasticity18. However, this hypothesis has never been tested in an intact brain in vivo due to the challenge of inducing synchronised pauses in sparsely distributed ChIs, aligned with phasic activity of dopamine neurons, within a physiologically meaningful timescale.In the current study, we tested the previously-proposed hypothesis29. This observation enables us to test for the first time whether the ChI multiphasic response is involved in dopamine-dependent plasticity. Using the inverted striatal local field potential (iLFP) as a read-out of spontaneous and electrically evoked excitatory activity in our anaesthetised preparation29, we are able to manipulate ChIs with cortical stimulation at the same time as we manipulate dopamine neurons. Here, we hypothesised that a temporal coincidence of a pause in the multiphasic activity of ChIs and phasic dopamine is required to induce long-term potentiation of corticostriatal synapses. Further, we propose that a marginal shift of the timing of phasic dopamine activity (a few hundred milliseconds) to overlap with excitation phases of ChI will switch the direction of long-term plasticity.We recently showed that the firing of ChIs is entrained to fluctuations in excitatory input30. Furthermore, the volume transmission of dopamine in the striatum will further enhance the opportunity for synchronised phasic dopamine activity to elevate dopamine levels at synapses irrelevant to the rewarded action. One possible mechanism for constraining synaptic change is to limit plasticity to those postsynaptic neurons depolarised during the action. In slice preparations, depolarisation of the postsynaptic neurons is necessary to form long-term plasticity31 and a conjunction of pre- and postsynaptic activity, plus a reward signal (a three-factor plasticity rule) has been proposed5. However, the role of depolarisation has not been adequately characterised in vivo. Here, we further hypothesise that depolarisation of the postsynaptic SPNs is necessary for the induction of long-term plasticity, and the function is to constrain dopamine-dependent plasticity to the targeted synapses.Another currently unresolved question in dopamine-dependent plasticity is how widespread changes in dopamine signalling in the striatum induce long-term plasticity only at those synapses involved in driving actions that lead to reward. Dopamine neurons have extensively arborised axons such that a single neuron can cover up to 5% of the striatumTo test these hypotheses, we undertook two distinct in vivo experiments where we manipulated both midbrain dopamine neurons and putative ChIs (pChIs) so that phasic dopamine was present at different phases of the ChI multiphasic response. In addition, in the second set of experiments we also manipulated the membrane potential of the postsynaptic SPN by injecting positive current intracellularly, to determine the optimal timing of depolarisation for long-term plasticity. Our results suggest that the optimal conditions for induction of in vivo long-term potentiation at corticostriatal synapses on SPNs are a coincidence of phasic activity in dopamine neurons, pauses in ChIs, and depolarisation of postsynaptic SPNs.Two experimental paradigms were used to investigate the integration and timing of glutamatergic, dopaminergic, and cholinergic signals in the striatum.32  was disinhibited by local injection of the GABA antagonist bicuculline . Then, to induce synaptic plasticity, cortical stimulation was paired with light stimulation. After 5 min of pairing, the SC was disinhibited locally by BIC and then pairing continued for another 10 min. The combination of these two manipulations either drove phasic dopamine activity to coincide with a pChI pause (the ‘Match’ group) or with pChI excitation (‘Mismatch’ group). Then, spike activity of SPNs in response to cortical test stimulation alone was continued for at least another 30 mins. We found that the spike activity of SPNs induced by cortical stimulation  remained potentiated only when the period of pChI pauses and phasic dopamine was coincident , light flashes were only paired with cortical stimulation for the 5 min prior to bicuculline injection and not subsequently, so pChI pauses were entrained by the cortical stimulation but dopamine neurons were not phasically-activated. We found a similar depression in the ‘Control’ group as the ‘Mismatch’ group Fig. . Thus, p36. Therefore, we used a second experimental paradigm to allow us to manipulate SPN depolarisation independently of SNc dopamine cell activation .We tested whether depolarisation of postsynaptic SPNs is required for LTP in vivo by using a paradigm with which we have previously demonstrated an association between synaptic plasticity and positive reinforcement4 we found that dopamine-dependent long-term potentiation (LTP) of synapses on recorded SPNs was induced when the neurons were depolarised during the SPN ‘down state’ using intracellular current injection, and when dopamine neurons were activated simultaneously. However, according to our recent investigation of the factors underlying the firing activity of ChIs29, the down state of SPNs not only corresponds to a period of minimum excitatory cortical input to the striatum37, but also to the time when ChIs exhibit a pause  or depolarisation of SPNs alone  applied during the pChI pause period, induced LTD.We further tested whether the combination of dopamine and the pause in ChIs, or the combination of SPN depolarisation and the pause in pChIs, is effective in inducing LTP. We found that either dopamine stimulation alone  may also contribute to the formation of the ChI pause in the ventral striatum44, indicating acetylcholine and dopamine system may interplay in a different manner in the ventral striatum than dorsal striatum.Our results suggest that the ChI pause may define a time window for phasic dopamine to induce long-term synaptic potentiation. We here entrained pChIs to pause with cortical electrical stimulation38. Thus, because phasic dopamine activity reduces at late stages of learning whereas the ChI pause remains intact, the resulting effect of the interplay between ChI activity and dopamine afferents at the striatal level may be dependent on the stage of learning. In addition, manipulating dopamine neurons to fire phasically can only induce synaptic potentiation if it coincides with the pChI pause but not the excitation phase, indicating that the ChI pause is the time window for synaptic potentiation.We further demonstrated that changes in pChI activity without phasic dopamine do not induce long-term potentiation at corticostriatal synapses , consistent with others’ findings45. To avoid blocking the tonic effects of dopamine, our control experiments instead altered the timing of phasic dopamine or omitted dopamine neuron stimulation altogether. We found that even shifting the timing of phasic dopamine activity marginally by a few hundred milliseconds is powerful enough to reverse the direction of the long-term plasticity. Also, when phasic activation of dopamine neurons was omitted, LTD or no change of synaptic efficacy was found. It should be noted that our study focused on mimicking the cell body activity in ChIs and dopamine neurons, as revealed earlier15. However, the release profile of dopamine may not precisely mirror the firing pattern of dopamine neurons due to the potential axonal release driven by ChIs24, and should be addressed in future work. Overall, these experiments indicate that the LTP we observed is dependent on phasic dopamine.We here also addressed how phasic dopamine contributes to synaptic plasticity without using dopamine receptor antagonists, which undesirably also block the effects of tonic dopamine. Indeed, a previous ex vivo study showed that tonic activation of dopamine receptors can reverse the direction of long-term plasticity induced by cortical stimulation46 and is consistent with previous ex vivo observations31. This suggests that depolarisation of postsynaptic SPNs may play a critical role in constraining long-term potentiation to synapses on neurons engaged in the action of retrieving rewards. While SPNs in the direct pathway and indirect pathway express distinct sets of dopamine and muscarinic receptors, these neurons may be potentiated to different degrees during learning, which is worth addressing in future studies. In summary, our study provides empirical data supporting the important temporal gating of corticostriatal synaptic plasticity by a physiologically-induced pause response in ChIs and depolarisation of the postsynaptic SPNs.Our results also provide further support for the requirement for depolarisation of the target postsynaptic SPNs for long-term potentiation in vivoAll procedures in this study were conducted in accordance with approvals granted by the University of Otago Animal Ethics Committee or in accordance with the UK animals scientific procedures act, 1986 with approval of the University of Bristol ethics committee. A total of 136 male Long-Evan rats for extracellular recording were used, yielding 258 putative spiny projection neurons, and 18 were successfully recorded for the full plasticity recording protocol. The intracellular recording was performed with 105 male Wistar rats, yielding 32 spiny projection neurons recorded for the plasticity protocol.Male Long–Evans rats (250–450 g) or Wistar rats (280– 50 g) were anaesthetised with urethane . During recording, the level of anaesthesia was monitored by continuous observation of the bandpass filtered EEG signal (0.01–500 Hz). Supplementary urethane was administered via an intraperitoneal catheter at any sign of EEG desynchronisation, indicating a lessening of depth of anaesthesia. The head was fixed in a stereotaxic frame  and core temperature maintained above 36 °C by a homoeothermic blanket and rectal probe . All wounds and pressure points were infiltrated with a long-acting local anaesthetic .For monitoring of the electroencephalogram (EEG), a hole was drilled in the skull above the left posterior cortex, and a silver wire electrode placed against the dura overlying the cortex and fixed in placed with dental cement. A flap of bone overlying the cortex was removed to provide access to the recording site in the left medial striatum, and a “well” of dental cement fashioned around the perimeter of the hole. All coordinates are given in millimetres in relation to Bregma and the midline.To implant a stimulating electrode into the medial agranular motor cortex, a round piece of skull overlying the right hemisphere (centred AP + 2.0 to +2.7 mm and ML −1.6 to −2.0 mm to Bregma) was removed. A concentric  or parallel-contact  stimulating electrode was implanted in the medial agranular motor cortex to a depth of 1.6 to 2.4 mm. Stimulating electrodes were connected to constant current electrical stimulators  Stimulus pulses applied to the cortex were biphasic . For experiments requiring substantia nigra stimulation, the medial contact of a parallel-contact bipolar stimulating electrode was implanted at interaural coordinates AP + 3.4 to +3.6; ML + 1.6; DV 2.1 to 2.3. Substantia nigra stimulation consisted of 50 biphasic pulses (0.5 ms duration) applied at 100 Hz (average current applied for each group 500 to 990 uA).In the extracellular recording experiments, visual stimuli  were delivered by a white LED (1500 mcd) that was placed 1–2 cm directly in front of the right eye of the animal. The left eye was covered. LED and electrical stimulating electrodes were connected to constant current electrical stimulators .The drug-filled pipettes were lowered to 4.0–4.2 mm from the brain surface into the deep layers of the superior colliculus (AP −6.5/ ML + 1.5 mm), and either supported by the IVM micromanipulator or secured with dental cement. Bicuculline  was injected into the superior colliculus at a rate of 400 nl/min.Extracellular single-unit recordings were made using 5–15 MΩ micropipettes. Electrodes were filled with 1 M NaCl solution with 2% neurobiotin . Only stable striatal neurons with wide average spike waveform (>1.1 ms), and slow spontaneous firing rate (<0.1 Hz), e.g. Supplementary Fig. Dopamine neurons were extracellularly recorded and juxtacellularly labelled (AP −5(±0.3) ML + 2(±0.3) DV −7/−8) using glass electrodes as described above. Single-unit data were acquired with an ELC-01MX headstage (NPI electronic), amplified 1000 times, and bandpass filtered at 300 to 5000 Hz (DPA-2FS filter). Depth of anaesthesia was monitored using the electrocorticogram (amplified 2000 times and bandpass filtered at 300 to 1500 Hz) from a stainless-steel screw implanted at AP +2, ML +2. Data were digitised at 25 kHz using a CED Power 1401 mkII.Intracellular recordings were made using 35–130 MΩ micropipettes with 1 M K-acetate internal solution, in some cases containing 3–4% biocytin. For intracellular recording, only stable neurons with a membrane potential more negative than –60 mV that displayed characteristic spontaneous fluctuations in membrane potential (>10 mV amplitude) and action potential firing were included in this study. Current–voltage relations were obtained by injecting hyperpolarising and depolarising current pulses through the micropipette, using an Axoclamp-2B amplifier (Molecular Devices) configured in current-clamp mode. Membrane potential fluctuations were recorded for periods of at least a minute after the cell had stabilised following impalement and at regular intervals of >15 min. All waveform data were digitised at 10 kHz by a Digidata 1200B or a Digidata 1322A (Molecular Devices), displayed using pClamp 8 software (Molecular Devices).After a stable single-unit recording was obtained from a putative spiny projection neuron, cortical stimulation (0.2 Hz) was applied throughout the recording, and the short latency (<30 ms) spike response was measured as the strength of the corticostriatal synaptic input. During the recording, firstly, a baseline of 10 min of spike activity was recorded. The cortical stimulation was then paired with light stimulation for another 5 min. Bicuculline was then injected locally to the deep layers of the superior colliculus. Visual stimulation was paired for another 10 min with the cortical stimulation. In the match group, visual stimulation was applied when the iLFP started to decrease, approximately 500 to 600 ms after cortical stimulation. In the mismatch group, visual stimulation was applied when the iLFP started to increase, approximately 250 ms after cortical stimulation. Therefore, the estimated light-induced phasic dopamine activity occurred either during the ChI pause (Match group) or excitation  of putative ChIs and SPN spikes were plotted using the cortical stimulation Figs. c, 2a or 47, and this used to trigger data acquisition and substantia nigra electrical stimulation, where relevant, early in the ensuing Down or Up state. A delay of 600 ms was imposed between intracellular current injection and substantia nigra stimulation in the dissociated group to ensure that the depolarisation was induced in the up state, and the substantia nigra stimulation occurred primarily in the ensuing down state. Intracellular current injection, when included in the protocol, was set 0.2 nA higher than the threshold for eliciting continuous action potential firing (range 0.5–2.0 nA). Membrane potential fluctuations and current–voltage relations were assessed before and after the plasticity-inducing protocol to ensure that changes in PSPs were not associated with changes in membrane characteristics. The experimental protocols used are illustrated in Fig. After a stable impalement was obtained from a spiny projection neuron, a baseline of 20 min of postsynaptic potentials (PSPs) was recorded before the plasticity-inducing protocol. Postsynaptic potentials were always elicited from the Down state. The transitions between membrane potential “states” were detected online using a locally-constructed functional clamp48. Briefly, the target cells were “driven” to spike by applying positive current through the recording pipette  for up to 15 min. At the end of intracellular recording, putative spiny neurons were filled intracellularly with biocytin by applying depolarising current pulses  via the recording micropipette.At the end of extracellular recording, the putative spiny neurons were actively filled with neurobiotin by a juxtacellular filling protocol49 and labelled cells were identified by light or fluorescent microscopy. In the extracellular recording experiments, 8 neurons, and in the intracellular recording experiments, 14 neurons, were recovered and verified histologically as spiny projection neurons. Juxtacellularly labelled dopamine neurons were identified as previously described  using Cy3-conjugated streptavidin , chicken anti-Tyrosine Hydroxylase primary  and Brilliant Violet 421-conjugated anti-chicken secondary antibodies 50.Vibratome sections (50–60 µm) were processed using standard histological procedures51.Positions of the substantia nigra electrodes for intracellular recordings were determined in unstained or cresyl-violet stained sections to be within 500 µm of the dopamine cell layer of the substantia nigra pars compacta. There were no systematic differences in electrode positions between groups. In groups where SN stimulation was not applied, electrodes were still fitted during surgery to control for the release of dopamine that may accompany acute electrode implantationData were analysed offline using built-in functions of Axograph 4.9 software and SPIKE2 v6 or v7. Statistical tests on data from single cells as well as on group data were performed in SPSS and Prism.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


T = 16 K and is virtually independent of temperature. In contrast, the AMR induced by the scattering-dependent extrinsic contribution was approximately 3% at T = 16 K but decreased to 1.5% at T = 155 K, which is the maximum temperature at which the AMR can be resolved using THz measurements. Our results experimentally quantify the temperature-dependent intrinsic and extrinsic contributions to AMR, which can stimulate further theoretical research to aid the fundamental understanding of AMR.Electrical conduction in magnetic materials depends on their magnetization configuration, resulting in various magnetoresistances (MRs). The microscopic mechanisms of MR have so far been attributed to either an intrinsic or extrinsic origin, yet the contribution and temperature dependence of either origin has remained elusive due to experimental limitations. In this study, we independently probed the intrinsic and extrinsic contributions to the anisotropic MR (AMR) of a permalloy film at varying temperatures using temperature-variable terahertz time-domain spectroscopy. The AMR induced by the scattering-independent intrinsic origin was observed to be approximately 1.5% at  The microscopic mechanisms of such MRs have been extensively studied and have so far been classified into either intrinsic or extrinsic origins7.Electrical conduction in magnetic metals depends on their magnetic state, which results in various magnetoresistances (MRs), such as anisotropic MR (AMR)8, in which the electrical conduction in transition metals is modelled as the sum of two separate currents of majority and minority spin electrons, with the following two assumptions: (i) s electrons are responsible for the conduction, owing to the relatively low mobility of d electrons; (ii) the large density of d states mainly accounts for the scattering rates of s electrons. This led to AMR being attributed to the magnetization-dependent scattering time, i.e., the extrinsic effect13, as the d electrons experience the SOI but the s electrons do not. Specifically, the conducting s electrons experience anisotropic scattering processes via spin–orbit-coupled d bands. This class of AMR theories take into account various SOI-induced effects on the scattering time, such as the mixing of majority and minority d states13, altering the atomic wave function of d orbitals12, and spin mixing processes including spin-flip scattering10.Among the various MRs, the AMR is one of the most fundamental spin–orbit–interaction (SOI)-induced transport phenomena in magnetic materials. The AMR describes the anisotropic charge conductivity and its dependence on the relative orientation of the current flow and the magnetization. Early theories viewed the AMR through Mott’s two-current modeld electrons also participate in electrical conduction15 through spd hybridization16. In other words, the conduction electrons are no longer intact to SOI; rather, their band structures are affected by SOI. Therefore, not only does the magnetization-dependent scattering time play a crucial role in the AMR, but the magnetization-dependent electronic structure, i.e., the intrinsic origin, is also critical. Recent theoretical works have emphasized the intrinsic effect on AMR by illustrating ballistic AMR owing to the anisotropic d bands17 and demonstrating the magnetization-dependent band structure owing to the orbital hybridization18. The existence of the intrinsic contribution has also been investigated experimentally. Hupfauer et al. demonstrated the crystalline AMR effect, originating from the difference in electronic density, through its magnetization orientation19, and Zeng et al. reported the intrinsic contribution caused by the magnetization-direction-dependent band crossing effect20.Recent theories have diverted from the assumptions of the Mott model and have identified the intrinsic mechanism of AMR, which arises from the scattering-independent band-structure effect. The resulting intrinsic relaxation is essential for transition metals since 21, in which they found that the anisotropy of a crystal structure could be an origin of intrinsic contribution to AMR. Despite the success, however, our understanding of AMR is still far from satisfactory. In particular, the temperature dependence of the intrinsic and extrinsic contributions to AMR remains elusive, despite the large temperature variance of AMR22. In this respect, investigating the temperature dependence of the intrinsic and extrinsic contributions to AMR is of crucial importance to understand the fundamental origins of the AMR.Recently, Nadvornik et al. have successfully disentangled the intrinsic and extrinsic contributions to AMR in polycrystalline thin films at room temperatureT = 16 K, which increased to . We find that the intrinsic contribution to AMR was virtually independent of temperature, while the extrinsic contribution to AMR decreased with increasing temperature, suggesting that the portion of intrinsic contribution to the total AMR increases with increasing temperature. Quantitatively, the intrinsic portion accounted for 23 can be used to investigate the intrinsic and extrinsic contributions to the AMR:21. Therefore, the intrinsic and extrinsic contributions to the AMR can be separately identified by measuring both spd hybridization, because the conduction of d-electrons can be neglected due to their localized characteristics and large effective mass24.As the AMR stems from changes in the longitudinal conductivity, the DC Drude modelThe independent probing of entclass1pt{minimaz-axis) and its transmission was measured. The polarization of the THz electric field lies along the y-axis . To measure the complex conductivity of sample, we prepared bare Si substrate as a reference which has almost identical thickness with that of the deposited sample. For this study, we prepared three Py films (#1—#3): #1 was used for angle dependent THz-TDS measurement  with 4 TPX lenses was used for THz measurement. Montana vacuum cryostat was used to control the temperature of sample. A standard glass windows were replaced with the TPX windows for THz experiment. The deposited film and reference substrate were installed simultaneously inside the cryostat using L-shaped sample holder. Two samples (deposited film and reference substrate) were attached to each plane of L-shaped holder that was attached at the ANC300 piezo rotator. By rotating the piezo rotator, we could selectively choose the deposited film or reference substrate without breaking the vacuum. An in-plane magnetic field was applied by using home-made vector electromagnet. The vector magnet is made of two axis electromagnets, each of which generates magnetic field of up to 100 mT. The directional magnetic field can be applied by adjusting the strengths of two orthogonal magnetic fields. The direction of the magnetic field is confirmed by sensing the magnetic field at sample position using Gaussmeter. To rule out any mechanical effects, every structure inside the vacuum chamber is made of OFHC  copper and brass which have very low magnetic susceptibility. Field-induced mechanical effects have been confirmed to be negligibly small . The error bars in Figs. For the case of experimental data Fig. , error bmentclass2pt{minimmentclass2pt{minimSupplementary Information.


Kaposi’s sarcoma (KS) is a rare tumour of uncertain nature. It may be a true cancer or an aggressive viral lesion, which can regress in many patients, and there is a real need for more research on the subject. This systematic review aimed to summarize the available evidence on somatic mutations and clonality within KS to assess whether KS is a neoplasm or not, concluding that knowledge is currently insufficient to determine whether KS is a clonal neoplasm (sarcoma), or simply an aggressive reactive virus-driven lesion.Background: It remains uncertain whether Kaposi sarcoma (KS) is a true neoplasm, in that it regresses after removal of the stimulus to growth (as HHV8) when immunosuppression is reduced. We aimed to summarize the available evidence on somatic mutations and clonality within KS to assess whether KS is a neoplasm or not. Methods: Medline and Web of Science were searched until September 2020 for articles on clonality or mutation in KS. Search strings were supervised by expert librarians, and two researchers independently performed study selection and data extraction. An adapted version of the QUADAS2 tool was used for methodological quality appraisal. Results: Of 3077 identified records, 20 publications reported on relevant outcomes and were eligible for qualitative synthesis. Five studies reported on clonality, 10 studies reported on various mutations, and 5 studies reported on chromosomal aberrations in KS. All studies were descriptive and were judged to have a high risk of bias. There was considerable heterogeneity of results with respect to clonality, mutation and cytogenetic abnormalities as well as in terms of types of lesions and patient characteristics. Conclusions: While KS certainly produces tumours, the knowledge is currently insufficient to determine whether KS is a clonal neoplasm (sarcoma), or simply an aggressive reactive virus-driven lesion. Kaposi sarcoma (KS) is an unusual neoplasm, which presents with clinical manifestations that range from those that are slowly progressive and confined to the skin, to an aggressive tumour that invades visceral organs. The clinicopathological characteristics are usually described in terms of the location of the lesions , and the clinical stage of the disease . Epidemiologically, KS presents in several forms: (a) a classic form predominantly in older men; (b) an endemic form in young men and children from central Africa; (c) an iatrogenic form secondary to treatment with immunosuppressive drugs, such as steroids; and (d) an epidemic, HIV/AIDS-associated form .KS was first described at the end of the 19th century as mainly occurring in elderly males from Mediterranean regions but was later reported in East and Central Africa with presentation in younger people . It was KS became the most common HIV-related “malignancy” worldwide, although HIV alone could not explain all of the cases. Its occurrence in patients on long-term immunosuppressive therapy and the predominance in males and HIV-negative immunocompetent individuals with increased risk for Sexual Transmitted Diseases (STDs) suggested the implication of another transmissible agent. Eventually human herpesvirus-8 (HHV8) was identified as the virus associated with KS in all different groups, including individuals in Eastern Europe, Africa and the United States .Depending on the clinical subtype, age-standardized incidence rates ranged from 0.3 in Eastern Asia to 8.5 ASR per 100,000 in Eastern Africa in 2020 . VariousA neoplasm is usually defined as “an abnormal mass of tissue, the growth of which exceeds and is uncoordinated with that of the normal tissues, and persists in the same excessive manner after cessation of the stimulus which evoked the change” . KS doesIntersecting with this established definition is the concept of clonality, which is not part of the long-accepted definition of a neoplasm and states that cancers evolve by an iterative process of clonal expansion . The dogThere are other tumours with similar issues. For example, transient neoplasia is an increasingly used concept, where diseases, such as nodular fasciitis, have a translocation but still spontaneously resolve in many cases . This isIt is likely to be viral in origin; however, there is still a debate about which virus is involved: HPV is the main suspect as in KS. There are other cellular proliferations that are oligoclonal and treated seriously but not regarded as cancers. Infectious mononucleosis  is driveWe know that most cancers start as a clonal proliferation from a single cell and acquire mutations with growth/progression, something that was described early by Knudson in his studies of retinoblastoma . HoweverKS is currently classified as a neoplasm by the WHO Classification of Tumours (WCT); however, this constitutes a controversial topic that continues to be reviewed periodically. Any future decision on its status will be informed by the best available evidence. The aim of this systematic review is to summarize any available published scientific evidence on KS with regard to somatic mutations and clonality in order to determine whether KS can be defined as a neoplasm or not. The results of this evidence synthesis will serve to inform future decisions of the WCT and detect possible evidence gaps.A systematic review was conducted to identify peer-reviewed articles of data on clonality or somatic mutations in KS following a protocol registered with PROSPERO (CRD42018087595). This review was performed following the PRISMA  guidelines and considering the recently published update of these guidelines in 2020 .Tailored conceptual strings of relevant keywords and database-specific terms were devised for the major concepts of Kaposi Sarcoma, Clonality and mutations. Conceptual strings were combined with Boolean operators using appropriate MeSH headings and filters to search the Medline and Web of Science databases for peer-reviewed articles published up to 01 September 2020, without date restrictions. The Cochrane library was also consulted, and the reference lists of relevant articles were checked for additional studies. The concepts and keywords used to inform the search strategy are detailed in Studies were excluded if they exclusively focused on: (1) Kaposi Sarcoma Herpes Virus (KSHV) mutations or viral clonality; (2) on cell lines/cultures; (3) describing tumour phenotype, proteins, receptors, vascular markers etc.; (4) mitochondrial genomes; or (5) the epidemiology or aetiology of KS; or if they were not original research, such as editorials, letters, narrative reviews and book chapters. Titles and abstracts were screened by two reviewers independently (I.C. and B.I.I.), and the full text of potentially eligible articles was assessed to decide final inclusion by both of them. The lists of included articles were compared and differences about the selected studies were resolved by consensus.All data were extracted by two researchers independently (B.I.I. and A.L.) into standardized data extraction forms. Discrepancies in data extraction were resolved by discussion and consensus. Relevant information was recorded from the selected publications, including the author(s), publication year, country where the study was performed, baseline population characteristics and demographics, study methodology, methods for clonality or mutation testing, main results on clonality, somatic mutations or chromosomal aberrations (only descriptive outcomes due to the nature of the studies) as well as COI disclosure. We registered frequency measures and ordered the rest of outcomes strategically in order to facilitate interpretation of the results. Data were recorded and compiled using Microsoft Excel.Risk of bias of included individual studies was performed using an ad hoc adapted version of the QUADAS2 tool for the Quality Assessment of Diagnostic Accuracy Studies . This asThe results were tabulated in Excel sheets and summary of findings tables were drafted to present the main outcomes for each of the included studies. Outcomes were grouped based on their focus: clonality, somatic mutations or chromosomal aberrations. A descriptive analysis was performed for a qualitative synthesis of the results. The performance of a meta-analysis to calculate an adjusted pooled estimate was ruled out because the descriptive nature of the retrieved studies means that a pooled summary of the data would not be useful. Additional important methodological heterogeneity between the studies from the reported outcome measures rendered formal quantitative synthesis inappropriate. Instead, the aim was to narratively assess and combine the studies in order to derive clinically meaningful conclusions about the nature of KS.Of the 3077 records identified and screened through the database searches, 25 publications were eventually deemed as relevant and retrieved for full text inspection and qualitative synthesis . Five ofResults of the included studies are summarised in Tornesello et al.  performeThe studies were performed using data from 13 different countries, and eight were international collaborations . The firClinical and morphological description of clonality studies: 34 (74%) of the 46 cases were HIV associated, 12 (16%) classic KS forms of all 17 (37%) studied nodular lesions, 3 (7%) were plaque lesion samples, and the 26 (57%) remaining cases were from 2 studies ,27, whicClinical and morphological description of mutation studies: in total, 84 29%) HIV associated cases were included, 146 (52%) classic KS and 2 (1%) cases of iatrogenic KS. For 49 (17%) cases, no clinical information was available since Nicolaides et al. , compris% HIV assClinical and morphological description of chromosomal aberrations studies: 58 (27%) of the 214 cases were HIV associated, 147 (69%) classic and 9 (4%) iatrogenic KS presentations. Only 3 studies ,37,39 reOutcomes assessing the clonal nature of KS provided by the 20 included studies  resultedDetermining clonality: outcomes on clonality in KS samples were mostly determined by monoclonal patterns of gene inactivation or methylation and obtained mixed results. Four case series ,25,26,27Detecting mutations: Ten studies aimed to detect mutations in KS lesions, focusing three on p53 mutations ,34,35 anDemonstrating chromosomal aberrations: Finally, of the five selected studies ,38,39,40Descriptions of clinical and morphological  descriptions, such as plaque, nodular and patch stage, were frequently incomplete but still showed an important diversity of case combinations when studying the different outcomes, as follows:We performed a systematic review of the peer-reviewed and published scientific literature on genetic alterations assessing the clonal nature of KS. The limited number of retrieved studies and the low-level of evidence of the retrieved studies ,43, togeThat said, there are several aspects of the pathogenesis of KS that are currently under investigation to determine the relevance of latency-associated nuclear antigen (LANA) and other HHV8 proteins in subverting normal endothelial cell biology to induce proliferation ,50 and tKRAS mutation of amplification in substantial numbers of advanced cases in which codons 13, 15, 16, 18 and 31 were implicated. A small number of patients with the TP53 mutation have been found and may be associated with progression [We retrieved only two studies with an appropriate study design for the investigation of such associations; however, both focused on epidemiological aspects of the disease and did not provide effect measures analysing the relationship of a mutation or clonality with KS. Nicolaides et al.  showed Kgression ,35. TP53gression . The abiClonality of the virus can be shown ,57 but iThey found that differences ‘strongly suggest that disseminated lesions represent multiple distinct primary expansions of HHV-8-infected spindle cells originating from different infectious events rather than metastatic proliferations’. It is known that EBV-induced lymphoproliferative disease starts as an oligoclonal proliferation and can lead to monoclonal diffuse large B-cell lymphoma with the adoption of additional genetic mutations; however, the same has not been shown for KS—although it remains a possibility.The chronic and slow evolution of classic-KS lesions, the partial reversibility of iatrogenic-KS lesions after diminution of immunosuppressive regimens and the complete epidemic-KS regression after anti-retroviral therapy seem more consistent with reactive proliferation ,26,38,40Detailed, and ideally longitudinal, NGS studies with examination of multiples of a range of early (cutaneous patch stage) and late  lesions in individual patients could provide evidence of clonal selection and progression. Such an approach would evaluate for neoplastic transformation of early lesions.In conclusion, insufficient scientific evidence exists to describe the nature of KS. Current knowledge is insufficient to determine whether KS is a clonal neoplasm, or simply an aggressive reactive virus-driven lesion. Although evidence from mechanistic studies that points towards a reactive virus-driven process that gives rise to a clonal neoplasm is mounting, direct evidence for the pathogenic pathway would allow an evidence-based assessment of KS neoplastic status with all the implications that this may have for clinical practice and patient care. Its current neoplastic classification as a sarcoma is questionable, and regarding it as a ‘Kaposi tumour’ may be more accurate.


Vibrio vulnificus has not been widely studied. The aim of this study was to investigate the inhibitory effect and germicidal activity of OEO on V. vulnificus and its possible inhibition mechanism. The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of OEO against four V. vulnificus strains (ATCC 27562 and three isolates from seafoods) were from 0.06 to 0.15 μL/mL. Compared with untreated bacteria, OEO reduced the concentration of intracellular adenosine triphosphate (ATP), hyperpolarized the cell membrane, increased the level of reactive oxygen species (ROS), and increased the concentration of intracellular malondialdehyde (MDA), but there was no obvious DNA damage at the OEO test concentration. It was indicated that OEO inactivated V. vulnificus by generating ROS which caused lipid peroxidation of cell membranes, thereby reducing the permeability and integrity of cell membranes and causing morphological changes to cells, but there was no obvious damage to DNA. In addition, OEO could effectively kill V. vulnificus in oysters at 25 °C, and the number of bacteria decreased by 48.2% after 0.09% OEO treatment for 10 h. The good inhibitory effect and bactericidal activity of OEO showed in this study, and the economy and security of OEO make it possible to apply OEO to control V. vulnificus contamination in oysters and other seafoods.Oregano essential oil (OEO) is an effective natural antibacterial agent, but its antibacterial activity against  Vibrio vulnificus is a Gram-negative food-borne opportunistic pathogen with slightly curved shape, extreme single flagella, low salinity and [V. vulnificus widely exists in estuaries and coastal areas all over the world, and is often isolated from oysters, fish, shrimp and other aquatic products [V. vulnificus is the leading cause of seafood related disease and is increasing in prevalence globally [V. vulnificus by ingesting raw or undercooked seafood or by contacting contaminated seawater in wounds. Food intake can lead to gastroenteritis and sepsis, and wound contact can lead to tissue necrosis near the wound [V. vulnificus infection cases often have acute onset, high mortality, high disability rate and great harm, which has important public health significance [nity and . V. vulnproducts . V. vulnglobally . Humans he wound . The morhe wound . At presificance  that hasificance .V. vulnificus in oyster, shrimp, crab and other seafood, so as to reduce the risk of foodborne diseases of V. vulnificus in these foods. Several prevention and control methods have been proposed, including gamma irradiation [V. vulnificus. In recent years, a variety of natural, environmental friendly and safe plant essential oils have attracted extensive attention because of their good anti-bacterial effects [It is very important to employ a safe and effective alternative method to control adiation , modifieadiation , and cheadiation . These madiation , but theadiation . Moreoveadiation . Chemicaadiation . Therefo effects .Origanum vulgare L.) is an aromatic plant with a wide distribution throughout the Mediterranean area and Asia [V. vulnificus have rarely been reported.Oregano , malondialdehyde (MDA), DNA damage, cell membrane integrity and cell morphology. In addition, the antibacterial activity of OEO against V. vulnificus in contaminated oyster samples was evaluated.Based on the above factors, the purpose of this study was to study the antibacterial activity and mode of action of OEO against v/v), which has no apparent effect on the growth of V. vulnificus.OEO  and dimethyl sulfoxide  were purchased from Sigma-Aldrich . Alkaline peptone water (APW) and 3% NaCl tryptone soya agar (TSA) were purchased from Land Bridge Technology Co. . All other chemicals and reagents were of analytical grade. Before each assay, OEO was dissolved in DMSO and vortexed for 30 s at room temperature. The final concentration of DMSO in all of the sample solutions (treatment and control samples) was 0.1% . The other three V. vulnificus strains  were obtained from the Chinese University of Hong Kong laboratory and were originally isolated from seafood collected from supermarkets. Only V. vulnificus ATCC 27562 was used for antibacterial mechanism analysis. ATCC 27562 and three isolates  were used for the experiment in fresh oysters. All strains were stored in 3% (w/v) NaCl APW with 20% (v/v) glycerol at −80 °C. Stock cultures were streaked in 3% NaCl TSA and grown at 37 °C for 14 h prior to each experiment. A loop of the strain was then inoculated into 30 mL APW and cultured in a shaking table (130 rpm) at 37 °C for 16 h to the late logarithmic growth stage. The bacterial culture was washed twice with phosphate-buffered saline , then subsequently adjusted to OD600 nm = 0.5 (~4 × 108 CFU/mL) with APW or PBS and standby.V. vulnificus strains was measured using the broth microdilution method as described by Clinical and Laboratory Standards Institute guidelines [5 CFU/mL. OEO was added to each well to achieve final concentrations of 0.48, 0.24, 0.12, 0.06, 0.03, 0.015 or 0 (control) μL/mL. APW broth containing 0.1% DMSO was used as the blank background sample. APW containing 0.1% DMSO dissolved with 0.1 mg/mL chloramphenicol was used as the positive control. The MICs were defined as the lowest concentration of OEO that resulted in no visible V. vulnificus growth within 24 h. In order to determine MBCs, 100 μL of bacterial solution was taken from each well showing inhibition, plated on 3% NaCl TSA agar and cultured for 48 h, and the lowest antimicrobial concentration that did not allow any bacterial growth was recorded as the MBC.The MICs of OEO against idelines , with mi6 CFU/mL to obtain the final concentrations of 1/16MIC, 1/8MIC, 1/4MIC, 1/2MIC and MIC. V. vulnificus ATCC 27562 with or without OEO was incubated at 37 °C and cell growth was determined by measuring OD600 nm every 1 h using a multimode plate reader  for 24 h. Three independent replicates of each experiment were performed.The method was followed to determine growth curve as described by Shi et al. , with slV. vulnificus ATCC 27562 was tested using the plate count method described by Ashrafudoulla et al. [6 CFU/mL, and then mixed with OEO to achieve the final concentrations of 0 (control), 1/4MIC, 1/2MIC, and MIC. The prepared samples were incubated at 37 °C for 0, 20, 40, 60, 90 and 120 min. Subsequently, 100 μL of each sample was spread onto 3% NaCl TSA plate and incubated at 37 °C for 14 h. This experiment was repeated three times independently.The bactericidal activity of OEO against a et al. , with so8 CFU/mL) was prepared according to the method described in g for 5 min, the supernatant was gathered and stored on ice to prevent ATP loss. Intracellular ATP was measured with an ATP assay kit . Briefly, the mixtures (125 μL ATP assay working solution and 125 μL supernatant/125 μL ATP gradient standard solution) were added to white, opaque, 96-well microtiter plates, and the supernatant fluorescence values were measured with a microplate reader . A standard curve was constructed based on ATP standard solutions  and their corresponding luminescence values to calculate the concentration of ATP in the sample cells. Three independent replicates were performed for each experiment.Intracellular ATP concentration determination was carried out as described by Sanchez et al. , with sl8 CFU/mL) was prepared according to the method described in 4(3)) was added to each well to a final concentration of 1 μmol/L. After sample incubation at 37 °C for 30 min, OEO was added to final concentrations of 0 (control), MIC and 2MIC. Fluorescence measured by a fluorescence microplate reader (Spectra Max M2) at excitation and emission wavelengths of 492 and 515 nm were measured at 1, 4, 8 and 12 min, respectively. Membrane potential was illustrated in terms of relative fluorescence units  − (FUcontrol − FUbackground)), with three independent replicates included in each experiment.The measurement method of membrane potential refers to that described by Yang et al. . Briefly8 CFU/mL) prepared according to According to Shivaprasad et al. , the flu6 CFU/mL. Cultures of V. vulnificus ATCC 27562 with and without OEO treatment (0 (control), 1/4MIC, 1/2MIC and MIC) were incubated at 37 °C for 30 min. After centrifugation to collect bacteria, the extract was added according to the proportion of 5 million bacteria to 1 mL. Subsequently, the samples were centrifuged for 10 min at 8000× g, and the supernatants were collected. The contents of MDA in the supernatants were determined according to the instructions in the lipid peroxidation MDA assay kit  by using a multimode microplate reader (Spectra Max M2) at 450 nm, 532 nm and 600 nm. MDA content (nmol/mL) = 5 × (12.9 × (ΔA532 nm − ΔA600 nm) − 2.58 × ΔA450 nm).The determination of MDA content was carried out as described by Cao et al. , with slV. vulnificus caused by OEO as described by Kim et al. [V. vulnificus (~4 × 108 CFU/mL) was treated with OEO (0 (control), MIC, 2MIC and 4MIC) at 37 °C for 2 h. The treated samples (75 μL) were mixed with 90 mL Comet Agarose, 5 mg/mL RNase A solution (SigmaeAldrich), 0.25% N-Lauroylsarcosine sodium salt solution (SigmaeAldrich) and 0.5 mg/mL lysozyme (Sigmae Aldrich). Then, 75 μL of the mixture was placed on a Comet slide and refrigerated at 4 °C in the dark for 15 min to solidify on the slide. Slides were immersed in 1× lysis buffer (pH 10) for 45 min and then in alkaline solution for 30 min in the dark at 4 °C. Subsequently, the slides were electrophoresed in alkaline running buffer at 12 V and 100 mA for 20 min. After electrophoresis, the slides were soaked and rinsed three times in distilled water, and then placed in pre-cooled 70% (v/v) ethanol. After 5 min, the ethanol was blotted dry and the slides were allowed to air dry naturally. Finally, 100 μL of Vista Green DNA dye was added to each well of the slides, and the samples were visualized by fluorescence microscopy  under a 100× oil microscope.The comet assay for detecting DNA damage of m et al.  and sligV. vulnificus ATCC 27562 was determined using two nucleic acid dyes, SYTO 9 (green) and propidium iodide , as described by Qi et al. [V. vulnificus (~2 × 108 CFU/mL) was treated with OEO (0 (control), MIC, 2MIC and 4MIC) and incubated at 37 °C for 30 min. The cell suspension was then centrifuged at 10,000× g for 5 min and resuspended in 200 μL of 0.85% (m/v) NaCl. Then, 0.5 μL of a mixed dye of PI and SYTO 9 was added to the bacterial resuspension solution and incubated for 5 min at room temperature. Untreated V. vulnificus served as a control sample. Finally, 5 μL of the sample was placed on a glass slide and observed at 400× magnification using a confocal laser scanning microscope .The cell membrane integrity of i et al. , with mi8 CFU/mL) obtained according to the method described in The FESEM assay was performed according to the method previously published by Tian et al. with slight modifications . Bacterig, 10 min, 4 °C) and washed twice with PBS. Afterwards, the cells were maintained in 2.5% (v/v) glutaraldehyde at 4 °C to fix the cells. The cells were harvested by centrifugation after 12 h and dehydrated with 30%, 50%, 70%, 80%, 90% and 100% ethanol for 10 min, shaken at each concentration. Finally, the samples were mounted on a FESEM stand for sputter-coated gold under vacuum and examined under a scanning electron microscope .Then after 2 h incubation at 37 °C, the cells were centrifuged . Later, equal volumes of the four bacterial suspensions were then mixed for use in the following assays. Fresh oysters cleaned under sterile conditions were immersed in 3% (v/v) NaClO for 20 min to remove the pre-existing bacteria. The oysters were then washed twice with sterile water to remove residual NaClO. The oyster meat was mixed with an equal amount of PBS and stirred to obtain an oyster homogenate (dilution 1:2). The mixed bacteria suspension was added into the oyster homogenate so that the concentration of V. vulnificus in the oysters was 106 CFU/g. Then, OEO was added to aliquots of a mixed solution of bacterial suspension and oyster homogenate to make final concentrations of OEO of 0 (control), 0.03%, 0.06% and 0.09% (v/v). The samples were incubated at 25 °C and collected at 0, 2, 4, 6, 8 and 10 h, then diluted with PBS and spread on 3% NaCl TSA plates and counted. (The results were expressed as log CFU/g.)Fresh oysters were purchased from the local supermarket . Suspensions of t-test and analyzed using IBM SPSS software . p < 0.05 was considered significant, p < 0.01 was considered extremely significant.All experiments were performed in triplicate independently. All data are presented as the mean ± standard deviation (SD); differences between means were tested using a Student’s V. vulnificus strains tested .The antibacterial effect of OEO on 2 = 0.999, the standard curve was shown in the V. vulnificus decreased extremely significantly (p < 0.01) as the OEO concentration increased , MIC, and 2MIC, and the relative fluorescence intensity increased negatively, which was extremely significant when compared with the control sample (p < 0.01). Moreover, as the concentration of OEO increased, the hyperpolarization of the cell membrane of V. vulnificus increased, and with the increase of time, the cell membrane hyperpolarization also showed an increasing trend.The effects of OEO on the membrane potential of V. vulnificus ATCC 27562 treated with OEO. As shown in the figure, the ROS level in the V. vulnificus cells increase with the enhanced concentrations of OEO. The ROS level of V. vulnificus cells was 2.15 ± 0.04 in the control sample, while the levels cells treated with OEO at 1/4MIC, 1/2MIC and MIC were 3.51 ± 0.39, 4.64 ± 0.45, 18.62 ± 0.55 after 30 min, respectively.V. vulnificus treated with OEO increased in a concentration-dependent manner. When V. vulnificus was treated with OEO at the concentration of 1/2MIC and MIC, the content of intracellular MDA increased significantly from 0.30 ± 0.09 nmol/mL to 0.67 ± 0.12 nmol/mL and 1.37 ± 0.27 nmol/mL, respectively (p < 0.01).The production of MDA was analyzed by the thiobarbituric acid method. As shown in V. vulnificus.The comet assay was used to study the effect of different concentrations of OEO on DNA damage. As shown in V. vulnificus cells emitted yellow or red fluorescence with little green fluorescence  reduced the total V. vulnificus counts in fresh oysters to 6.39 ± 0.05, 4.16 ± 0.03, and 3.39 ± 0.10 log CFU/g, respectively. Among them, 0.06% and 0.09% (v/v) treatments reduced the number of bacteria by 29.6% and 48.2% after 10 h.An artificially contaminated oyster model was constructed to evaluate the bacteriostatic effect of OEO against d medium . At 0 h,V. vulnificus with MIC values ranging from 0.06 to 0.15 μL/mL (50 to 140 μg/mL) . Based on the above information, in this study, the antibacterial activity of OEO against V. vulnificus was superior to most of these natural compounds in the literature. Early in our study, the MIC of carvacrol, the main component of OEO, against V. vulnificus ATCC 27562 was determined to be 50 μg/mL. Except carvacrol, OEO contains a variety of antibacterial active components, and the combination of two or more components may produce synergistic effects and improve the antibacterial activity [In this study, OEO exhibited inhibitory activity against four strains of 0 μg/mL) . The inho et al.  determin90 μg/mL . In addi90 μg/mL  determinr et al. , OEO wasactivity . CompareV. vulnificus compared with the control sample (p < 0.01) (Cronobacter sakazakii treated with 0.4 mg/mL coenzyme Q0 decreased extremely significantly after 20 min (p < 0.01). The decrease of intracellular ATP concentration may be caused by the increase of membrane permeability after treatment with natural products, which leads to the release of ATP [V. vulnificus may change the existing active system to cope with environmental pressure when treated with OEO, which have been shown to affect the steady-state changes of the ATP secretion efflux pump [ATP is one of the most important energy-related molecules in bacteria and is closely related to the cellular function, growth and survival of microorganisms . The res < 0.01) . Similare of ATP . Moreovelux pump  or the Alux pump .V. vulnificus cells in a concentration- and time-dependent manner  during 20 days of storage at 4 °C. Meanwhile, Hernandez et al. [Salmonella enteritidis and E. coli in air dried beef. The current study showed that the inhibitory effect of OEO against V. vulnificus was better in APW medium (igh risk . Thus, a oysters . Similarz et al.  discusseW medium  than in W medium . This maV. vulnificus, as presented in V. vulnificus in fresh oysters. Therefore, OEO has potential for use in oysters and other seafoods industry as a natural antibacterial agent to reduce the risk of V. vulnificus infection. Therefore, OEO has the potential to be used as a natural antibacterial agent in the oyster and other seafood industries to reduce the risk of V. vulnificus infection.In conclusion, our study reported the good antibacterial activity and mode of action of OEO against


Kounis syndrome (KS) has been defined as acute coronary syndrome (ACS) in the context of a hypersensitivity reaction. Patients may present with normal coronary arteries (Type I), established coronary artery disease (Type II) or in-stent thrombosis and restenosis (Type III). We searched PubMed until 1 January 2020 for KS case reports. Patients with age <18 years, non-coronary vascular manifestations or without an established diagnosis were excluded. Information regarding patient demographics, medical history, presentation, allergic reaction trigger, angiography, laboratory values and management were extracted from every report. The data were pulled in a combined dataset. From 288 patients with KS, 57.6% had Type I, 24.7% Type II and 6.6% Type III, while 11.1% could not be classified. The mean age was 54.1 years and 70.6% were male. Most presented with a combination of cardiac and allergic symptoms, with medication being the most common trigger. Electrocardiographically, 75.1% had ST segment elevation with only 3.3% demonstrating no abnormalities. Coronary imaging was available in 84.8% of the patients, showing occlusive lesions (32.5%), vascular spasm (16.2%) or normal coronary arteries (51.3%). Revascularization was pursued in 29.4% of the cases. In conclusion, allergic reactions may be complicated by ACS. KS should be considered in the differential diagnosis of myocardial infarction with non-obstructive coronary arteries. It is well known that the cardiovascular system is heavily involved in allergic reactions with potential symptoms including hypotension, arrhythmias and ventricular dysfunction ,2. One nWe searched PubMed until 1 January 2020 for case reports and case series of patients presenting with KS. Using “Kounis [TIAB]” as our search parameters, we identified 517 unique publications that we subsequently reviewed. After excluding those without case reports, not carrying a definite KS diagnosis, including patients < 18 years old, or not being in English, we found a total of 247 papers presenting 276 patients. We additionally included another 12 case reports not originally diagnosed as KS but identified as such by letters to the editor of the respective journals. Overall, we were able to detect 288 cases of KS in prior literature .After carefully reviewing all individual reports, we created a patient level database that was used for the present analysis. Multiple variables were collected for every case. The syndrome type was either specified by the original report or defined according to KS classification if enough information was available. Age, gender, pre-existing medical conditions and allergic reaction trigger were extracted for all of the patients. Regarding the clinical presentation of the syndrome, all initial symptoms were registered in our database and categorized into cardiac and allergic. Electrocardiographic findings on presentation were also available. We captured initial management  as well as medical therapy on discharge. In cases that coronary angiography or other coronary imaging studies were pursued, we compiled data on the coronary anatomy, pathophysiologic mechanism identified (clean coronaries vs. atherosclerosis vs. spasm) and revascularization strategy, if applicable. The initial and follow-up ejection fractions were included when available. If echocardiographic results were described but no specific percentage was mentioned, this was manually calculated by the authors. Finally, we assembled all of the accessible information on cardiac, allergic and inflammatory biomarkers. A detailed list of all collected parameters is summarized in The collected findings are reported as the mean or percentage for continuous and categorical variables, respectively. Only the patients with available data were included in the calculation of every parameter. In terms of biomarkers, the results were reported as elevated or not elevated rather than exact numbers to account for normal range variability between different labs. All descriptive statistics were analyzed using IBM SPSS Statistics.Of the 288 patients with KS that were analyzed, 71.2% were male and 28.8% were female and the average age was 54.1 years. The most common type was KS type I, while KS type III was the least frequent. Past medical history of a prior allergic reaction was documented in 31.4% and prior CAD in 21.9% of cases. Traditional cardiovascular risk factors including hypertension, diabetes mellitus, smoking and dyslipidemia were only present in a small fraction of patients . With reAs part of the standard acute coronary syndrome work-up, electrocardiography and cardiac biomarkers were available for almost all of the patients. The most common finding was ST elevation while only 3.3% of patients had a normal EKG and troponin elevation was seen in 77.5% of patients. Despite this impressive clinical presentation, 50.6% of the subsequently performed catheterization procedures failed to reveal any changes in the coronary arteries and hemodynamically significant lesions due to atheromatous or thrombotic lesions requiring revascularization were only found in one third of the cases. The most frequently involved vessels were the left anterior descending and the right coronary artery .Inflammatory cells and biomarkers that are frequently implicated in allergic reactions include tryptase, eosinophils and immunoglobulin E (IgE). Amongst cases with reported biomarker values, elevation of tryptase, eosinophils and IgE was found in 80.6%, 58.0% and 75.7% of cases, respectively.12. With regards to the allergic reaction, multiple patients received steroids, epinephrine, calcium channel blockers and/or antihistamines but again no reliable pattern was identified.Finally, the long-term management of patients with KS was variable. Antiplatelet therapy following the hospitalization was consistent with the results of the cardiac catheterization, with 41.2% getting discharged on aspirin and 35.1% on a P2YThe main findings of the present review include: (1) allergic reactions may be complicated with acute coronary syndrome, (2) many patients with KS present in a young age and have no risk factors of CAD or history of prior allergic reaction, (3) even though most of the patients present with ST-elevation myocardial infarction, only a small fraction will require coronary revascularization, (4) many inflammatory biomarkers including tryptase, eosinophils and IgE have been found to be elevated and(5) no algorithm for the management of KS is currently available.Allergic reactions have multiple manifestations, including skin and mucosal surface changes, gastrointestinal tract upset, respiratory distress as well as cardiovascular symptoms. Our review, as well as previous studies, demonstrate that hypersensitivity may be complicated by acute coronary syndrome and myocardial ischemia . This fiInflammatory cells, including eosinophils, macrophages and mast cells, play a significant role in hypersensitivity reactions. They initiate the inflammatory cascade by releasing mediators, including leukotrienes, thromboxane, IgE, tryptase, histamine and cytokines, that act on multiple targets located on various organ systems . At the A wide variety of KS triggers has been previously described . In our Clinical presentation and management of patients with KS also appears to be of a particular interest. Chest pain was the most common presenting symptom and most patients also exhibited allergic symptoms such as arash, swelling and itching. Despite this, cases without cardiac symptomatology found to have ischemia either by EKG changes or biomarker elevation have been reported . On the Regarding the management of KS, we observed wide variability between cases, likely stemming from the absence of predetermined algorithms. Optimal treatment consists of concomitant management of myocardial ischemia and the allergic reaction. This can be clinically challenging since medications treating one condition may exacerbate the other . The useWith regards to managing the allergic component, we found that corticosteroids were the most used medication on admission, and given their limited cardiac side effects, their use in KS appears to be safe. Antihistamines that were the second most utilized class are also safe in patients with ACS but must be used with caution since rapid intravenous administration may lower blood pressure . FinallyThe management of ACS in the acute phase should follow the existing guideline recommendations primarily based on electrocardiographic criteria, since no specific recommendations are currently available ,28. In oFinally, patients with KS type I presenting with ST-segment elevation myocardial infarction in the absence of obstructive coronary disease in KS more likely suggests resolved epicardial spasm and/or resolved or ongoing acute microvascular spasm/disturbance. Although the pathophysiology, course, treatment and prognosis require further investigation, we think that KS Type I meets the criteria for myocardial infarction in the absence of obstructive coronary artery disease (MINOCA) and should be incorporated in future guidelines. Such patients should also be further evaluated and treated according to the current MINOCA recommendations .Important limitations should be considered when interpreting our results. Stemming from previously published case reports, our findings are subject to publication bias. There was no uniformity in data presentation across different included reports, resulting in a significant number of missing variables in the created database. Coronary angiography, which is required for definite classification of KS type, was not performed in some cases. We included all cases of KS identified as such by the authors or letters to the editor of the journals. Despite this, given that KS represents an underdiagnosed entity, we may have missed other cases published in literature but not accordingly labeled.In conclusion, hypersensitivity reactions regardless of the initial trigger may be complicated by ACS, and KS appears to be an underdiagnosed entity. Management should be tailored to the underlying pathophysiology according to the most recent guidelines for ACS management. KS type I should be considered in the differential diagnosis of MINOCA.