Laryngeal granulomas are benign lesion that rarely occurs after surgery of laryngeal cancer. Until now there has not been standard treatment for it.The patient was diagnosed with laryngeal neoplasm one and half a month ago. Endoscopic low-temperature plasma knife in the radical excision of left vocal cord was performed under the general anesthesia. Postoperative histopathological examination confirmed left vocal cord tumor was highly differentiated invasive squamous cell carcinoma (SCC). Then the patient suffered unexplained intermittent dyspnea which persisted nearly 1 month after the surgery. Laryngoscope examination showed granulation formation on the glottis.The patient was diagnosed with laryngeal granuloma 1 month after the surgery of laryngeal cancer.The patient received resection of the laryngeal mass, and pathological examination confirmed the granuloma. Postoperative radiotherapy (RT) was performed within 24 hours after surgery.The patient was followed up for 3 years after surgery and the laryngeal granuloma and laryngeal cancer did not recur during follow-up. The symptoms of intermittent dyspnea disappeared and a satisfactory outcome was achieved.Usually for primary laryngeal granulomas, surgical treatment alone is not enough, because it is easy to relapse. RT within 24 hours after operation can significantly reduce the recurrence of laryngeal granuloma. However, laryngeal granulomas rarely occur after surgical resection of laryngeal cancer. To date, this is the first reported case of glottic granuloma occurring after laryngeal neoplasm operation.Laryngeal granulomas are uncommon benign disease with no exact cause yet. SCC of the glottis is performed with surgery or RT depending on the extent of the disease.Laryngeal cancer is the second most common type of head and neck malignancy worldwide, with estimated 151,000 new cases and 82,000 deaths annually in the world. However, surgical excision is associated with high recurrence rates.,7 Low-dose postoperative RT has been proved capable of decreasing the recurrence rate of primary granuloma. As for laryngeal granuloma after laryngeal surgery, there is no standard treatment for this rare disease. We report this rare disease and use preventive dose radiotherapy (RT) within 24 hours after surgery to decrease the recurrence of laryngeal granuloma and laryngeal cancer.For primary granuloma, surgery remains as the standard treatment for laryngeal granuloma.2A 66-year-old woman was admitted to hospital with unexplained intermittent dyspnea and hoarseness that had persisted for 1 month. The Karnofsky score was 90. The patient had no history of chronic diseases and family medical history. The left vocal cord and right vocal cord tumor resection were performed by endoscopic low-temperature plasma knife one and half a month ago. The postoperative histopathology showed the tumor cells were an invasive, highly differentiated SCC on the left vocal cord and squamous dysplasia on the right vocal cord, which conformed to the typical features of well-differentiated invasive squamous cell  with mechanically injured vocal cord mucosa and demonstrated that the cause of laryngeal granuloma may be the combination of 2 factors: gastric reflux and mechanical injury. Martins RH clarified that Intubation granulomas occur more frequently after prolonged intubation, especially in females. Besides the aforesaid factors, voice abuse is also the frequent factor for laryngeal granuloma. In our case, the patient is a female and suffered prolong intubation during the radical surgery of laryngeal cancer, both of which may contribute to the formation of laryngeal granuloma.In our PubMed search, there are some cases of primary vocal granuloma, albeit rare. But our case is the first reported case of glottic granuloma occurring after laryngeal neoplasm operation. When it comes to laryngeal granuloma, Clawsen first described laryngeal granulomas resulting from endotracheal intubation, and started to draw others’ attention to the study of laryngeal granuloma in 1932.,12 Surgery is still the predominant treatment for granuloma. However, patients with granuloma submitted to only sugery have high recurrence rates, ranging from 50% to 92%.,13 Duan Hong-Gang studied that the repeated operation had no significant effect on recurred patients. The treatment of laryngeal granuloma had to be treated by surgery together with other treatments. Duan Hong-Gang found that combination surgery with proton pump inhibitors (PPIs) can reduce the recurrence from 50% to 38.4%. Yongli's survey had demonstrated the effectiveness of surgical removal followed by immediate radiation therapy in the management of refractory vocal process granuloma (VPG). In Yongli's study, low-dose RT combined with surgery resulted in a recurrence rate of 0% during the follow-up examinations of the next 3 to 6 years. With recent studies, postoperative RT with low-dose has showed great advantages in treating non-malignant disease.,16 In our case, postoperative RT was necessary to be done.Clinical treatments of granuloma, according to many academic literature, may consist of surgical excision, reflux treatment, voice therapy and the other approaches targeting predisposing factors.,17 During Several hours after the surgery, the granulation began to form. The main components of the granulation were the immature fibroblasts, the unstable collagen fibers and the new blood capillaries. All of them are sensitive to the radiation. The postoperative RT within 24 hours can effectively depress the reprolification of the lesion. Our patient was treated with RT immediately after surgery of laryngeal granuloma.The pathology of laryngeal granuloma is hyperplasic fibrous tissue, the formation of which is same as benign proliferation of normal tissues, such as skin keloid. For T1 glottic SCC, the optimal treatment strategy is not well-defined. In a single-institution retrospective analysis of 244 patients with T1–2 glottic SCC, the 5-year RFS was higher for patients who received RT as any part of their treatment (either definitive or adjuvant) than for those who underwent surgery alone . Especially, in patients with T1 primaries, the 5-year RFS rates with primary RT vs. surgery were 83 and 75%, respectively (P = .05). In our case, we should both reduce the risk of recurrence rate of the laryngeal cancer and eliminate laryngeal granuloma concurrently. As for the first case of this kind, the dose of postoperative RT needed to be safely handled. After many discussions, the patient was given 50 Gy, namely, preventive dose, which cannot only decrease the recurrence of laryngeal granuloma and laryngeal cancer, but also prevent secondary malignant mucosa caused by high-dose RT. Now the symptoms including intermittent dyspnea and hoarseness have disappeared and the patient is currently alive with no evidence of recurrence.With rare reported accidents, there is no consensus in the literature regarding the best dose for laryngeal granuloma happening after laryngeal cancer. Usually, the low-dose RT (10–30Gy) is sufficient for unspecified laryngeal granuloma.Conceptualization: Jingyi Wu.Funding acquisition: Lijuan Ding.Investigation: Jingyi Wu, Lijuan Ding.Methodology: Lijuan Ding.Project administration: Lijuan Ding.Resources: Lijuan Ding, Lihua Dong.Software: Yu Wu, Lijuan Ding, Lihua Dong.Supervision: Lijuan Ding.Validation: Lihua Dong.Visualization: Lihua Dong.Writing – original draft: Jingyi Wu.Writing – review & editing: Jingyi Wu, Tongchao Jiang.


Mycobacterium bovis strain heterogeneity on disease transmission dynamics; possible impacts of concurrent endemic infections on the disclosure of truly infected animals; climatological differences and change coupled with environmental contamination. We further argue that control and eradication of this complex disease may benefit from an ecosystem level approach to management. We hope that this perspective can stimulate a new conversation about the many factors potentially impacting bTB eradication schemes in Britain and Ireland and possibly stimulate new research in the areas identified.Successful eradication schemes for bovine tuberculosis (bTB) have been implemented in a number of European and other countries over the last 50 years. However, the islands of Britain and Ireland remain a significant aberration to this trend, with the recent exception of Scotland. Why have eradication schemes failed within these countries, while apparently similar programs have been successful elsewhere? While significant socio-economic and political factors have been discussed elsewhere as key determinants of disease eradication, here we review some of the potential ecological and epidemiological constraints that are present in these islands relative to other parts of Europe. We argue that the convergence of these potential factors may interact additively to diminish the potential of the present control programs to achieve eradication. Issues identified include heterogeneity of diagnostic testing approaches, the presence of an abundant wildlife reservoir of infection and the challenge of sustainably managing this risk effectively; the nature, size, density and network structure of cattle farming; potential effects of  Mycobacterium bovis is a zoonotic disease, primarily affecting livestock, which is of economic importance to the European Union (EU) due to its impact on trade. Indeed, at the inception of the European project, as the European Economic Community (EEC), the first legal initiatives were taken to combat the disease in 1964 with the drafting of council directive 64/432/EEC (Bovine tuberculosis (bTB) caused by M. bovis  in GB has been doubling at a rate of every 10 years. Prior to the Foot and Mouth Disease epidemic of 2001, the doubling rate was every 5.2 years’  to detect skin thickness increases indicative of infection , a comparative test is used  / cervical intradermal test (CIT) is used, involving the inoculation of nfection . Convers is used . The sinc metric . The SITc metric : SIT senc metric . It is pc metric  despite In continental European countries, which do not have the same problem as Britain and Ireland, bTB testing programs can have radically different outcomes. For example herd de-population upon the detection of reactor animals was common in France  – effectDeployment of gamma interferon, as a higher sensitivity ancillary test, in Britain and Ireland is variable – in Northern Ireland, participation in testing has been voluntary, with no statutory powers in place to remove test positive animals . In the Other European countries such as Switzerland also use the practice of taking inconclusive reactors from skin test positive herds ; howeverCompartmentalisation of regions within European countries, based on relative prevalence has been used to contain infection and prioritise resources . However It is conceivable that the program differences discussed above have compounded the ongoing problem Britain and Ireland have with bTB, however it is currently difficult to quantify the magnitude of these impacts.In summary, these differences in tuberculin potency, application of skin test formats and heterogeneity in downstream choices in program management could have had a divergent effect on the bTB outcomes observed in Britain and Ireland vs Continental Europe. However, it is extremely difficult to untangle their relative importance , especially against the background of such differing epidemiological  contexts. Future research efforts, including “big data”, could assess differing interventions in settings with similar prevalence. Such approaches are predicated on better data harmonisation and sharing.Odocoileus virginianus; . Research suggests that both the size of herds [e.g.,  of the bacillus. The fact that Eu1 strains were exported during the time of Empire suggests this lineage may have been at the very least, common for a considerable period of time in Britain and Ireland. Therefore any speculation on a fitter phenotype evolving within Britain and Ireland may be moot. Allen et al. , with the immunological response compromising tests used to disclose infected animals (Mycobacterium species such as M. avium paratuberculosis (MAP or Johne’s disease) can confound the immunological diagnosis of bTB through cross-reactivity  infection has been associated with a negative impact on the disclosure of bTB using the experimentally-infected cattle model and SCIT testing  at the cooler temperature, whilst results for differing soil types were inconclusive , it has been noted that there is considerable heterogeneity in cell wall content as detected by Fourier Transform Infra-Red Spectroscopy  territory within Britain – it is part of the British isles, badgers reside there  especially in the lowlands, there is a significant cattle industry, and similar tests and testing regimes have been employed as in the rest of the British Isles. However, the relative magnitude of these characteristics is worth dwelling upon.−2  is high risk and future climate changes may exacerbate this problem . The conScotland is on a quadrennial testing regime, with herds tested at least once every four years , therefoAn argument could be made that Scotland reduced historic bTB levels to a low enough level to allow cattle measures alone to achieve freedom. Perhaps then, the historical reduced wildlife and cattle densities were then sufficient to act as a bulwark against recrudescence and the establishment of endemic disease? In fact, GB was very close to eradication in the 1980s before bTB re-emerged significantly in the 30 years since . During In this article we have attempted to propose potential reasons why the British and Irish experience in eradicating bovine tuberculosis has been so fraught compared to that of other jurisdictions in recent times. Our suggestions have arisen from a broad comparative approach which contrasts landscape, ecological, animal husbandry and molecular epidemiological characteristics within Britain and Ireland to those primarily observed on the wider European Continent. We note, with caution, that correlation is not causation. However for all proposed factors, we have endeavoured to present a coherent narrative, supported by published evidence, which links each to pertinent aspects of bTB epidemiology. We do not propose that these potential factors are exhaustive, merely that they may be worthy of further investigation, and individually or collectively may constitute novel hypotheses that go some way to explaining the comparative lack of progress in bTB eradication in these islands.M. bovis maintenance, a number of endemic infections that could impact on the diagnosis and transmission of bTB, an evolutionary lineage of the pathogen unique to Western Europe and a large, highly connected, dense network of farms where the movement of infected animals could be facilitated, partially due to the limitations of the statutory test at the individual level.Our hypothesis is that owing to their history, ecology and geography, Britain and Ireland may occupy a “goldilocks zone” for bovine TB. Factors highlighted in this review include the presence of a sufficient wildlife reservoir, a potentially amenable environment for As regards further investigation, we propose a wider scale comparison of all listed factors across Britain and Ireland, and their association with risk of bTB persistence and other pertinent epidemiological outcomes, contrasted to territories / regions with lower bTB prevalence. The latter may help to ascertain if any of the factors have a significant impact on bTB eradication efforts and also to quantify their relative importance. The latter type of investigation could be achieved in two ways:Aggregating all retrospective information for the listed factors across multiple patches of interest across Britain and Ireland into a single data resource that could contrast intra-regional differences and find potential associations and effects – in effect a meta-analysis.Prospectively, across Britain, Ireland and Western European countries, identify regions with varying burdens of disease and actively measure / catalogue the stated factors for statistical analyses.A caveat is that both strategies would bring their own inherent problems. Both would require harmonisation of retrospectively and prospectively collected data, to control for differences in bTB eradication scheme administration and data collection methods.M. bovis found in Britain and Ireland lacks diversity, intra-national comparison of potential effects of strain variation would be difficult to detect, since everything seems so genetically similar.However, efforts to survey broader vistas of the bTB landscape may make these efforts more worthwhile, identifying novel mechanisms amenable to control. There may have been a tendency to restrict one’s horizons when investigating bTB persistence in Britain and Ireland – a parochial approach, that whilst understandable with a complex disease affecting many herds and animals on a national scale – may miss some important epidemiological drivers. Owing to potential intra-national homogeneity in the characteristics of risk factors, their relative importance at a wider scale could be masked – for example: since the lineage of A potential criticism of our focus on some of these factors, is that even if they did have a significant effect on bTB epidemiology, that effect may be very small and therefore, any intervention would potentially not be practical or cost efficient. However, in the absence of firm evidence either way, this criticism could appear to be somewhat pessimistic. The reproductive index (R0) for bovine TB between cattle in Britain has been estimated to be low – 1.1 . BetweenAA and AB came up with the concept for the manuscript and drafted it. RS made additions to the text, edited existing text and gave advice.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To determine whether fibrin clot properties are associated with clinical outcomes following acute coronary syndrome (ACS).P < 0.01] and CV death alone . Similarly, each 50% increase in maximum turbidity was associated with increased risk of CV death . After adjustment for other prognostic biomarkers , the association with CV death remained significant for lysis time  but not for maximum turbidity. These associations were consistent regardless of randomized antiplatelet treatment . Neither lysis time nor maximum turbidity was associated with major bleeding events. Plasma samples were collected at hospital discharge from 4354 ACS patients randomized to clopidogrel or ticagrelor in the PLATelet inhibition and patient Outcomes (PLATO) trial. A validated turbidimetric assay was employed to study plasma clot lysis time and maximum turbidity (a measure of clot density). One-year rates of cardiovascular (CV) death, spontaneous myocardial infarction (MI) and PLATO-defined major bleeding events were assessed after sample collection. Hazard ratios (HRs) were estimated using Cox proportional hazards models. After adjusting for CV risk factors, each 50% increase in lysis time was associated with CV death/spontaneous MI [HR 1.17, 95% confidence interval (CI) 1.05–1.31; Fibrin clots that are resistant to lysis independently predict adverse outcome in ACS patients. Novel therapies targeting fibrin clot properties might be a new avenue for improving prognosis in patients with ACS. Intensive antithrombotic therapies, including potent P2Y,There is marked overlap between risk factors for ischaemic and bleeding events.12 inhibitor.,,Following ACS, treatment includes aspirin and a P2Y,Patients with thrombotic conditions demonstrate unfavourable fibrin clot structure.The majority of previous studies in this area used a cross-sectional retrospective design and large-scale longitudinal studies are lacking. We, therefore, aimed to study fibrin clot properties in plasma samples collected from ACS patients at hospital discharge and explore the relationship between those characteristics and subsequent clinical outcomes.,The PLATelet inhibition and patient Outcomes (PLATO) trial was an international multi-centre, double-blind, randomized controlled trial of ticagrelor compared with clopidogrel in 18 624 moderate- to high-risk ACS patients. The study design and results have previously been published.,Human thrombin was obtained from Merck Biosciences, recombinant tissue plasminogen activator from Technoclone, calcium chloride dehydrate and Tris from Fisher Scientific, and sodium chloride from Sigma Aldrich. High-throughput turbidimetric analysis was performed in flat-bottomed, polystyrene 96-well plates (Greiner) using a dedicated Multiskan FC (Thermo Scientific) plate reader. Permeation buffer solution  was used for dilution. Twenty five μL aliquots of plasma (in duplicates) were mixed with 75 μL lysis mix and clots were formed by adding 50 μL activation mix . After shaking for 2 s, plates were read at 340 nm every 12 s at 37°C until lysis in all samples was achieved. Quality control samples were included in all plates. This method has previously been validated.,Plasma samples obtained at randomization were used to determine other biomarker levels, as previously reported.2 tests. The primary outcome of interest was the composite of CV death and spontaneous myocardial infarction (MI). Secondary outcomes were CV death alone, spontaneous MI alone, stroke, all-cause mortality, definite or probable stent thrombosis according to Academic Research Consortium criteria, PLATO-defined major bleeding, and PLATO-defined bleeding unrelated to coronary artery bypass graft surgery (CABG) .Biomarker levels were natural log-transformed before analysis. Baseline patient characteristics, medical history, and biomarkers were compared across quartile groups of each of the fibrin variables. Continuous data are presented as medians and interquartile ranges and compared using Kruskal–Wallis tests. Categorical data are presented as numbers and percentages and compared using χTable 1 and A total of 4354 patients were included in this study. The prevalence of DM, hypertension, CKD, and female sex significantly increased with increasing lysis time quartile group. Although the differences were not significant, the highest quartile group of lysis time appeared to have the highest prevalence of PAD and CHF.Patients in the shortest lysis time quartile group tended to be older but the absolute difference in mean age was small. Strong associations were also observed between lysis time and other biomarkers: with increasing quartile group, the levels of troponin T, NT-proBNP, CRP, and white blood cell count increased. Growth differentiation factor-15 and cystatin-C levels were also highest in the highest quartile group.Similar to lysis time, the prevalence of DM was highest in the highest quartile group of maximum turbidity. There was a higher percentage of female patients in the lowest maximum turbidity quartile group. The relationship between maximum turbidity and the other biomarkers was also pronounced see Table S1.Neither lysis time nor maximum turbidity appeared to be affected by haemoglobin or haematocrit.P < 0.001).Maximum turbidity and lysis time had a modest correlation  patients had CV death, 183 (4.2%) patients had spontaneous MI, and 41 (0.94%) patients had stroke. There were 145 all-cause deaths (3.3%). Amongst those treated with PCI (4335), 38 (0.88%) had definite or probable stent thrombosis. PLATO-defined major bleeding occurred in 256 (5.9%) patients with 96 (2.2%) patients having non-CABG-related major bleeding.Figure 1). This was primarily driven by higher rates of CV death in the highest quartile groups . After adjustment for CV risk factors (Model 1), the highest quartile group of lysis time was associated with increased risk of CV death/spontaneous MI  and CV death alone . As a continuous variable, each 50% increase in lysis time was associated with increased risk of CV death/spontaneous MI  and CV death alone . This association remained significant for lysis time after adjustment for inflammatory and prognostic biomarkers (see P = 0.024) but this association was no longer significant after adjustment for inflammatory and prognostic biomarkers . Neither lysis time nor maximum turbidity was able to predict major bleeding events (see P > 0.7) . There was also no significant impact of fibrin clot properties on the association between randomized treatment and major bleeding . Similarly, the association between fibrin clot properties and CV death was present irrespective of subtype of ACS presentation . A subset of patients received low-molecular weight heparin on either the day of sampling or the day before (see P > 0.25).There was no significant impact of fibrin clot properties on the CV mortality reduction with ticagrelor compared with clopidogrel  for Model 1 + lysis time vs. 0.69 (0.642–0.741) for Model 1 only, P < 0.001.Model performance to predict the composite outcome of CV death/sMI significantly improved when lysis time was added to a clinical predictive model: C-index 0.67 (0.637–0.703) for Model 1 + lysis time vs. 0.665 (0.631–0.698) for Model 1 only, Take home figure). Given that antithrombotic therapy is mainly centred around antiplatelet agents following the acute phase, our data support the hypothesis that at least a subgroup of patients might benefit from additional therapy that aims at improving lysis potential. For example, further work could explore whether anticoagulant therapy, in combination with a platelet P2Y12 receptor antagonist, offers an advantage over dual antiplatelet therapy in those with adverse fibrin clot properties. Other novel therapies that specifically target proteins implicated in impaired lysis, such as complement C3 or plasmin inhibitor, is another approach that might have less impact on haemostasis, particularly if the aim was to normalise lysis potential.We have shown, in a large population of ACS patients treated with contemporary therapies and followed up for up to 1 year, that fibrin clot properties independently predict the risk of spontaneous MI and CV death following initial in-hospital management. Importantly, lysis time predicted worse outcome after adjusting for other established or new prognostic biomarkers, thus indicating the potential for a further biomarker that provides insight into prognosis following ACS. The association between lysis time and the levels of the established biomarkers also raises the possibility that variability in fibrin clot properties might contribute to the association of these other biomarkers with thrombotic events . It is established that internal fibrinolytic activity has a circadian rhythm largely driven by variations in plasminogen activator inhibitor—1 activity.Despite strong relationships with clinical risk factors, particularly DM, and inflammatory and other prognostic biomarkers, the resistance of fibrin clots to lysis independently predicts CV death following ACS. These findings suggest that novel therapies targeting fibrin clot properties might be a new avenue for improving clinical outcomes in patients with ACS.European Heart Journal online.Supplementary Figure 1Click here for additional data file.Supplementary TablesClick here for additional data file.


Amddc and Amth) encoding enzymes involved in dopamine synthesis in queens. The distribution of cells expressing Amddc in the brain revealed that soma clusters of dopaminergic cells were similar between castes at 7–8 days after pupation, suggesting the upregulation of Amddc expression in some cells in queens rather than addition of cell clusters. In contrast, genes encoding dopamine receptors were downregulated in queens or showed similar expression levels between castes. The expression of genes encoding dopamine transporters did not differ between castes. These results reveal the developmental process of caste-specific dopaminergic systems during metamorphosis in the honey bee, suggesting caste-specific behavior and division of reproduction in this highly eusocial species.Caste-specific differences in the dopaminergic systems of social insects assist in maintaining caste-specific behavior. To determine how caste differences in the honey bee occur during metamorphosis, a number of comparative analyses between castes were performed including comprehensive quantification of: levels of dopamine and its metabolite in the brain, the gene expression levels of enzymes involved in dopamine biosynthesis and conversion as well as expression levels of dopamine receptors and a dopamine transporter. Dopamine levels standardized to the protein contents of a whole brain at the day of eclosion in queens were 3.6-fold higher than those in workers. Dopamine levels increased until eclosion (7 days) in queens, whereas those in workers increased until 5–6 days before eclosion and then maintained until eclosion (10 days). These caste-specific dopamine dynamics in the brain were supported by the higher expression of genes ( Polyphenism is a form of phenotypic plasticity involving discontinuous morphological and physiological expression seen widely in animals. This occurs through different developmental processes for adaptation to different environments, such as temperature-dependent sex differentiation in reptiles and fish –3, alterThe caste-specific morphology and physiology in adults maintains the caste-specific behavior and division of reproduction. Various morphological differences of the central nervous system including the structure of brain subcompartments  –14 and tOne caste-specific physiological character in the adult honey bee brain is the dopaminergic system ,18. DopaThere are several reports on the dynamics of dopamine levels ,37 and eThe present study focuses on caste differences in dopaminergic systems in the pupal brain of the honey bee. Developmental processes for dopamine biosynthesis, dopamine signaling, and dopamine conversion or uptake are considered to be important to understand caste-specific dopaminergic systems. Therefore, we compared the levels of dopamine and its metabolite in the brain, as well as the gene expression levels of enzymes involved in dopamine biosynthesis and conversion, dopamine receptors, and a dopamine transporter between castes, and determined how the caste-specific dopaminergic systems in each adult brain are generated.Apis mellifera) were used to collect pupae and adult queens and workers. Queen pupae and adults were reared by a standard queen-rearing method with queenright colonies  and DOPA decarboxylase [Amddc]) or degradation (dopamine N-acetyltransferase [Amnat]), a dopamine transporter gene (Amdat) and four dopamine receptor genes  were selected as targets for quantitative PCR (qPCR)  were examined with sets of primers [www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) (Amdop1 and Amgpcr19 were applied from [Three genes encoding enzymes involved in dopamine biosynthesis (tyrosine hydroxylase [R (qPCR) . Four re primers  (S1 Tabllus.cgi) , but thoied from . StandarAmact, Amrp49, Amef1a1, and Amtbpaf) was evaluated using the software BestKeeper [Amtbpaf were the most stable in BestKeeper and were not significantly different among the developmental stages that we examined . Therefore, we normalized the expression levels of target genes by using expression levels of Amtbpaf. For quantification of the relative gene expression levels, the ΔΔCq method was adopted. For each sample, the ΔCq (target Cq–Amtbpaf Cq) were calculated, and then the ΔCq were normalized relative to an average ΔCq of control samples (adult workers) (ΔΔCq). The values of ΔΔCq were used for the calculation as 2-ΔΔCq. The ΔΔCq method requires an amplification efficiency of nearly 1.0 for both the reference gene and target genes. The amplification efficiencies were 1.052 in Amtbpaf and ranging from 1.018 to 1.128 in the target genes -labeled riboprobes for Amddc. Although neuropiles may still be developing in the brains of worker pupae 7–8 days after pupation, the basic structure of the brain 6 days after pupation is that of an adult brain [in situ hybridization were performed as described previously [Amddc were synthesized by in vitro transcription with a DIG RNA Labeling Mix . The primer sequences for the riboprobe synthesis were as follows: Fw: 5’-GTGATGTTGGACTGGCTAGG-3’, Rv: 5’-TGGATGTCTTTGCTCTCGCT-3’. Sense probes were used as a negative control in each experiment. The intensity and brightness of the micrographs were processed with Photoshop .To compare the soma clusters of dopaminergic cells in the brain between same-aged queens and workers, the brains of newly emerged queens (7 days after pupation) and worker pupae 2–3 days before eclosion (7–8 days after pupation) were stained by lt brain ,48, suggeviously . The senThe amount of dopamine and NADA in the brains were compared between queens and workers among different days before eclosion by a one-way ANOVA following the post hoc test  for multiple comparisons. The relative expression of dopamine-related genes in the brains was also compared between castes among different days before eclosion by using the same statistical test.In each caste, the dopamine levels per brain differed significantly among the days before eclosion that we examined . In queens, dopamine levels in the brain 6–7 days before eclosion were on average 0.956 pmol/brain and were highest on the day of eclosion (37.247 pmol/brain on average) during the pupal stage . HoweverTo examine any caste differences in dopamine levels in the brain, the levels were normalized to the protein content of a whole brain and then compared with the day of eclosion. In both queens and workers, the dopamine levels per protein (mg) in the brain differed significantly across the pre-eclosion times examined . In queens, the NADA levels per brain 6–7 days before eclosion were on average 2.01 pmol/brain and were highest on the day of eclosion (31.386 pmol/brain on average) during the pupal stage . In contAmth and Amddc), in the degradation, or conversion, of dopamine into NADA (Amnat), and a dopamine transporter (Amdat) were quantified and compared between castes across pupal development until eclosion.The relative expression of genes encoding enzymes involved in the biosynthesis of dopamine , but did not differ between castes at 2–3 and 4–5 (5–6) days before eclosion , but not at 4–5 (5–6) days before eclosion  and a dopamine/ecdysteroid receptor gene (Amgpcr19) were quantified and compared between castes as above.The relative expression of dopamine receptor genes  days before eclosion and on the day of eclosion  and workers (7-day-old pupae) were similar  in the brain increased significantly in early stage pupae (i.e. 2–5 days old) of both queens and workers. Dopamine levels in queens increased until eclosion, whereas those in workers increased until 5–6 days before eclosion. On the day of eclosion, dopamine levels in queens were 3.6-fold higher than those in workers. These caste-specific dynamics in dopamine levels can be explained by the expression of Amdat would be expressed more in queens than in workers. However, Amdat expression did not differ between castes and became elevated in response to eclosion in both queens and workers. The functional role(s) of Amdat during the pupal stage remain unknown.The dopamine transporter is a membrane protein involved in the uptake of dopamine from presynaptic dopaminergic cells or other cells. Given that the levels of dopamine and NADA were increased in queen brains, active dopamine release and conversion in queens would be expected. Therefore, we also expected that the dopamine uptake system would be active, and that the dopamine transporter gene Amddc in the brain revealed that the positions of somata in the dopaminergic cells of queens and workers were similar during stages when the levels of dopamine were the highest. Although dopamine levels in the brain were higher in queens than in workers, there were no additional clusters of cells expressing Amddc in queen brains. Therefore, some of these dopaminergic cells might differentially express Amddc in the brains of queens. It is also possible that the increased dopaminergic cell number in queens accounts for the higher dopamine levels. However, the number of dopaminergic cells was not determined in this study and the possibility remains to be tested. Several somata in dopaminergic cells located in the posterior medulla of a pair of optic lobes were identified as novel dopaminergic cells. These cells were detected in the brains of both queens and workers. However, we do not know whether the cells are neurons or glial cells, or what functional role the cells have in the brains. More detailed investigations of the morphology of the cells is required.The distribution of cells expressing Amdop1, at 2–3 days before eclosion and emerged adults in terms of Amdop2, and at 2–3 days before eclosion in terms of Amdop3. It was interesting that both D1-like (Amdop1 and Amdop2) and D2-like (Amdop3) receptors were depressed in queens compared with workers, although the actions of these types of receptor are excitatory and inhibitory at the cellular level, respectively [Amgpcr19, which encodes a dopamine-ecdysteroid membrane receptor, its expression did not differ significantly between castes. Although the hemolymph ecdysteroid titer in both queens and workers changes significantly during metamorphosis [Amgpcr19 was stable during this stage, suggesting that activation of this receptor by ecdysteroid or dopamine does not differ between castes.The expression of dopamine receptor genes differed between castes. Queens showed reduced expression of genes encoding dopamine receptors compared with workers from 5–6 days before eclosion to newly emerged adults in terms of ectively . These dectively –56. D1-lectively . Althougectively ,13. For orphosis , the expAmth and Amddc during the late pupal stages. In Drosophila melanogaster, experimental transient activation of dopaminergic neurons in brains of late stage pupae increased locomotor activity and visual responsiveness in adults [The roles of dopamine during the pupal stage in the honey bee are largely unknown. There are several reports implicating dopamine after eclosion in virgin queens, which have high levels of dopamine in the brain ,31. Appln adults . Thus, aThe promotion of reproduction including ovarian activity and oviposition in adult females is an important role of dopamine in solitary –27 and eAmddc in the brain were similar between castes, suggesting that the expression of Amddc is upregulated in some cells in queens but not by the addition of cell clusters. In contrast, dopamine receptor genes were downregulated in queens or showed similar expression levels between castes, suggesting the influence of excessive dopamine production in the brain of queens. These caste-specific ligand–receptor characters might generate the caste-specific behavior and intensive division of reproduction seen in these highly eusocial honey bees.The present study revealed caste differences in the dopaminergic system during metamorphosis in the honey bee. The caste-specific dopamine dynamics in the brain might derive from the differential expression of genes encoding enzymes involved in dopamine biosynthesis. The distribution of cells expressing S1 FigAmddc riboprobes. Dotted lines indicate the midline of the brain. Each section corresponds to photos in Sequential frozen sections were arranged in parallel for antisense and sense (EPS)Click here for additional data file.S2 FigAmddc riboprobes were observed in the zone in both queen and worker (arrows), whereas no somata were stained with sense Amddc riboprobes. Dotted lines indicate the midline of the brain. Scale bars: 0.1 mm.The soma clusters stained with antisense (EPS)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.


Ziphiidae (deep-diving cetaceans), mimics decompression sickness (DCS) in human divers  Red List, being increasingly threatened worldwide by several anthropogenic and natural factors, often acting synergistically with each other, as in the case of infectious agents and immunotoxic environmental pollutants , high lenoxae like morbilliviruses, herpesviruses, Brucella ceti, B. pinnipedialis, and Toxoplasma gondii, just to cite a few  to “recipient” species like fin whales   (Ziphius cavirostris) (Phoca vitulina) (Lutra lutra) (Delphinapterus leucas) population residing in the St. Lawrence River Estuary, Canada, in which neoplastic disease was found in association with high tissue burdens of organochlorines, heavy metals, and benzo-a-pyrene. A direct and an indirect carcinogenic activity could have both been involved in the development of the aforementioned neoplasms, together with an antitumor immune surveillance deficiency  , sperm wephalus) , Cuvier'rostris) , and eveitulina)  and Euraa lutra) . Within a lutra) . A defeca lutra) . Deepenia lutra) . These hficiency .ad hoc immunologic tools, specifically developed and/or validated also for marine mammals, are urgently needed.A critical component of all studies on the comparative immunology of marine mammals is that the vast majority of the laboratory reagents available on the market have been developed and “validated” only in “conventional” species. Therefore, Zalophus californianus), cetacean host–pathogen interaction(s), and the comparative immunopathology of CeMV infection in free-ranging dolphins. In this respect, we would like to express our most sincere feelings of appreciation and gratitude to Prof. Miki Nakao, chief editor of Frontiers in Immunology, who enthusiastically accepted our proposal to host this monographic collection in such an outstanding and prestigious Journal, as well as to Dr. Tara Sugrue, Research Topic team leader, Dr. Carmen E. Flores Nakandakare, and all the eminent scientists, associate editors, and reviewers who provided their valuable contributions for the present Research Topic.The aforementioned issues represent the focus, along with several others, of the present Research Topic on the comparative immunology of marine mammals, hosting eight original contributions from leading scientists and scientific groups worldwide. In more detail, the monograph's contents address T-helper lymphocytes and other cellular and molecular effectors of cetaceans' immune response, acute-phase proteins and class II major histocompatibility complex in marine mammals, along with antioncogenic virus immune surveillance in California sea lions (GD wrote the first draft of the present Guest Editorial, which was subsequently revised by MC as well as by ES and SM, who provided their valuable and precious comments, remarks, and suggestions. The final Editorial's draft and the current Editorial's text were entirely agreed and approved by all the 4 Guest co-Editors of the present Research Topic, dealing with the Comparative Immunology of Marine Mammals.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


EPM2A gene, encoding the dual phosphatase laforin, or the EPM2B gene, encoding the E3-ubiquitin ligase malin. Laforin and malin form a functional complex that is involved in the regulation of glycogen synthesis. Thus, in the absence of a functional complex glycogen accumulates in LBs. In addition, it has been suggested that the laforin-malin complex participates in alternative physiological pathways, such as intracellular protein degradation, oxidative stress, and the endoplasmic reticulum unfolded protein response. In this work we review the possible cellular functions of laforin and malin with a special focus on their role in the ubiquitination of specific substrates. We also discuss here the pathological consequences of defects in laforin or malin functions, as well as the therapeutic strategies that are being explored for LD.Lafora disease  is a rare and fatal form of progressive myoclonus epilepsy (PME). Among PMEs, LD is unique because of the rapid neurological deterioration of the patients and the appearance in brain and peripheral tissues of insoluble glycogen-like (polyglucosan) inclusions, named Lafora bodies (LBs). LD is caused by mutations in the  Lafora disease  is a rare and fatal form of progressive myoclonus epilepsy (PME). The term PME comprises a group of neurological disorders characterized by the presence of focal and generalized seizures, myoclonus and progressive neurodegeneration, accompanied with cerebellar signs and dementia . Among PIn this work, we review the possible cellular functions of laforin and malin, the two main proteins defective in LD, with a special focus, since malin is an E3-ubiquitin ligase, on their role in the ubiquitination of specific substrates. We also discuss here the pathological consequences of defects in laforin or malin functions, as well as the therapeutic strategies that are being explored for LD.EPM2A, encoding laforin, a glucan phosphatase, and EPM2B/NHLRC1, encoding malin, an E3-ubiquitin ligase [EPM2A gene, whereas 40–50% carry mutations in the EPM2B gene [http://projects.tcag.ca/lafora. Until June 2018, this database identified 105 modifications in the EPM2A gene  and 85 modifications in the EPM2B gene . In both genes, modifications are scattered throughout the complete sequence.LD is a very rare disease with a prevalence of less than 4 patients per 1,000,000 individuals . It is an ligase ,9,10. AcM2B gene ,7. In boEPM2A mutations are more prevalent, in others, like Italy, mutations in the EPM2B gene are more common [EPM2A, one of the most frequent mutations is R241X, whereas in the case of EPM2B, P69A is the most frequent mutation described in the literature [The geographic distribution of the mutations varies depending on the country. While in some countries, like Spain, e common . In the terature ,12.EPM2A or in EPM2B only partially affects the function of the corresponding protein, or that the presence of genetic or environmental modifiers influences the regular progression of the disease [Patients with mutations in laforin or malin are neurologically and histologically indistinguishable , suggest disease ,14,15,16EPM2A gene, containing four exons and located on the chromosome 6q24 of the human genome, which produces a major mRNA with an ORF of 993 nts, according to the ENSEMBL database. It is expressed in all tissues, but particularly in brain, skeletal muscle, heart, and liver [Laforin is encoded by the nd liver . Laforinnd liver , the CBMnd liver , being tnd liver ,21,22. Ind liver ,23,24.Laforin contains also a C-terminal DSP A. This dThe laforin crystal structure was recently described  Figure B. It conEPM2B gene, located on chromosome 6q22.3 of the human genome, which produces a major mRNA with an ORF of 1185 nts, according to the ENSEMBL database. It is expressed in brain, heart, skeletal muscle, liver, and pancreas [Malin is encoded by the single exon pancreas . Malin, pancreas   and in vitro assays have demonstrated that malin is able to interact with UBE2D1, UBE2D2, UBE2D3, and UBE2E1, but not with UBE2C and UBE2L3 [As an E3-ubiquitin ligase, malin is able to ubiquitinate specific substrates. To perform this function, malin interacts with specific E2-conjugating enzymes. Forty-one different E2-conjugating enzymes have been described thus far in the human genome  thd UBE2L3  and R6  Ad UBE2L3 ; (iv) thd UBE2L3 ; (v) pyrd UBE2L3 ; (vi) std UBE2L3 .Branchiostoma floridae [A phylogenetic study suggested that malin gene is exclusively found in vertebrate genomes and in the cephalochordate floridae . This difloridae . These rfloridae .The same report indicates that malin shares an evolutionary relationship with TRIM32, an E3-ubiquitin ligase that belongs to the tripartite motif-containing (TRIM) protein family . Both maWhen overexpressed, malin is prone to aggregate. The co-chaperone CHIP (C-terminus of Hsc70 Interacting Protein) and the chaperone Hsp70 stabilize malin . In factPatients with mutations in laforin or malin are neurologically and histologically indistinguishable . TherefoIn this complex laforin could, for example, recognize and recruit putative substrates to be ubiquitinated by malin for degradation . In factOverexpression studies with tagged version of laforin suggested that laforin has a cytoplasmic distribution, mostly associated with endoplasmic reticulum markers ,50. HoweAs mentioned above, the hallmark of LD is the accumulation in brain and peripheral tissues of poorly-branched glycogen-like inclusions named LBs. These structures also contain higher levels of phosphomonoesters on the glucose residues ,52. BothAlthough the results described above have been confirmed by different labs, they are largely based on results obtained under conditions of overexpression of laforin and malin in cell cultures using recombinant proteins. In contrast, the levels of some glycogen related proteins, such as glycogen synthase or R5/PTG, do not increase in animals of 3–6 months of age, as it would be expected by the lack of a functional laforin-malin complex ,52,67. H+ and glutamate uptake [In any case, the animal models of LD clearly show an accumulation of insoluble glycogen-like structures (polyglucosans) in different tissues . These Le uptake ,69,70. Ie uptake .Glucose enters the cell through specific glucose transporters. In the case of brain, the main glucose transporters are GLUT1 and GLUT3, expressed predominantly in astrocytes and neurons, respectively. The amount of these transporters at the plasma membrane, which is regulated by cycles of endocytosis and exocytosis, determines the efficiency of glucose uptake. It has been described  that oveIn addition to carbohydrate moieties, LBs contain up to 6% of proteinaceous material, including glycogen synthase, laforin, chaperones, autophagic components, proteasome subunits, advanced glycation end products, etc. ,62,72. MMoreover, due to the autophagy impairment, processes that require a functional autophagy machinery are also affected in cellular and animal models of the disease. In this sense, we have recently described a defect in mitophagy, most likely due again to defects in autophagosome formation  Table 2Table 2. Finally, we have also reported that oxidative stress is affected in the absence of a functional laforin-malin complex , since wWe and others have described that in animal models of LD there is a transformation of astrocytes into GFAP+ reactive astrocytes ,56. In aThe presence of reactive astrocytes and microglia is associated with pro-inflammatory conditions. In agreement with this, we have described that in the brain of animal models of LD there is an increase in the levels of pro-inflammatory mediators such as TNFα, IL-6 and IL-1β, chemokines, cytokines, and other inflammatory markers . The estWhat triggers the inflammatory process? The existence of an interrelationship between epilepsy and inflammation has been widely documented ,87,88. OIt has been reported that malin interacts with dishevelled2, a key component of the Wnt signalling pathway. Dishevelled2 is a cytosolic phosphoprotein whose activation results in the inactivation of the degradation pathway of β-catenin, resulting in the accumulation of this protein and in its translocation to the nucleus, where it activates the transcription of a specific set of genes. Overexpression of malin enhances degradation of dishevelled2 and inhibits Wnt signalling through the downregulation of β-catenin protein levels and, thus, of the expression of β-catenin target genes. Since malin enhances both K48- and K63-linked ubiquitination of dishevelled2, its degradation could occur through both proteasomes and autophagy  Table 2Table 2. Processing bodies (PBs) are cytoplasmic structures that house the RNA decay machinery . It has been reported that malin, when overexpressed, is recruited to the PBs, where it interacts with Dcp1a and promotes its degradation via the ubiquitin proteasome system . Since dThere is currently no proven therapy for LD. The initial symptoms of seizures and epilepsy are treated with regular anti-epileptic drugs , but patients soon become resistant to them . Based oIf the hallmark of the disease is the accumulation of insoluble poorly branched glycogen-like structures (LBs), it seems logical to think that any action aimed to decrease glycogen synthesis could be beneficial. This assumption was elegantly demonstrated by different authors, which created mice models combining the absence of laforin or malin and of R5/PTG or glycogen synthase. Obviously, these mice synthesized no or only small amounts of glycogen, because of the lack of key regulators of glycogen synthesis, but in addition and more interestingly, these double knock-out mice showed no symptoms of LD ,89,90,91Since proteostasis is affected in LD, other groups have analyzed the possibility of using different drugs to improve proteostasis. In this sense, since autophagy is impaired in LD, we and others checked whether inducers of autophagy, such as inhibitors of the autophagy regulator mTORC1, could be beneficial for treating this disease. However, the use of rapamycin, everolimus or temsirolimus had no effect ( and unpuFinally, additional compounds for the treatment of LD have been tested by other authors. One example is provided by sodium selenate , a compoIt has been recently described that the use of perampanel, an antagonist of post-synaptic AMPA receptors, has beneficial effects for LD in terms of decreasing the frequency of seizures and improving neurocognitive processes ,100. TheLD is a devastating pathology that leads to the death of the patients after around ten years from the beginning of the first symptoms. In recent years, many data have accumulated trying to understand the molecular basis of the pathophysiology of LD. Although several relevant discoveries have been made, the primary defect that triggers LD is still controversial. For example, we still do not understand why LD cells accumulate more glycogen than normal cells and why this polysaccharide is poorly branched and hyperphosphorylated. We also do not know how proteostasis is affected, how neuroinflammation is initiated, or how epilepsy is triggered. We hope that in the near future, we could provide answers to these yet unsolved questions. In the meantime, different strategies are being explored to obtain a treatment that slows down the progression of the disease and diminishes its symptoms.


Ultrasound is a workhorse in clinical routine and often the first imaging modality with that a patient gets in contact. Its strengths are real time imaging capability, low costs and lack of radiation exposure. Due to its considerably high user dependence and limited reproducibility, it is not the first choice for whole body screening examinations but favorably suited for monitoring arterial predilection sites for the development of plaques, stenoses, and aneurysms. Here, the assessment of blood flow by Doppler techniques facilitates the differentiation between the vascular wall and the blood. This can be further improved by using gas-filled microbubbles acting as an intravascular contrast agent. The sensitivity of ultrasound to microbubbles is very high and even a single microbubble can be detected. This fact qualifies contrast-enhanced ultrasound imaging for more than just the visualization of the blood. By binding targeting ligands to the surface of microbubbles, they can be turned into molecular imaging probes and used to assess changes in the vascular wall that not yet have an anatomical correlate. There is a long history of molecular ultrasound imaging of the vascular wall including the assessment of thrombosis, inflammation and sclerosis , and theMost of the previously mentioned markers are also upregulated on arteriosclerotic plaques. However, there is still an intense debate, which marker is capable of predicting plaque growth and instability. This information cannot be derived from morphologic imaging and would open important early therapeutic perspectives before stenoses and occlusions occur.In our recent paper, we evaluated the suitability of the junctional adhesion molecule A (JAM-A) as an early biomarker of vascular inflammation and plaque development using molecular ultrasound imaging and multiphoton microscopy with fluorescent poly(n-butyl cyanoacrylate) (PBCA) microbubbles . JAM-A bIn summary, we could confirm JAM-A as an interesting biomarker of early vascular inflammation, that may indicate vascular areas exposed to acutely alternated flow and thus links mechanical stress to molecular regulations. Besides for molecular ultrasound imaging, which is more suited to evaluate defined predilection sites, JAM-A targeted probes may also be considered for PET or SPECT imaging, then enabling an efficient whole body staging and the monitoring of therapy, e.g. using statins or inflammatory agents .


He had a history of non–ST-elevation myocardial infarction 2 weeks earlier, for which he had been admitted to another center and treated medically. Electrocardiography showed an ST elevation in lead Val leads . Right pR to V6R . AccordiR to V6R . It appe


An additional supplementary file, named S7 FigTo allow for simultaneous detection of proteins of different molecular weights, membranes were at times cut horizontally approximately 25 kD below and above the expected molecular weight, prior to incubation with antibodies. In the instances the proteins had similar molecular weights (as for coilin and WRAP53) two parallel membranes were blotted and analyzed. However, for IP experiments that test the binding between immunoprecipitated and co-precipitated proteins during different conditions, blotting is required on the same filter to ensure that the loading does not impact the interpretation of the results. In such cases, antibodies that were raised in different species were used (rabbit for WRAP53 and mouse for coilin) to minimize the risk of cross-reaction, and the co-precipitated protein was always blotted first, followed by detection of the immunoprecipitated protein itself.(EPS)Click here for additional data file.


PLOS ONE policy. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.There is additional information that needs to be included in the Competing Interests statement. The correct Competing Interests statement is as follows: NG, DJA, PLB, DDH, CN and ASP were employees of the Marine Stewardship Council at the time of this study. TEE, KS, and ADMS were unpaid members of the MSC's Technical Advisory Board. KS was an unpaid member of the MSC’s Board of Trustees. SRV, TAB, JKB, JCD, CC, OD, RH, AEL, RLS, SS, SJT, JTT and NEW did not have direct affiliation with MSC at the time of this study. The groups combined their initially separate but duplicative analyses as part of a National Center for Ecological Analysis and Synthesis (NCEAS) working group. KS additionally declares the following roles: Commissioner with the Australian Fishery Management Authority (since 2004), member of the Lenfest Task Force to develop standards for management of fisheries targeted on forage fish species (2008–2012), independent advisor to the Corporate Social Responsibility Committee of the China Fisheries Group (2011–2015), and consultant to the Food and Agriculture Organization of the United Nations, authoring a 2008 review of fisheries ecolabelling schemes. Funding was received by RH in the five-year period prior to the publication of this article from sources including fishery/seafood corporations and industry groups, as well as environmental groups and foundations Click here for additional data file.


Klinefelter syndrome (KS) is the most frequent type of congenital sex-chromosomal disorder caused by at least one extra X chromosome and commonly treated with lifetime testosterone therapy. Ulcerative lesions on lower extremities may occur as a complication of KS. The pathogenesis of ulcers in KS patients has not been clarified on a molecular level. Here we present a case of leg ulcers exacerbation associated with the administration of a high dose of acetylsalicylic acid in a 63-year-old KS patient with karyotype 47,XXY undergoing testosterone replacement therapy for the last 20 years. The appearance of the ulcer on the patient's leg occurred during one week of high oral acetylsalicylic acid intake (1.2 g daily). The patient was advised to return to his standard daily dose of 0.1 g of acetylsalicylic acid and significant improvement of his leg ulcer was observed after two weeks. We hypothesize that testosterone-mediated nitric oxide balance in KS patient is perturbed under the condition of acute high-dose acetylsalicylic acid administration. We propose that small standard doses of approximately 0.1 g/day of acetylsalicylic acid have no apparent effect on nitric oxide status, whereas higher doses may cause dysregulation of nitric oxide production and/or utilization, creating conditions which may cause the appearance of leg ulcers in the KS patients. Klinefelter syndrome (KS) is the most frequent type of congenital sex-chromosomal disorder affecting approximately 1:660 newborn males, and it is a rather common cause of infertility, hypogonadism and learning disability [The occurrence of leg ulcers as a symptom of KS (6‒13%) appears to be related to occurrence of a single pathophysiological condition or a combination of several conditions such as chronic venous insufficiency, arterial dysplasia in lower extremities, platelet hyperaggregability, factor V Leiden mutation, and decrease in fibrinolysis due to elevated levels of plasminogen activator inhibitor-1 (PAI-1). However, the underlying molecular mechanism is not completely identified nor understood .Due to the lack of reliable evidence, there is no consensus regarding the effect of oral intake of acetylsalicylic acid on venous leg ulcers . We hereThe patient is a male born in 1956, diagnosed with KS at the age of 16. Regardless of a fact that KS is commonly associated with learning disabilities, the patient in question earned a Bachelor's degree in Economics at the age of 26. From 1982 to 2000 he was supplemented with human chorionic gonadotropin , which was a recommendation of late professor and an expert in the management of KS treatment, Bruno Lunenfeld. In 2000, he becomes unresponsive to that therapy and began supplementation with testosterone-undecanoate  four times yearly (approximately every three months). The additional medications that he uses regularly are angiotensin-converting enzyme inhibitor Enalapril  and acetylsalicylic acid in a daily dose of 0.1 g/day as standard prescribed therapy for the management of high arterial blood pressure. Compiled from personal health record in the past 10 years, the patient had an average serum value for testosterone of 3.87 ± 1.14 ng/mL before injection of testosterone.In the past, the patient has experienced several episodes of leg ulceration often associated with an open bleeding wound that occurred simultaneously with the consumption of a high dose of acetylsalicylic acid. These problems started in mid-2014 after the patient voluntarily began taking increased doses of 400 mg of acetylsalicylic acid per day at times when he felt sick. One of the culminations happened on September 2018 when he took acetylsalicylic acid tablets  three times a day, without prior consultation with his assigned physician. After a few days, an ulcer on his left leg re-appeared and worsened  are presented below, with normal/reference value or range given in brackets.Laboratory evaluations showed a normal complete blood count, and blood group ABO & RhD B-. There were no abnormalities in the prothrombin time, partial thromboplastin time or plasminogen level. Fasting serum glucose was normal at 5.2 mM (3.9‒5.4 mM), as well as hemoglobin A1c level at 5.4% , indicating glucose homeostasis. The analysis of fasting serum lipid profile revealed a normal high-density lipoprotein level of 1.4 mM , elevated triacylglycerides at 2.7 mM , total cholesterol at 7.5 mM  and low-density lipoprotein level was on upper limit at 4.9 mM .The endocrine evaluation showed total testosterone at 5.7 ng/mL (3.0-10.0 ng/mL), follicle-stimulating hormone at 13.2 U/L (2.0-15.0 U/L) and luteinizing hormone at 6.5 U/L (0.4-15.0 U/L) within reference values, suggesting that testosterone supplementation is efficient in this patient. Plasma levels of protein C at 122.5 U/dL (70.0-140 U/dL), protein S at 101.4% (67.5-139%), D-dimer at 64.8 ng/mL (<250.0 ng/mL), antithrombin III at 98% (80-120%) and fibrinogen at 340 mg/dL (200-375 mg/dL), as well as the serum levels of coagulation factors VII of 118% (70-140%), VIII of 115% (50-175%), IX of 110% (70-120%), XI of 101% (70-120%) and XII of 115% (60-140%) were all within the normal range. The level of serum homocysteine was 17.3 μM (5.5-16.2 μM), and the C-reactive protein level was normal at 0.9 mg/L (<5 mg/L). These results indicate a low risk for deep vein thrombosis in our patient.The chromosomal analysis confirmed KS diagnosis with a non-mosaic 47,XXY karyotype. The patient has heterozygous 4G/5G PAI-1 genotype with normal plasma PAI-1 concentration of 30 ng/mL (3-72 ng/mL). Genetic mutations for factor II (G20210A), factor V Leiden (R506Q) and MTHFR (C677T) genes were not detected. Levels of tested antibodies were all within reference ranges, including lupus anticoagulant at 2.1 U/mL (<25 U/mL), anticardiolipin M at <2.0 U/mL (<13 U/mL) and G at 4.7 U/mL (<20.0 U/mL), antiphospholipid M at 1.3 U/mL (<15 U/mL) and G at 1.0 U/mL (<15 U/mL), and finally β2-glycoprotein I antibodies M at 1.7 U/mL (<5 U/mL), G at 1.0 U/mL (<5 U/mL) and A at 3.0 U/mL (<5 U/mL).Taken together, we concluded that the blood analysis of biochemical and immunological parameters suggests that the patient's leg ulcer was not related to deep vein thrombosis and metabolic syndrome.In this report, the high-dose acetylsalicylic acid oral intake was associated with the (re)appearance of leg ulcers in the KS patient. There is only one study so far in which acetylsalicylic acid treatment was associated with leg ulceration in KS, where, surprisingly a reduction in the size of leg ulcer was achieved with a low-dose therapy (0.3 g twice a week for one month), although the initial improvement was not maintained long-term .When it was first described 1942), KS was not related to leg ulceration . Almost 42, KS waLeg ulceration in patients with KS is typically attributed to hypostasis, deep vein thrombosis and venous insufficiency as well as to platelet hyperaggregability. Such a relation was first described in a study published by Norris et al. in 1987 who emphasized the significance of supplemental androgen therapy in the context of ulcers improvement . Seven yIn the study of Depaire-Duclos et al. in 1997, a KS patient was found to have heterozygous factor V Leiden (R506Q) mutation and, contrary to other cases described above, supplemental androgen therapy did not show a beneficial effect on his leg ulcer . The firGoto et al. suggested that formation of the leg ulcers in KS patients is attributed not only to the abnormalities of PAI-1 activity and platelet hyperaggregability but also to immunological defects due to androgen deficiency . They deBased on the literature data, it can be concluded that KS patients have an increased risk of thromboembolic events with a high prevalence of recurrent venous ulcers, vein insufficiency, and both recurrent venous and artery thromboembolism with a higher risk of deep venous thrombosis or pulmonary embolism, compared to general population. According to the recent data analysis from 2016), there is no clear evidence of the impact of hormone replacement therapy on the risk of venous thromboembolism in patients with KS [, there iThe proper concentration of gaseous nitric oxide at the location of wounded tissue seems to be essential for ulcer healing . Nitric The exact biochemical pathways and mechanisms for the complex interplay between aspirin, testosterone and nitric oxide, both generally and in this particular condition (KS), have not been thoroughly elucidated. The same can be said for the systemic effects of individual substances. For instance, the antihypertensive effects of aspirin are currently a widely debated clinical issue. Although it has been suggested that aspirin lowers blood pressure and could be used for preventing hypertension, the latest guideline indicates that low-dose aspirin (75-100 mg daily) should be used infrequently in this regard, because of lack of net benefit .Based on the acquisition and analysis of clinical parameters, genetic profiling, and corresponding available literature evidence, the authors propose that a sudden and short-lived increase in nitric oxide concentration after administration of high doses of acetylsalicylic acid may create conditions for the appearance of leg ulcers in KS patients on a lifetime testosterone supplementation therapy. Further research on dose-dependent molecular cross-talk between testosterone, acetylsalicylic acid, and nitric oxide and its redox species, as well as the pharmacogenetic approach, are currently underway in our laboratories.


Zm-INVINH4 is specifically expressed in maize sink tissues, such as the base part of the leaves and young kernels. Moreover, drought stress perturbation significantly induced Zm-INVINH4 expression, which was accompanied with a decrease of cell wall invertase (CWI) activities and an increase of sucrose accumulation in both base parts of the leaves 2 to 7 days after pollinated kernels. In summary, the results support the hypothesis that INV-related sink growth in response to drought treatment is  caused by a silencing of INV activity via drought-induced induction of Zm-INVINH4 protein.Invertases (INVs) play essential roles in plant growth in response to environmental cues. Previous work showed that plant invertases can be post-translationally regulated by small protein inhibitors (INVINHs). Here, this study characterizes a proteinaceous inhibitor of INVs in maize (Zm-INVINH4). A functional analysis of the recombinant Zm-INVINH4 protein revealed that it inhibited both cell wall and vacuolar invertase activities from maize leaves. A Zm-INVINH4::green fluorescent protein fusion experiment indicated that this protein localized in the apoplast. Transcript analysis showed that  Zea mays L. ssp. Mays is one of the world’s most important cereal plants. Sucrose as the transport form of carbohydrates in maize is synthesized in source-leaf mesophyll cells and transported through the phloem to develop sink organs by “mass flow” [ss flow” ,2. Adjusss flow” . The souss flow” ,5. An orss flow” ,7.Acid invertase, a sucrose-cleaving enzyme which produces hexose sugars from sucrose, regulates the plant primary carbon metabolism and effects on plant growth and development ,9. In maAcid invertases display multiple regulatory mechanisms at the transcriptional and post-transcriptional levels in response to environmental stimuli, such as drought , temperaDrought effecting source-sink regulation is a major factor limiting plant distribution and productivity. In maize, the pre- and early post-pollination phases of female reproductive development are critical for sufficient pollination and seed set. The process is extremely sensitive to drought ,19. DrouPreviously, an invertase inhibitor, named Zm-INVINH1, localized to the embryo surrounding the region during early kernel development, has been found, and its enzyme activity was identified in vitro . HoweverHere, this research presents an apoplast-localized proteinaceous inhibitor of INV in maize, named Zm-INVINH4, which has inhibitory activity by reducing maize invertase activity and is specifically expressed in the base part of leaves and young kernels after drought stimulus, accompanied by the decrease of cell wall invertase (CWI) activities and the increase of sucrose accumulation. Results also suggest that Zm-INVINH4 may modulate the INV-related source to sink growth and the development in maize under the drought stress regime.Zm-INVINH4 (GenBank accession no: NM_001159249). It is located in chromosome 2 with 642 nucleotides in the open reading frame . One putative maize invertase inhibitor sequence was identified. The sequence has a similar size and shared sequence homology with monocot INVINH proteins and was named ng frame .A deduced amino acid sequence of Zm-INVINH4 was aligned with previously reported genes from other species using the CLUSTAL W2 program . The resActinidia deliciosa PMEI (AAZ20131). A comparison of these with the closest homologs in the Poaceae species reveals a higher degree of similarity of PMEIs between maize and Poaceae than other species.Phylogenetic and molecular evolutionary analysis was conducted with MEGA 5.05 . Amino aIn addition, Zm-INVINH4 clustered most closely with rice apoplastic invertase inhibitors and the Arabidopsis cell wall and vacuolar invertase inhibitor 2 . FurtherZm-INVINH4 was cloned from maize leaves and young kernels. The protein was 20.36 KD with a pI value of 7.81, which is in accordance with other INVINH-related proteins. There was an apparent signal peptide sequence with 21 amino acid residues in Zm-INVINH4 protein.Full-length cDNA of the E. coli expression system. The recombinant Zm-INVINH4 with 6xHis-tag showed the expected molecular mass of 24 kDa on the SDS-PAGE gel , two vacuolar invertase genes (Zm-Ivr2 and Zm-Ivr3), and Zm-INVINH4 , one vacuolar invertase gene (Zm-Ivr2), and two inhibitor genes (Zm-INVINH1 and Zm-INVINH4) were found to be constantly induced in young maize kernels during the drought treatment  proteins ,29. TherZm-INVINH4 is expressed specifically in maize sink tissues, such as young kernels and base part of leaves under drought stress, indicating that Zm-INVINH4 is a drought-response invertase inhibitor. The question is, which invertase can be inhibited by Zm-INVINH4?In kernels, from 4 to 10 days after pollination, the current study showed both cell wall and vacuolar invertase activities were decreased in response to drought stress, correlating with sucrose accumulation .Zm-INCW1, Zm-INCW3, and Zm-INCW5 were almost undetectable. On the contrary, Zm-INCW2 was expressed significantly in kernels. Previously, Zm-INCW2, a kernel-specific cell wall invertase, has been implicated in early maize floral development, and especially in the ovary expansion [Zm-INVINH1 is specifically expressed in kernels between 4 and 7 days after pollination [The cell wall invertase (CWI) is essential for sucrose partitioning and seed and fruit development. In maize, cell wall invertases have been implicated in early maize floral development, and especially ovary expansion and seed filling ,31,32. Fxpansion ,31,32. Tlination , howeverlination .Zm-INVINH4 expression, the results suggest that the cell wall invertase Zm-INCW1, a vacuolar invertase Zm-Ivr2 and a putative vacuolar invertase Zm-Ivr3, might bind Zm-INVINH4 to form INV/INVINH complexes, following the time course during the drought treatment. Zm-INVINH4 finally prohibited the cell wall and vacuolar invertase activities  activities and an increase of sucrose accumulation. This research will help to unravel how Zm-INVINH4 may modulate the invertase (INV)-related source to sink growth and the development in maize under the drought stress regime. Moreover, this research may motivate new strategies on how to improve productivity and avoiding drought-induced seed abortion in maize.Zea mays L.  was grown in the greenhouse on soil at 25 ± 2 °C under long day conditions . Drought treatments were carried out on the three weeks growing seedlings (they were not watered for two weeks) or on the manual pollination stage after flowering for one month. ABA treatment was performed on three weeks seedlings with a final ABA concentration of 100 μM for two weeks. For monitoring gene expression and invertase activity, samples at different times, as indicated in the results, were collected.−2 s−1). The 4th leaves from one month potato plants were used for further experiments.Potato  was grown in the greenhouse at 25 ± 2 °C under long day conditions . cDNA synthesis was performed immediately after DNase  treatment, using AMV—Reverse Transcriptase . Full-length cDNAs of Zm-INVINH4 were cloned from maize leaf cDNA by PCR , using Phusion High-Fidelity DNA polymerase with a GC buffer  and corresponding primers presented in E. coli. The cDNA of Zm-INVINH4 was cloned without signal peptide into the pETG10A vector, which leads to the expression of the protein in fusion with a c-terminal 6× His-tag. DNA fragments were purified using the NucleoSpin Extract II Kit , according to the manufacturer’s instructions. A purified product was ligated using T4 DNA ligase , with incubation at 14 °C for 16 h. A ligation product was transformed into E. coli competent DH5α cells by electroporation.Recombinant Zm-INVINH4 protein: For transient expression of the Zm-INVINH4 protein in the potato leaves, the coding region was cloned into the pB7WG2 vector in the downstream of the 35S promoter  were assessed using GeNorm algorithms, and the relative gene expression level was calculated by normalizing the geometric mean of the reference genes, according to a previously described method [qPCR analysis was performed with the Rotor-Gene Q system (Qiagen) using SYBR Green  to monitor dsDNA synthesis. Thermal cycling conditions were identical for all primer pairs: 95 °C/6 min, followed by 40 cycles of 95 °C/20 sec–58 °C/20 sec–72 °C/20 s, followed by a melt cycle from 50 to 95 °C. To determine the primer efficiency, serial dilutions of the templates were conducted for all primer combinations. Each reaction was performed in triplicate, and the amplification products were examined by agarose gel electrophoresis and melting curve analysis. The expression stability of reference genes . Purification of recombinant protein followed the procedure of Eufinger [Plasmid construct for expression of Zm-INVINH4 protein (see above) was transformed into Eufinger .Agrobacterium tumefaciens strain C58C1 harboring Zm-INVINH4 overexpression plasmids  were washed and concentrated by a Centrifugal Filter  with 50 mM NaOAc buffer pH 5. Protein concentrations were determined by a Bradford assay . Different aliquots were incubated with 100 mM sucrose  in 50 mm NaOAc buffer, pH 5.0 at 37 °C for different time intervals. After incubation, the reaction was stopped by heating at 95 °C for 5 min. Released hexoses were determined by a coupled spectrophotometric enzyme assay as described in Link et al. [Extraction of soluble and cell wall-bound proteins from plant tissues was carried out following the protocol described in Link et al. . Bound pk et al. . All enzE. coli extracts (see above) by His-tag affinity chromatography [The recombinant inhibitor protein Zm-INVINH4 was purified from tography . For inhtography . All enzMicroscopic analyses were carried out using a confocal laser scanning microscope . The following excitation and detection wavelength were used: GFP, excitation at 488 nm, detection at bandpass 505–530 nm; RFP, excitation at 543 nm, detection at bandpass 560–615 nm.t-test using SPSS 20.0 software  was used. For further details of statistical analysis see figure legends.Gene expression analysis via qPCR and invertase activity measurement (plant extracts or recombinant proteins) were performed at least 3 independent experiments, with 3 technical replicas for each experiment. For statistical analysis, a Student’s


Thirdhand smoke has been a growing topic for years in China. Thirdhand smoke (THS) consists of residual tobacco smoke pollutants that remain on surfaces and in dust. These pollutants are re-emitted as a gas or react with oxidants and other compounds in the environment to yield secondary pollutants.Collecting media reports on THS from major media outlets and analyzing this subject using topic modeling can facilitate a better understanding of the role that the media plays in communicating this health issue to the public.The data were retrieved from the Wiser and Factiva news databases. A preliminary investigation focused on articles dated between January 1, 2013, and December 31, 2017. Use of Latent Dirichlet Allocation yielded the top 10 topics about THS. The use of the modified LDAvis tool enabled an overall view of the topic model, which visualizes different topics as circles. Multidimensional scaling was used to represent the intertopic distances on a two-dimensional plane.We found 745 articles dated between January 1, 2013, and December 31, 2017. The United States ranked first in terms of publications (152 articles on THS from 2013-2017). We found 279 news reports about THS from the Chinese media over the same period and 363 news reports from the United States. Given our analysis of the percentage of news related to THS in China, Topic 1 (Cancer) was the most popular among the topics and was mentioned in 31.9% of all news stories. Topic 2 (Control of quitting smoking) was related to roughly 15% of news items on THS.2.5), and control and restrictions are the major concerns of the Chinese media reporting on THS. The Chinese press still needs to consider fuller reports on THS based on scientific evidence and with less focus on sensational headlines. We recommend that additional studies be conducted related to sentiment analysis of news data to verify and measure the influence of THS-related topics.Data analysis and the visualization of news articles can generate useful information. Our study shows that topic modeling can offer insights into understanding news reports related to THS. This analysis of media trends indicated that related diseases, air and particulate matter (PM Thirdhand smoke (THS) is an important public health issue and has been an increasingly popular topic for decades in China since its first mention in 2009 . AggregaGiven the increasing interest in THS, the mass media has focused on delivering and communicating information on this topic to public audiences. Both in China and abroad, there have been media reports related to THS. According to search results of news articles mentioning THS, the United States ranks first in terms of topic mentions. However, previous studies have shown that fewer people are aware of the harms of THS than that of secondhand smoke . BecauseMultimodal data modeling combines information from different resources. Topic modeling refers to statistical models in which unstructured data are structured in accordance with latent themes to deal with multimodal data. Latent Dirichlet Allocation (LDA) is the most popular form of topic modeling and is a generative probabilistic modeling method for converting visual words into images and visual word documents -9. TopicWe used the Wiser database for Chinese news content (from the Wise News website) and the Factiva database (from the Dow Jones website) to retrieve the international news articles. The Wiser database is an ever-growing, professional Chinese media content database that contains more than one million data entries. It is currently the best source for Chinese media content given its large volume of data and timely updates. Factiva is an international news and information database that includes nearly 33,000 premium sources such as licensed publications, influential websites, blogs, images, and videos. To get a general idea of the topic of THS, we conducted a preliminary investigation of the Factiva database and retrieved articles dated between January 1, 2013, and December 31, 2017. The Wiser database includes data published only within the last five years. However, many of the articles we flagged merely noted the topic of smoking without further elaborating on the issue of THS. We next narrowed down the entries to only those newspaper sources pertaining to THS; we believe that articles from newspapers are much more reliable than those from other sources. We used LDA to further analyze the Chinese news articles.An LDA topic model is a model with a three-level hierarchical Bayesian model. The basic assumption of this model is a combination of words belonging to different topics . LDA sugData processing  was condMultiple studies have been conducted related to the choice of LDA topic number and the explanations of each topic. In previous research related to topic number, 10-30 topics were assigned nearly the same log-likelihood measure. Therefore, we adopted the number 10 for the topic parameter. By analyzing key words, topic content was generated accordingly. If we considered only the statistical measures, the results might not be interpretable by humans . Therefo2.5), and control and restrictions  of the all-news stories were related to the air and PMIn comparing the Chinese and American news, we found an enormous variation between reports on the same topic in terms of the following three aspects. The Chinese news focused on children and the elderly as victims, whereas the American news focused more on pregnant women. The Chinese media discussed a number of measures and restrictions the government or authorities took or should be taken related to smoking, while the American press focused more on how individuals could eliminate risks. Furthermore, Chinese news articles were, on average, shorter than American news articles.2.5, and tobacco control and restrictions.Thirdhand smoke has been a growing topic for years in China. Topic modeling offers insights into understanding news reports related to THS. This study collected media reports from the United States and China and analyzed them using topic modeling. Specifically, we retrieved Chinese reports about THS from major newspaper and press outlets in China and compared this information with English-language news on the same subject from the United States. Our results revealed that the American press was more concerned with THS than the Chinese. In the Chinese media, three major concerns emerged: links of THS to disease, air and PMOur results indicate that the media has served a public health function by publishing reports that warn the general public about THS-related dangers such as lung cancer, asthma, tumors, and other diseases linked to tobacco. Furthermore, these articles emphasize the possible risks of THS and the susceptible populations  in indoor environments. Smoking cessation and relevant research are also reported. Therefore, the media do communicate the risks associated with smoking, as well as information about prevention and smoking cessation.2.5-related reports, the media attempts to connect sensational concepts with the topic, even if such concepts are not quite related. Therefore, professionalism and credibility are of vital importance. In addition, new media platforms enable the rapid spread of news that is more emotional or personal, or possibly false, which is a serious concern. Aided by new media platforms such as WeChat, Weibo, and Jinri Toutiao (the biggest new media platforms in China), information that may include misleading or exaggerated concepts can quickly be disseminated on personalized newsfeeds.For air quality and PMIn terms of topics related to tobacco control and restrictions, the Chinese media emphasizes control and restrictions more than the United States. The American media focuses on helping smokers quit the habit.Topic modeling is a new method that reveals the major topics in media reports and singles out several key concerns and findings related to the topics. Data analysis and the visualization of news articles can generate useful information. However, there is a limitation to be noted in our study. We included only major media databases, which might omit some news content from new media, such as WeChat posts. Therefore, we may have missed some news stories.Thirdhand smoke is an important public health issue. Collecting media reports on THS from major media outlets and analyzing this subject using topic modeling can facilitate a better understanding of the role that the media plays in communicating this health issue to the public. We conclude that the Chinese press still needs to consider fuller reports on THS rather than simply reporting sensational headlines and needs to show more professionalism by not publishing articles that lack scientific evidence. We recommend that additional studies be conducted related to sentiment analysis of news data to verify and measure the influence of topics revealed from the reports. For example, scientists could measure the educational function of the media for public health or study the influence of misleading information about THS generated by news reports.


However, the safe cumulative dose of pegylated liposomal doxorubicin is unclear.Pegylated liposomal doxorubicin plays an important role in the treatment of patients with severe refractory human immunodeficiency virus (HIV)-associated Kaposi sarcoma (KS). High cumulative doses of conventional doxorubicin exceeding 500 mg/m2. The patient’s left ventricular ejection fraction remained unchanged from baseline during treatment. After he died as a result of cachexia and wasting, caused by recurrent sepsis and advanced KS, an autopsy specimen of his heart revealed little or no evidence of histological cardiac damage. We also conducted a literature review focusing on histological changes of the myocardium in patients treated with a cumulative dose of pegylated liposomal doxorubicin exceeding 500 mg/m2.A 40-year-old Japanese man with HIV infection presented with pain, edema, and multiple skin nodules on both legs which worsened over several months. He was diagnosed with HIV-associated KS. He received long-term pegylated liposomal doxorubicin combined with antiretroviral therapy for advanced, progressive KS. The cumulative dose of pegylated liposomal doxorubicin reached 980 mg/m2) cumulative doses of pegylated liposomal doxorubicin may be used without significant histological/clinical cardiac toxicity in patients with HIV-associated KS.This case report and literature review suggest that high (> 500 mg/m Kaposi sarcoma (KS) is a major life-threatening complication associated with human immunodeficiency virus (HIV) infection. KS is caused by human herpesvirus 8 (HHV-8)/KS-associated herpesvirus (KSHV) infection, which also causes primary effusion lymphoma and HIV-associated multicentric Castleman disease. The incidence of HIV-associated KS has decreased and prognosis has improved dramatically since the development and introduction of antiretroviral therapy (ART). It has been reported that 35% of all patients with HIV-associated KS have an advanced form of the disease, such as visceral involvement, tumor-associated edema or ulceration, or extensive oral KS .Systemic chemotherapy combined with ART is recommended for advanced KS, and pegylated liposomal doxorubicin is the first-line drug used for this purpose. However, there is an emerging problem of patients who do not respond to the combination of chemotherapy and ART. A questionnaire surveillance study of HIV-associated KS in Japan showed that approximately 10% of patients had KS that was refractory to pegylated liposomal doxorubicin therapy combined with ART . In pati2. The risk of developing congestive heart failure (CHF) increases dramatically with cumulative doses of doxorubicin > 400 mg/m2 [2 is not recommended because of the risk of developing CHF [2.The main concern regarding long-term use of doxorubicin is dose-dependent cardiac toxicity. Based on a combined index of signs, symptoms, and decline in left ventricular ejection fraction (LVEF), cardiac toxicity is estimated to occur in 3–20% patients receiving cumulative doxorubicin doses of 300–500 mg/m00 mg/m2 . Therefo00 mg/m2 . Howeverping CHF . However2 of pegylated liposomal doxorubicin, and had no histological evidence of myocardial damage on autopsy. We also review the literature on other patients treated with cumulative doses of ≥500 mg/m2, and who received myocardial biopsy. We acknowledge that we have reported the present case as a clinical image demonstrating 18F-fluorodeoxyglucose-positron emission tomography and gallium-67 scintigraphy findings of KS lesions in the lower extremities [Here, we present a case of severe, refractory HIV-associated KS treated with a high cumulative dose of pegylated liposomal doxorubicin after showing no response to second-line therapy with paclitaxel. The patient eventually died as a result of wasting and cachexia caused by recurrent sepsis and advanced KS, after receiving a cumulative dose of 980 mg/m6 leukocytes in whole blood samples.A 40-year-old Japanese man, who had had sex with men, presented with pain, edema, and multiple skin nodules on both legs which had worsened over several months. Although he had been diagnosed with HIV infection 20 years previously, he had not sought medical treatment. On admission, he was afebrile. His legs had hardened with numerous black nodules on dark skin. Multiple lymph nodes were palpable, including cervical, submandibular, submental, supraclavicular, and axillary lymph nodes. Breath sounds in the left lung were decreased. A chest X-ray showed bilateral pleural effusion. His CD4 T-lymphocyte count was 170 cells/μL and his HIV-1 RNA load was 68,000 copies/mL. His serum C-reactive protein level was 3.1 mg/dL and he had an HHV-8 DNA load of 6500 copies/102 every 2 weeks was initiated for KS. His LVEF before initiation of chemotherapy was 60%. After 16 courses of pegylated liposomal doxorubicin, the cumulative dose was 320 mg/m2. His pleural effusion had decreased, and leg edema and skin nodules had gradually improved. His LVEF was monitored and did not decrease during chemotherapy.A cytological analysis of pleural effusion revealed no atypical lymphocytes suggestive of primary effusion lymphoma. Biopsy specimens of the skin nodules and submental lymph node revealed KS, identified by the presence of whorls of spindle-shaped cells and positive immunostaining for HHV-8 and D2–40. There was no clinical or pathological evidence suggestive of multicentric Castleman disease. The patient was diagnosed with HIV-associated KS with a tumor Stage 1, immune system Stage 1, and systemic illness Stage 1, according to the AIDS Clinical Trials Group Oncology Committee staging criteria . He met 2 every 2 weeks). However, his right pleural effusion increased rapidly after two courses of paclitaxel. Repeated cytology of pleural fluid revealed no atypical lymphocytes. As an echocardiogram indicated that his LVEF had not changed significantly from baseline (62% from 60%), we switched back to pegylated liposomal doxorubicin treatment. At this point, his right pleural effusion began to gradually decrease again. His skin nodules became less marked, but he developed recurrent blisters on his thighs and knees, which required application of topical antiseptics such as silver sulfadiazine cream. He did not experience any palmar-planter erythrodysthesia, thrombocytopenia, or severe neutropenia during the treatment. Cardiac scintigraphy using 123I-β-methyl-P-iodophenyl-pentadecanoic acid (BMIPP), performed after 42 courses of pegylated liposomal doxorubicin, showed no myocardial metabolic defect , breast cancer (5 patients), or ovarian cancer (2 patients). The median cumulative pegylated liposomal doxorubicin dose among these patients was 708 mg/m2 . The Billingham scores of all patients were under 1.5, indicating that no patient had significant histological myocardial damage.It has been proposed that LVEF may not accurately indicate cardiotoxicity in adult patients receiving high cumulative doses of pegylated liposomal doxorubicin . We therKICS is a recently described syndrome that is characterized by severe inflammatory cytokine dysregulation attributed to lytic activation of HHV-8/KSHV. Proposed diagnostic criteria for KICS require that all of the following conditions be met: (1) At least two symptoms, laboratory, or radiographic abnormalities ; (2) evidence of systemic inflammation (elevated C-reactive protein); (3) elevated KSHV viral load in peripheral blood mononuclear cells; and (4) exclusion of multicentric Castleman disease . Since K2 is not necessarily associated with significant clinical/histological cardiac damage. Because alternative therapies are limited, long-term and multi-course pegylated liposomal doxorubicin treatment is occasionally required for refractory HIV-associated KS. Further studies are warranted to identify the cumulative dose of pegylated liposomal doxorubicin that is the threshold for clinical/histological cardiac toxicity in patients with HIV-associated KS.In conclusion, both our patient and previous reports suggest that a cumulative dose of pegylated liposomal doxorubicin exceeding 500 mg/m