The adsorbed magnetic molecules with tunable spin states have drawn wide attention for their immense potential in the emerging fields of molecular spintronics and quantum computing. One of the key issues toward their application is the efficient controlling of their spin state. This review briefly summarizes the recent progress in the field of molecular spin state manipulation on surfaces. We focus on the molecular spins originated from the unpaired electrons of which the Kondo effect and spin excitation can be detected by scanning tunneling microscopy and spectroscopy (STM and STS). Studies of the molecular spin-carriers in three categories are overviewed, i.e., the ones solely composed of main group elements, the ones comprising 3d-metals, and the ones comprising 4f-metals. Several frequently used strategies for tuning molecular spin state are exemplified, including chemical reactions, reversible atomic/molecular chemisorption, and STM-tip manipulations. The summary of the successful case studies of molecular spin state manipulation may not only facilitate the fundamental understanding of molecular magnetism and spintronics but also inspire the design of the molecule-based spintronic devices and materials. Magnetic molecules have attracted extensive interest during the past few decades due to their potential applications as high-density memories, displays, sensors, and so forth ,2,3,4. RI/dV) spectra of the molecules, which refers to scanning tunneling spectroscopy (STS). Specifically, for the magnetic molecular systems absorbed on surfaces, Kondo resonance and spin-excitation-induced inelastic electron tunneling are frequently observed by STS measurement, and they are taken as the key evidence for analyzing the spin information of the systems, such as spin state and magnetic interactions [I/dV spectra.The investigation of the molecular spins absorbed on surfaces has been facilitated by the rapid development of surface science techniques over the past few decades, especially scanning tunneling microscopy (STM) whose ultra-high spatial resolution in real space makes it one of the most powerful tools for directly charactering the surface-confined molecular systems at angstrom scale ,17,18,19ractions ,28,29,30ractions . Thus, tractions ,33. It iThis review focuses on the manipulation of the electron spins of surface-confined molecules, aiming at overviewing the recent progress achieved by molecular-level investigations based on STM and STS. The main body of the review is divided into three parts, each of which summarizes the studies addressing one of the categories of magnetic molecules, i.e., the molecules solely composed of main group elements, the 3d-metal complexes, and the 4f-metal complexes. The examples presented show the application of various methods for controlling the spin state of the molecular systems, including chemical reactions, reversible atomic/molecular chemisorption, and STM-tip manipulations.Ranging from small inorganic radicals such as NO , open-shI/dV spectra  molecule  molecules, are also of interest in terms of molecular magnetism. Pc, a widely used macrocyclic chelating ligand, possesses four indole rings bridged by four –N= groups with their two –NH– and two –N= groups pointing toward the center of the molecule. The coordination of Pc with metal atoms usually takes place via the dissociation of the two H atoms in the –NH– groups of the indole rings, leading to a –2 valence state of the coordinated Pc ligand. Therefore, the stable metal phthalocyanine (MPc) molecules prefer to form between Pc and the metals that can be stabilized in their +2 oxidation state, such as the 3d-metals. As a comparison, for the main-group metals that not tend to stabilize in their +2 state, e.g., K (+1), Al (+3), and Ti (+4), the synthesis of the Pc-coordinated complexes and the following characterization and manipulation of their spin-related properties are challenging.2Pc on Au(111). The formation of the new species was confirmed by its distinct appearance in STM images  upon the tip-induced dehydrogenation . Deprotospectrum a was intspectrum b, which 2 dosage caused the conversion of the original MnPc molecules into the structures with a depression at the center  showed a step-shaped feature at zero bias (red curves in I/dV curves were found for H-MnPc (blue curve in I/dV spectra between the Kondo-featured and featureless curves were achieved by controlled H2 dosages which led to a MnPc to H-MnPc conversion and tip-pulses which resulted in the H detachment of H-MnPc (S = 3/2 state of MnPc on Au(111). The exchange interaction between the localized spin of the Mn ion and the substrate electron gas gave rise to the experimentally observed Kondo resonance. As for H-MnPc, the calculations unraveled an almost unchanged number of d-electrons of the Mn ion but redistributed effective charges in d orbitals compared with MnPc. The latter was responsible for the reduction in the net spin of the molecule from S = 3/2 for MnPc to S = 1 for H-MnPc. Moreover, H attachment also resulted in a larger Mn–substrate separation and thus weakened Mn–substrate coupling for H-MnPc. Both factors contributed to the suppression of the Kondo effect in H-MnPc. In all, the chemisorption of atomic H served as an efficient approach to tuning the spin state of MnPc on Au(111) reversibly. The similar strategy was also employed for the manipulation of the spin state of MnPc on Bi(110) between S = 1 and S = 1/2 states where CO instead of atomic H was used as the adsorbate [Due to the irreversibility of the tip-induced chemical conversions, the above-described strategy usually shows a one-way effect to the molecular spin systems. As another frequently used strategy, the chemical adsorption of atoms or small molecules provides a reversible approach to tuning the molecular spins. One example involving the MnPc/Au(111) system was reported by Liu and coworkers . MnPc moe center b. This vf H-MnPc c. Furthedsorbate .n (n = 4–7) electronic configuration serve as the spin centers. Upon external stimuli such as temperature, light irradiation, pressure, or electric field [Spin crossover (SCO) complexes are a family of coordination compounds featuring switchable spin-state bistability in which transition metals with 3dic field ,71,72,73S = 0) state and a HS (S = 1) state through reversibly changing the molecular structure between the ruffled and flat conformations. The two different molecular conformations were stabilized by the coordination and non-coordination of the central Ni with an axial ligand, respectively. A number of other cases have been overviewed in a recent review [Investigations of SCO phenomena of individual molecules at molecular scale have been widely carried out by STM. One recent example was reported by Kobke et al.  showing t review .I/dV measurements and theoretical calculations. The dark Ni atoms exhibited featureless dI/dV spectra near the Fermi level (blue curve in S = 0 (LS) state. As a comparison, zero-bias anomaly was found for the bright Ni atoms  state. As a consequence, the Ni atoms in the coordination chains were at a HS (S = 1) or a LS (S = 0) state alternately along the chains, giving rise to a unique spin-state ordering known as the antiferroelastic phase. With a magneto-structural dependence, the Ni atoms in the chains could play as the SCO centers when being excited by the tip-pulse located upon. Interestingly, it was found that such SCO behavior, i.e., the spin-state switching between HS and LS states, of multiple Ni atoms in the same coordination chain could take place collectively  and triple-decker (Ln2Pc3) complexes, are a promising class of single-molecule magnets (SMMs) [2 species, focusing on their self-assembly, electronic structures, magnetic properties, and so on [2 systems [2 molecules can be directly visualized by STM [2 systems by tuning the unpaired π-electrons residing on the Pc ligands [Lanthanide (Ln) Pc molecules, e.g., double-decker (LnPcs (SMMs) . Their lnd so on ,92,93,94 systems ,85,89,93d by STM ,89,91,94 ligands ,93.2 on Au(111) by rotating the upper Pc ligand with controlled current pulses [2, that is, the Tb ion and an unpaired electron in the ligand π orbital. Although the magnetic signature of the former was invisible by STM in this case, the Kondo effect of the latter was captured by spectroscopic investigations. The dI/dV measurement revealed the site dependence of the Kondo feature: the Kondo peak was prominent around the lobes of the molecule but was dramatically attenuated at the center  between the two Pc ligands of 45° whose center appeared brighter in the STM images  had an S = 1/2 spin originated from the unpaired π-electron. However, the rotation of the upper ligand to θ = 30° led to the rearrangement of the frontier molecular orbitals, which resulted in the charge transfer from the surface, therefore quenching the molecular spin and hence the Kondo state. Komeda and coworkers reported the switch-on and off of a π-electron spin of TbPct pulses . There ae center a. This sM images b, the KoM images d. Once pe to 30° c. Meanwh2 in the double-barrier tunneling junction (DBTJ) consisting of vacuum and CuO film (2 molecules. The adsorption of DyPc2 on the ultrathin CuO film on Cu(110) led to four types of molecules, that is A, B, and C in the assembled structures  was attributed to the spin excitation of the molecule from ms = −1/2 to ms = +1/2 which was originated from the Zeeman splitting of the degenerated ms = ±1/2 spin states at B = 8 T (B = 0 and 8 T (red and blue curves in 2 in the DBTJ, the manipulation of the charge state could hence lead to the change in the spin state. It was demonstrated that the charge state of DyPc2 in this system could be tuned by applying different biases of the DBTJ, which would give rise to the switching-on and off of the ligand spin of DyPc2.The investigation of DyPcCuO film a by Zhanructures b and theructures b inset.  B = 8 T d. Theref2 and DyPc2. Examples are presented to show the efficient effect of several spin state tuning strategies, including chemical reactions, reversible atomic/molecular chemisorption, and STM-tip manipulations, in different on-surface molecular systems. The employment of STM and STS in these studies enables the exploration of the spin-related phenomena and properties at molecular level by detecting the Kondo effect or spin excitation of the molecular systems. These case studies contribute to the fundamental understanding of molecular magnetism and spintronics.In this review, we summarize the recent investigations of spin state manipulation of surface-confined molecules in three categories, i.e., molecules composed of main group elements such as verdazyl radicals, ReA and AlPc, molecules comprising 3d-metals such as 3d-MPcs and the spin crossover coordination chains, and molecules comprising 4f-metals such as TbPcTo make further steps toward the practical application of the magnetic molecules as spintronic and quantum computing devices, the studies focusing on the magnetic molecular systems located on carbon-based substrates or nanostructures are of great significance. On the one hand, the work by Cervetti et al. concerning the molecular-spin–graphene hybrids has demonstrated that the interaction between magnetic molecules and graphene has an essential influence on the quantum dynamics of molecular spins . Further


First presentation of the Kaposi sarcoma (KS) on the penis is not prevalent, and it was reported in 2%‐3% of the cases that mostly occurred in the HIV‐positive patients. Here, we report a case of primary KS on the glans penis in an HIV‐negative patient. First presentation of the Kaposi sarcoma (KS) on the penis is not prevalent, and it was reported in 2%‐3% of the cases that mostly occurred in the HIV‐positive patients. Here, we report a case of primary KS on the glans penis in an HIV‐negative patient. He presented with erythematous nodular penile lesions of 4 months' duration. There was no history of fever, weight loss, or any other symptoms. He also denied any history of systemic disease. Our patient was married with no history of extramarital relationship.In his physical examination, there were some reddish, nontender nodules of 5‐10mm on the ventral and dorsal sides of glans penis , edema (21.2%), bleeding (14.9), and itching (3.9%).4Kaposi sarcoma should be considered as a differential diagnosis for erythematous nodular penile lesion. A rare presentation of KS may be present as a single lesion on the penis without any known risk factors. Therefore, histological evaluation is recommended in the patients with described penile lesions.None declared.FA: served as the main author and contributed to data acquisition and manuscript preparation. MT: contributed to data acquisition and manuscript writing. FB‐Z and ZA‐K: contributed to performing laboratory tests. RMR: served as the corresponding author and designed and supervised all the aspects and contributed to manuscript editing. All authors: contributed sufficiently and met the criteria for authorship.


Similarly, each 50% increase in maximum turbidity was associated with increased risk of CV death/MI  and CV death alone . The relationship between lysis time and the combined outcome of CV death and MI remained significant after adjusting for multiple prognostic vascular biomarkers (p = 0.034). Neither lysis time nor maximum turbidity was associated with major bleeding events. Impaired fibrin clot lysis predicts 1-year CV death and MI in diabetes patients following ACS.Hypofibrinolysis is a key abnormality in diabetes but the role of impaired clot lysis in predicting vascular events and mortality in this population is yet to be determined. We aimed to investigate the relationship between fibrin clot properties and clinical outcomes in patients with diabetes and recent acute coronary syndrome (ACS). Plasma samples were collected at hospital discharge from 974 ACS patients with diabetes randomised to clopidogrel or ticagrelor in the PLATO trial. A validated turbidimetric assay was employed to study fibrin clot lysis and maximum turbidity. 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 determined using Cox proportional analysis. After adjusting for CV risk factors, each 50% increase in lysis time was associated with increased risk of CV death/MI (HR 1.21; 95% confidence interval [CI] 1.02–1.44;Clinical Trial Registration URL:http://www.clinicaltrials.gov. Unique identifier NCT00391872. Up to 30% of patients presenting with acute coronary syndrome (ACS) suffer from diabetes mellitus.12inhibitors (ticagrelor/prasugrel) post-ACS improved outcomes in patients with diabetes without an apparent penalty of increased major bleeding compared with clopidogrel.Potent P2YClinical characteristics, such as the extent of coronary artery disease, history of recurrent events or renal impairment, and elevated CV biomarkers could help guide intensity of treatment,Cross-sectional studies have repeatedly shown a relationship between coronary artery disease and dense fibrin networks that are resistant to lysis.The PLATelet inhibition and patient Outcomes (PLATO) trial was an international multi-centre, double-blind, randomised controlled trial of ticagrelor compared with clopidogrel in 18,624 moderate-to-high-risk ACS patients.27.5 mM) and clotting was initiated with thrombin (0.03 U/mL). Fibrin clot maximum turbidity (a measure of fibrin clot density) and lysis time were determined using a Multiskan FC (Thermo scientific) plate reader in all 974 plasma samples taken at hospital discharge and 820 plasma samples taken at 1 month. All laboratory analysis was performed blinded to clinical outcomes, treatment allocation and other biomarker levels.This was performed using a turbidimetric assay as previously described.p-Values < 0.05 from two-tailed tests were considered statistically significant.p-Values were not adjusted for multiple testing. All statistical analyses were performed at Uppsala Clinical Research Centre using R statistics software .Biomarker levels were natural log transformed before analysis. Continuous data are presented as medians and interquartile ranges and compared using Kruskal–Wallis tests, Wilcoxon tests or multivariable linear regression models, as appropriate. Categorical data are presented as numbers and percentages and compared using chi-square tests. The primary outcome of interest was the composite of CV death and spontaneous myocardial infarction (MI). Secondary outcomes were CV death alone, MI alone, PLATO-defined major bleeding and all-cause mortality. Cox-proportional hazards models were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). HRs are expressed per 50% fibrin variable level increase. We adjusted for clinical risk factors (model 1) and for known prognostic biomarkers. Model 1 included all clinical risk factors, including randomised treatment, age, gender, body mass index (BMI), smoking history, hypertension, dyslipidaemia, chronic kidney disease (CKD), ST-elevation ACS and previous MI, congestive heart failure, revascularisation, ischaemic stroke or peripheral artery disease. Prognostic biomarkers were added sequentially to adjustment model 1. Model 2 included adjustment for model 1, white cell count and C-reactive protein (CRP). Model 3 included adjustment for model 1 clinical risk except for CKD, white cell count, CRP and cystatin C. Model 4 included adjustment for model 3 risk factors, troponin T and N-terminal pro B-type natriuretic peptide (NT-proBNP). Model 5 included all model 4 risk factors and growth differentiation factor-15. Restricted cubic splines were used to visually assess the relationship between fibrin clot properties and clinical outcomes. Interactions between prognostic value of fibrin clot parameters and each of randomised treatment, treatment strategy and treatment with low-molecular-weight heparin (LMWH) (within 2 days of sampling) were assessed using restricted cubic splines. Mean ± standard deviation fibrin clot properties at hospital discharge and 1 month were compared using Wilcoxon signed-rank test.After adjustment for risk factors and clinical characteristics (model 1), fibrin clot lysis time and maximum turbidity were significantly higher in patients with diabetes compared with those without. The majority of other prognostic and inflammatory biomarkers were significantly higher in patients with diabetes .r = 0.37,p < 0.001).The correlation between fibrin clot maximum turbidity and lysis time was weak (Platelet count and low-density lipoprotein (LDL) cholesterol levels increased with increasing fibrin clot lysis time but showed no relationship to maximum turbidity. There was no significant difference in the proportion of patients receiving ticagrelor across the four lysis time quartile groups.r = 0.29,p < 0.001). In contrast, the relationship between fibrin clot maximum turbidity and CRP appeared more linear . Both lysis time  and maximum turbidity  were significantly, though weakly, correlated with troponin T.The correlation between lysis time and the inflammatory biomarker CRP was weak (Glycated haemoglobin (HbA1c) also significantly increased with increasing lysis time  but no During follow-up, 48 patients (4.9%) had CV death, 72 (7.4%) had MI, 67 (2.9%) had major bleeding and 21 (2.2%) had non-coronary artery bypass graft-related major bleeding.The probability of the combined outcome of CV death and MI was higher with increasing values of lysis time . This wSimilarly, the probability of the combined outcome of CV death and MI was higher with increasing maximum turbidity . The hip = 0.034).After adjustment for clinical characteristics and CV disease risk factors (model 1), each 50% increase in lysis time was associated with increased risk of CV death/MI  and CV death alone . Similarly, every 50% increase in lysis time was associated with increased risk of all-cause mortality after adjustment for clinical risk factors in model 1  and this remained significant after adjustment for inflammatory biomarkers . After p = 0.15).Similarly, each 50% increase in maximum turbidity was associated with increased risk of CV death/MI  and CV death alone . These relationships lost significance after adjustment for prognostic biomarkers. There was a numerical increase in bleeding in the lowest quartile group of maximum turbidity but this failed to reach statistical significance (p > 0.1)  .Similarly, the prognostic value of fibrin clot lysis time was consistent regardless of diabetes status but the magnitude of relationship appears to be higher in patients with diabetes .p = 0.64) despite a drop in overall maximum turbidity .Fibrin clot lysis time remained prolonged at 1 month (864 ± 406 seconds) compared with hospital discharge (863 ± 445 seconds) (For the first time, in a large cohort of ACS patients with diabetes, we have shown prolonged fibrin clot lysis to predict CV death and MI after adjustment for clinical risk predictors as well as traditional and novel prognostic biomarkers. We have also shown fibrin clot lysis time to remain prolonged at 1 month following ACS, despite a drop in fibrin clot turbidity. Being a functional assay, our methodology takes into account quantitative and qualitative changes in various clotting factors, thus giving an overall assessment of fibrin-related thrombosis risk. The independent association between impaired lysis and worse ischaemic outcomes may indicate that some patients may benefit from further optimisation of anti-thrombotic therapy, for example, by dropping dual anti-platelet therapy and replacing it with a combination of a single anti-platelet agent and an anticoagulant, since anticoagulant therapy potentiates fibrinolysis.Despite the large number of patients included in our study, the study lacks sufficient power to detect an independent relationship between individual components of the primary endpoint and fibrin clot properties. However, the trend of association with both CV death and MI is consistent.Diabetes and prolonged lysis are both associated with many high-risk patient characteristics and prognostic biomarkers. We have adjusted for many potential confounders and although the association between prolonged fibrin clot lysis and CV death/spontaneous MI remained significant, including many confounders in adjustment models is a limitation. Reassuringly, the results are very similar with the many different models we tested for.Another limitation of our methodology is that we studied fibrinolysis in plasma samples rather than whole blood. This may exclude the cellular effects on the fibrinolytic pathway. However, our results provide a ‘proof-of-concept’ of the importance of intrinsic fibrinolysis in predicting outcomes.The very weak correlation between fibrin clot lysis potential and troponin levels indicates a possible pathophysiological link with worse outcomes that is independent of the size of MI. Fibrin clot maximum turbidity had a stronger relationship with CRP as well as troponin and NT-proBNP. This is to be expected as turbidity reflects fibrin clot density, which is more closely related to fibrinogen levels, and fibrinogen levels are well known to increase with inflammation, such as may be provoked by large MIs.Although fibrin clot turbidity is only marginally higher in the diabetes population compared with individuals without diabetes, lysis time is significantly longer in the former group, consistent with previous studies.LDL cholesterol levels and platelet count increased with increasing lysis time. These observations are intriguing and support previously reported effects of cholesterol and increased platelet reactivity on fibrinolysis.Increased incorporation of anti-fibrinolytic proteins into the fibrin clot represents another mechanism for impaired fibrinolysis in diabetes.The relationship between impaired fibrin clot lysis and female gender is consistent with previously observed results in other cohorts of high-risk vascular patients with type 2 diabetes.Adverse fibrin clots that resist lysis predict CV death and MI in ACS patients with diabetes despite contemporary therapies. The relationship between high-risk vascular conditions and impaired lysis provide potential mechanistic insights into recurrent events. The weak correlation between fibrin clot lysis potential and troponin indicates that the association with worse outcomes is relatively independent of the magnitude of MI. Developing strategies to improve lysis tendency may help improve prognosis in high-risk ACS patients and future research in this area is warranted.


EPHX2 and CYP450 on renal and vascular function, plasma levels of EETs and peripheral blood monuclear cell sEH activity was assessed in 79 kidney transplant recipients explored at least one year after transplantation. Additional experiments in a mouse model mimicking the ischemia–reperfusion (I/R) injury suffered by the transplanted kidney evaluated the cardiovascular and renal effects of the sEH inhibitor t-AUCB administered in drinking water (10 mg/l) during 28 days after surgery. There was a long-term protective effect of the sEH SNP rs6558004, which increased EET plasma levels, on renal allograft function and a deleterious effect of K55R, which increased sEH activity. Surprisingly, the loss-of-function CYP2C9*3 was associated with a better renal function without affecting EET levels. R287Q SNP, which decreased sEH activity, was protective against vascular dysfunction while CYP2C8*3 and 2C9*2 loss-of-function SNP, altered endothelial function by reducing flow-induced EET release. In I/R mice, sEH inhibition reduced kidney lesions, prevented cardiac fibrosis and dysfunction as well as preserved endothelial function. The preservation of EET bioavailability may prevent allograft dysfunction and improve cardiovascular disease in kidney transplant recipients. Inhibition of sEH appears thus as a novel therapeutic option but its impact on other epoxyfatty acids should be carefully evaluated.This study addressed the hypothesis that epoxyeicosatrienoic acids (EETs) synthesized by CYP450 and catabolized by soluble epoxide hydrolase (sEH) are involved in the maintenance of renal allograft function, either directly or through modulation of cardiovascular function. The impact of single nucleotide polymorphisms (SNPs) in the sEH gene  Ischemia–reperfusion injury, innate and adaptive immune activation and calcineurin inhibitors promote vascular toxicity and renal endothelial dysfunction which play a key role in chronic allograft nephropathy6. In addition, cardiovascular disease remains a leading cause of allograft loss and mortality in kidney transplant recipients7. Therefore, the development of new therapeutic strategies that could preserve kidney allograft function directly and/or through vascular protection is of crucial importance.While the incidence of acute allograft rejection has decreased over the last decades, chronic allograft nephropathy has only been marginally improved and represents the major cause of long-term dysfunction in kidney transplantation11. After their synthesis, EETs are metabolized to the less active dihydroxyeicosatrienoic acids (DHETs) by the ubiquitous enzyme soluble epoxide hydrolase (sEH), encoded by the EPHX2 gene11. Inhibitors of sEH have thus been developed to increase EET bioavailability and protective effects were observed in animal models of diseases11. Two sEH inhibitors moved to the first phases of clinical development, showing no toxicity13, but still remain unavailable for human use.Epoxyeicosatrienoic acids (EETs) are arachidonic acid derived eicosanoids synthesized by cytochrome P450 enzymes (CYP450) in endothelial and renal cells that induce vasodilation, natriuresis and prevent inflammation and apoptosisCYP450 and EPHX2 genes are highly polymorphic and some single nucleotide polymorphisms (SNPs) are known modulators of their enzymatic activities towards EET biosynthesis and degradation at least in vitro11. The determination of the effects of these SNPs on patients’ clinical characteristics represents a common way to assess the interest of modulating EET pathway in disease. To date, only the presence of the EPHX2 3-untranslated region (3′-UTR) polymorphism of the recipient was identified as a modifying factor of kidney allograft function15. However, the controversial results reported are difficult to interpret especially since the effect of this SNP on sEH expression or activity remains unclear15. In contrast, no impact of the frequent EPHX2 exonic SNPs i.e., R287Q and K55R that are known to affect sEH activity, has been observed in these studies15. The reasons for these unexpected findings are not known but simultaneous presence of different EPHX2 SNPs with opposite effects on sEH activity could be evoked16. Furthermore, although the modifications of the bioavailability of immunosuppressive agents induced by CYP450 SNPs have been extensively investigated17, only one study suggested that the presence of CYP450 loss-of-function (LoF) polymorphisms of the donors worsened the outcome of renal transplantation18. Moreover, no study has evaluated the impact of EPHX2 and CYP450 SNPs on vascular function in kidney transplant recipients.EPHX2 and CYP450 of kidney transplant recipients affect renal and vascular function taking into consideration their effects on EET metabolism. Moreover, experiments in a mouse model mimicking the ischemia–reperfusion (I/R) injury suffered by the transplanted kidney were performed to directly evaluate the cardiovascular and renal effects of blocking sEH in this context.Thus, the aim of this study was to carefully assess whether genetic variations in https://clinicaltrials.gov under the unique identifier NCT02555566.A total of 79 patients with a kidney transplantation performed in Rouen University Hospital and followed since at least 1 year were included in this study. Medical records of these patients were reviewed to obtain age at transplantation, donor age, immunosuppressive agents and concomitant treatments, comorbidities and plasma creatinine at 3, 6 and 12 months after kidney transplantation, allowing the determination of estimated glomerular filtration rate (eGFR) using the MDRD equation. All patients were Caucasians in order to limit ethnic variability regarding genetic investigations and aged between 18 and 75 years old. Patients with a previous renal graft or acute renal rejection demonstrated by kidney biopsy were excluded from the study. The study was approved by the local Ethics Committee (Committee for the Protection of Persons of Normandy), and all participants gave written informed consent. The study was conducted in accordance with the Principles of Good Clinical Practice and the Declaration of Helsinki and was registered at 20.Measurements were performed in the morning after a fat-free breakfast without tea or coffee while subjects were in a supine position, in a quiet air-conditioned and thermostated room (22–24 °C). Subjects were asked to refrain from smoking from the previous evening. Blood pressure and heart rate were measured on the dominant arm using a brachial cuff oscillometric device (OMRON HEM-705CP). Radial internal diameter and blood flow were measured on the non-dominant arm, using a multiarray high-resolution echotracking and Doppler 23. The hand skin temperature was modified by use of a water-filled thermo-controlled device . Briefly, the hand was introduced in the thermo-controlled tank by use of a thin watertight glove fixed to the device. The device was then filled with water and the temperature was fixed to 34 °C for 20 min. Then, hand skin heating was performed by gradually increasing the water temperature from 34 to 44 °C with each level of temperature maintained for 7 min. Radial artery endothelium-independent dilatation was assessed using 0.3 mg sublingual glyceryl trinitrate .Radial artery endothelium-dependent flow-mediated dilatation was assessed during a progressive and sustained increase in blood flow induced by hand skin heatingEPHX2 and CYP450 genotyping from whole blood. Genomic DNA was isolated using a QIamp DNA Blood Mini Kit  according to manufacturer’s instructions. Genotyping was carried out by Taqman allelic discrimination assays  on a 7500 Fast Real-Time PCR System . EPHX2 genotyping consisted in the assessment of the presence of the common exonic SNPs of EPHX2 K55R (rs415907953) R287Q (rs751141) and of the most prevalent intronic SNPs 3′UTR (rs1042032) and rs6558004. rs6558004 was in fact identified by a bioinformatic analysis and allows to study 19 intronic SNPs with a strong linkage disequilibrium (LD > 0.90) and a minor allele frequency higher than 15% , 2C9*2 (rs1799853), 2C9*3 (rs1057910), 2C19*2 (rs4244285) and 2J2*7 (rs890293) SNPs and of the gain-of-function (GoF) CYP2C19*17 (rs12248560) polymorphism was assessed. All these CYP generate EETs from arachidonic acid and the presence of their SNPs were shown to be associated with some cardiovascular diseases26.A 4-F catheter was inserted into the forearm cephalic vein, when accessible, allowing blood sampling in the venous return at 34 and 44 °C. Blood sampling was drawn at 34 °C to measure plasma creatinine and to perform  K55R rs45907953 R3H]-trans-1,3-diphenylpropene oxide-based radioactivity assay27. The sEH activity was normalized to total protein level measured by the BCA method.In addition, peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by density gradient centrifugation for further determination of sEH protein activity using a  years after kidney transplantation. Clinical characteristics of patients are summarized in Table .1–16.1 yEPHX2 and CYP450 SNPs was in agreement with Hardy–Weinberg equilibrium based on the MAF from the HapMap-CEU population listed at https://www.ncbi.nlm.nih.gov/snp/ , while their short-term impact appears less marked . In contrast, kidney weight was reduced in I/R mice treated with vehicle while this reduction was not significant in I/R mice treated with t-AUCB , which are also produced by the CYP450-sEH pathway but from linoleic acid, in K55R carriers39. This was observed in some populations but not all39, suggesting that other factors besides sEH catalytic activity play a role in the diseases associated with this SNP37. None of the analyses clearly identified the 3′-UTR SNP as a predictor of eGFR although a trend for a poorer renal function was observed in the Leaps analysis 3 months post-transplantation. This result could be explained either by the absence of impact of the 3′-UTR or by its association with rs6558004 or with R287Q, which may be also protective, thus masking its possible deleterious effect. In a previous study, haplotype construction, including R287Q and K55R, also showed a trend for a deleterious impact of the 3′-UTR SNP on the outcome of deceased-donor renal transplantation in Caucasians15. To note, the limited number of subjects included in the present study may have lea to only detect the greatest effects of EPHX2 SNPs on renal function.First of all, for the sEH, the Leaps analysis and haplotype construction both showed that, in addition to the classical predictors of eGFR, the presence of rs6558004 is associated with a higher eGFR and this impact appeared in particular more marked at the time of the exploration visit up to 16 years after transplantation. Interestingly, haplotype construction did not show a decreased sEH activity in carriers of the rs6558004 intronic mutation but these patients had higher plasma level of 8,9-EET, which was identified as the only EET regioisomer with a protective effect on the glomerular filtration barrierCYP450 SNPs assessed affected allograft function in the short term and only one, CYP2C9*3, appeared to positively affect allograft function in the long term. This unexpected finding is observed while no apparent modifications in EETs or DHETs are observed. We cannot exclude that the presence of this SNP in only 8 patients may have led to a misleading type II error. However, hypothetically, this LoF polymorphism may reduce the bioavailability of other epoxyfatty acids derived from omega-3 that are known to be efficiently produced by CYP2C9 and have recently been shown to potentiate experimental kidney injury40. The weak impact of CYP450 polymorphisms on allograft function was also reported in one study showing that only CYP2J2*7 SNP of the donor but not of the recipient negatively affects the outcome of renal transplantation18.Furthermore, none of the 23, the reduced EET production in response to the increase in blood flow may promote endothelial dysfunction of conduit arteries in kidney transplant recipients. This mechanism may contribute to the deleterious impact of LoF CYP450 SNPs on cardiovascular outcomes in this population41. Furthermore, patients carrying R287Q had a higher magnitude of flow-mediated dilatation but also of endothelium-independent dilatation to GTN. This surprising result had been previously observed in one study evaluating the impact of EPHX2 polymorphisms at the arteriolar level42. Indeed, endothelium-independent dilatation of forearm resistance arteries in response to local infusion of sodium nitroprusside was enhanced in R287Q carriers and the authors speculated that this effect was related to a long-term preventive effect of increasing EET bioavailability against vascular remodelling42. In support of this hypothesis, carriers of the only GoF SNP CYP2C19*17 also had increased endothelium-dependent dilatation. Modification in sEH activity was not detected in carriers of R287Q but the low number of subjects and the association of this SNP with K55R in some patients may explain this result. In addition, no change in EET and DHET levels was observed in R287Q and CYP2C19*17 carriers. As previously discussed for K55R, the absence of modifications in circulating EETs or DHETs bioavailability may not reflect tissue levels and, in R287Q carriers, only the circulating EpOME-to-DiHOME ratio but not EET-to-DHET ratio was increased as expected44.At the vascular level, the presence of CYP2C8*3 and CYP2C9*2 SNPs seemed to exert opposite effects on endothelium-dependent flow-mediated dilatation but the strong associations between both precludes the direct analysis of their real impact. However, carriers of these LoF polymorphisms had no change in EET or DHET basal bioavailability but displayed a reduced variation in EET + DHET during the sustained endothelial stimulation induced by heating. Given the major role of EETs in sustained flow-mediated dilatation45. As expected, plasma creatinine dramatically increased shortly after I/R and largely improved with time. However, plasma creatinine did not fully recover and kidney lesions were present at sacrifice, reflecting the particular severity of the model. This is notably due to the genetic background used, which is known to be particularly prone to kidney injury47, the duration of ischemia that was longer than generally performed in mice28 and the global clamping of renal vascular pedicle, with both artery and vein28. In accordance with the severity of this model, endothelial vasomotor alterations, LV fibrosis and cardiac diastolic and systolic dysfunction developed, as previously demonstrated in subtotal nephrectomy46.Then, the interest of inhibiting sEH in this setting was further investigated with a pharmacological approach in mice subjected to kidney I/R. Indeed, I/R is a major determinant of short and long-term allograft dysfunction in kidney transplant recipients, in addition to allo-immune and nephrotoxic mechanismst-AUCB. This model of I/R is characterized by rapid recovery of kidney function, and the transient increase in creatinine was not significantly affected by t-AUCB. These results suggest that the pharmacological blockade of sEH may be more effective in preventing the deleterious mechanisms associated with renal I/R that contribute to allograft dysfunction, especially inflammation, than those involved in the development of chronic kidney disease. As recently reviewed48, the renoprotective effects of sEH inhibition appear mostly believed to be ascribed to the increased renal levels of EETs with respect to their potent anti-inflammatory and antifibrotic actions. In the present work, the impact of t-AUCB may be related at least partly to the antiapoptotic effects of EETs that have been shown to protect tubular cells against I/R and its consequences through inhibition of NF-κB and MAPK activation and restoration of glycogen synthase kinase 3β (GSK3β) phosphorylation49. However, one should keep in mind that blockade of sEH may also exerts detrimental effect in this context by potentiating the bioavailability of epoxyfatty acids derived from omega-3 that, at the opposite, potentiates tubular cell apoptosis by worsening the reduction in GSK3β phosphorylation40. Furthermore, cardiovascular explorations showed for the first time that t-AUCB also prevented the cardiac dysfunction and remodelling as well as the renal endothelial dysfunction induced by I/R. Such beneficial effects are of particular interest in kidney recipients who are also more likely to experience cardiovascular complications than the general population. Of importance to define future human therapeutic strategy, it should be interesting to know whether this beneficial effect of sEH inhibition on renal inflammation and cardiovascular complications persists in the long-term in particular even after stopping drug administration.Interestingly, kidney lesions, especially interstitial inflammation, were clearly reduced at day 28 after I/R in mice treated with the sEH inhibitor 49.Altogether, the indirect evidence from the human genetic association study and the results obtained in animal experiments support the preservation of EET bioavailability as a novel therapeutic option to prevent allograft dysfunction and cardiovascular complications in kidney transplant recipients, in particular through inhibition of sEH. A possible limitation of sEH inhibition that emerges in the context of kidney diseases is that the concomitant potentiation of other epoxyfatty acids with sEH inhibition, especially those derived from omega-3, may hamper the beneficial renal impact of EETs. More targeted strategies such as the use of EET analogues alone or combined with sEH inhibition, may further optimize the promising benefits of modulating CYP450-sEH pathway in kidney transplantationSupplementary Information 1.


RET gene fusions in patients with NSCLC has become clinically important. As the number of biomarkers to be tested in NSCLC grows, it becomes increasingly important to optimize and prioritize the use of biopsy tissue, in order to both continue to allow accurate histopathological diagnosis and also to support concurrent genomic profiling to identify perhaps relatively uncommon genetic events. In order to provide practical expert consensus guidance to optimize processes facilitating genomic testing in NSCLC and to overcome barriers to access and implementation, a multidisciplinary advisory board was held in New York, on January 30, 2019. The panel comprised physicians involved in sample procurement , surgical pathologists specializing in the lung, molecular pathologists, and thoracic oncologists. Particular consideration was given to the key barriers faced by these experts in establishing institutional genomic screening programs for NSCLC. Potential solutions have been devised in the form of consensus opinions that might be used to help resolve such issues.For patients with advanced non–small cell lung cancer, genomic profiling of tumors to identify potentially targetable alterations and thereby inform treatment selection is now part of standard care. While molecular analyses are primarily focused on actionable biomarkers associated with regulatory agency-approved therapies, there are a number of emerging biomarkers linked to investigational agents in advanced stages of clinical development will become approved agents. A particularly timely example is the reported data and US Food and Drug Administration approval of highly specific small molecule inhibitors of the proto-oncogene tyrosine-protein kinase receptor RET indicate that testing for tumor  EGFR and BRAF and chromosomal rearrangements resulting in ALK, ROS1, and NTRK1–3 gene fusions. Recently the US Food and Drug Administration (FDA) has approved therapies targeting RET fusions, RETVMO (selpercatinib) and GAVRETO  as well as MET exon 14-skipping variants, TABRECTA (capmatinib) in NSCLC. Additionally, emerging targets such as HER2 insertions, and KRAS mutations add to the list of important biomarkers that may be included in testing protocols. Genomic alterations are almost exclusively present in non-squamous carcinomas, including in ~30% of adenocarcinomas overall when considering biomarkers with approved targeted therapies. Actionable activating alterations in proto-oncogenes commonly result in constitutive signaling through an intrinsic protein kinase, which drives oncogenesis. Specific inhibitors targeting the kinase products of such genes have now been synthesized and have either gained regulatory approval or are in clinical development.Most patients with non–small cell lung cancer (NSCLC) present with advanced stage unresectable disease requiring treatment with systemic therapies. Genomic profiling of tumors to identify potentially targetable alterations and/or eligibility for immune checkpoint inhibition has resulted in improved clinical outcomes in particular patient subgroups and is now part of standard care , 2. In NRET gene fusions in lung cancer, and activating point mutations in medullary thyroid cancer  was reported with the FDA approval of, selpercatinib, while a response rate of 85%  was reported for treatment naive patients with RET-fusion positive advanced NSCLC treated with selpercatinib  in treatment naive patients with RET-fusion positive advanced NSCLC   .In order to provide practical expert consensus guidance to optimize genomic testing in NSCLC, in light of approved and developing clinically relevant biomarkers, and to overcome barriers to access and implementation, a multidisciplinary advisory board was held in New York, on January 30, 2019. The panel comprised physicians involved in sample procurement , surgical pathologists specializing in the lung, molecular pathologists and thoracic oncologists. Particular consideration was given to the key barriers faced by these experts in establishing institutional genomic screening programs for NSCLC. Potential solutions have been devised in the form of consensus opinions that might be used to help resolve such issues.In order to maximize the level of biomarker testing, a shift in practice is required from a biopsy being viewed primarily as a tool for histological diagnosis to one where it is utilized as a route to complete pathologic staging, histological diagnosis, molecular profiling of the tumor, and ultimately a patient-specific treatment decision. This is of critical importance as unless molecular profiling is specified upfront and that message reaches the surgical pathologist and/or cytopathologist, then much or all of the available tissue may be used for diagnostic approaches, including immunohistochemistry. Therefore, close communication between the oncologist, surgeon, pulmonologist, interventional radiologist and pathologist is required during the entire specimen acquisition process.Both fine needle aspirates (FNA) and percutaneous core biopsies can provide sufficient material for diagnosis and molecular profiling and both may be procured from the same procedure. Investigators have shown that these approaches have similar overall diagnostic value in relation to identifying lung malignancies in patients with lung lesions, with no significant difference in procedural complications . LikewisConsideration should also be given to which lesion to biopsy. In particular, those that can provide adequate tissue and best define the stage of the tumor should be chosen as the target lesions. An accessible metastatic site or suspicious mediastinal node would pathologically define the highest stage, while also generally providing sufficient tumor material for molecular profiling. The use of endobronchial ultrasound using FNA needles, if carefully performed, has been documented to provide adequate tissue for molecular and immunologic analyses , 33.With regard to percutaneous biopsy, the size of the lesion is not the only consideration, but also the location in the lung. In patients with multiple bilateral lesions who have had prior thoracic surgery, the visceral and parietal pleura often scar together, reducing the chance of pneumothorax. Large, anteriorly located, peripheral pleural based, upper lobe lesions are perhaps the simplest to biopsy. The technique to avoid crossing multiple fissures which results in an increased rate of complications often requires placing patients in the prone position during the procedure. This is less well-tolerated compared with patients positioned supine. Peripheral lesions have been shown to decrease the incidence of crossing large central vessels that can result in intraparenchymal hemorrhage and hemoptysis. Anatomically, upper lobe lesions are preferred as the lung apices move less with respiration than lesions located near the diaphragm.Lung biopsies have among the highest rate of complications of all radiologic procedures with the main associated adverse event being pneumothorax, reported in over 50% of patients in some series. However, the incidence of this potential complication can be considerably reduced through the use of a tract sealant system or blood patch –36. The Other rarer complications arising from lung biopsies include hemoptysis, air embolization, tract seeding, and death. Tract seeding is exceedingly rare, with descriptions mostly limited to case reports; the reported frequency is 0.06% .To quickly assess a core sample and make a determination not only as to whether diagnostic material is present but also to make a more extensive assessment of whether or not it is going to be adequate for molecular studies, rapid on-site evaluation (ROSE) is essential. In particular, ROSE can allow for the immediate designation of material for molecular analysis, minimizing the rate of tissue insufficiency and by extension, rebiospy . The metIn addition to cell block preparations from FNA procedures which are routinely utilized for molecular testing, many laboratories can also perform testing on cytological smears prepared and stained onsite which may improve chances for successful molecular diagnosis.EGFR, BRAF, HER2, ALK, ROS1, RET, NTRK1–3, or MET) or associated resistance mechanisms in cfDNA may be potentially informative, a negative plasma-based assay should be regarded as uninformative, with a chance of false negative results, and testing should be subsequently performed by a tissue-based analysis. The modest diagnostic sensitivity reported for cfDNA analyses is most likely a consequence of heterogeneous DNA shedding in patients with solid tumors, which may vary according to tumor stage and type. A more recent study has shown that the use of liquid biopsies may result in a greater number of biomarkers tested vs. current standard of care  DNA may provide an alternative to the analysis of NSCLC when, as indicated in current NCCN guidelines, a representative tumor tissue sample is not available for molecular studies , 38. Howr tests) . On a prBoth FNA and core needle biopsies may provide adequate tissue for histologic and molecular diagnosis, and both may be procured from the same procedure; 4–5 cores using a 20 gauge needle should be attempted to minimize the need for re-biopsyThe most appropriate lesion for biopsy (whether in the lung or a metastatic lesion) should be chosen to allow for full staging and diagnosis while optimizing patient experience and minimizing patient adverse eventsWhile reportedly common and generally treatable, risk of pneumothorax can be significantly reduced by the use of tract sealants or a blood patchOnsite evaluation of the specimen by an experienced cytopathologist or equivalently trained professional can maximize tissue acquisition, improve diagnostic yield, aid in tissue triage for molecular studies and avoid re-biopsycfDNA testing can be considered if tissue acquisition is infeasible but its limited sensitivity, particularly for fusions, must be consideredThe precision oncology diagnostic workflow is summarized in Samples from patients with known and suspected NSCLC intended for molecular diagnosis should be prospectively identified within the histology workflow. This highlights the need for interdisciplinary communication at the time of samplingOnce identified for molecular testing, specific processes to maximize tissue availability should be deployed, with the understanding that these may add cost and time for the providing laboratory and that constraints on the utilization of these processes may be reasonableEGFR, ALK, ROS1, and perhaps KRAS to exclude further testing and for clinical trial eligibility), and driver negative tumors are then subjected to broader NGS profiling. In general, single gene tests, such as polymerase chain reaction (PCR)-based assays for EGFR mutations , or fluorescence in situ hybridization (FISH) or immunohistochemistry for ALK fusions, can be carried out more quickly , and are on an individual basis cheaper than NGS approaches. However, if multiple single marker assays are performed, the overall cost may exceed that of NGS analysis, which may consequently be more cost effective  and 5′/3′ differential expression technology . In addiffective . In addiROS1, NTRK2, NTRK3, NRG1, and RET in particular. The recent tumor type agnostic approvals of larotrectinib and entrectinib for patients with tumors harboring NTRK gene fusions makes the detection of patients with TRK fusion NSCLC an emerging clinical priority , the risk of tissue exhaustion and the overall cost effectiveness of NGS in NSCLC makes it the method of choice to screen tumors for actionable driversROS1) and splice alterations Analysis of both DNA and RNA by NGS can improve sensitivity for fusions  analysis and those which can be used for highly multiplexed parallel assessments such as 5′/3′ differential expression assays and NGS.Techniques that have been used to detect or imply the presence of RET fusions due to low sensitivity and highly variable specificity, when compared with molecular methods. Studies have shown that the sensitivity of RET IHC ranges between 55 and 65% with specificities of 40–85%, leading clinical guideline authors to recommend additional studies before consideration is given to the use of IHC for the identification of tumors harboring RET fusions  approaches reported to date appear to be of limited value in relation to the detection of  fusions , 11, 50.RET and other fusions is to use a 5′/3′ differential expression assay   –55. As shnology) . Assays l method , 54. HowAs increasing numbers of clinically relevant predictive biomarkers are characterized across multiple disease settings, it is likely that NGS will become the key diagnostic tool to inform treatment decisions in advanced cancers. NGS assays can be DNA- or RNA-based and can be targeted to a panel of genes relevant to treatment decisions, either through an amplicon or hybridization capture design. In relation to the detection of gene fusions, amplicon-based enrichment strategies require the 5′ partner gene and breakpoints to be known, whereas hybridization capture approaches allow for the detection of novel fusion partners. A more recent technology, anchored multiplexed PCR, combines these approaches, with a targeted capture at one end of the fusion RNA to provide specificity, and a second adapter ligated to the other end of randomly sheared nucleic acid strand, to enable the detection of all fusions, regardless of the specific partner.ROS1, NTRK2, and NTRK3 gene fusions, which are predictive of response to specific kinase inhibitors, including in NSCLC  design are the recommended modalities for fusion detection in general, inclusive of It is likely that in a notable proportion of cases, the pathologist will conclude that the tissue available for analysis is insufficient to run all of the tests requested by the oncologist. In these situations, triage of biomarker testing may be required. With poorly differentiated lung tumors, often a substantial proportion of the available material is needed to investigate a large panel of potentially informative immunohistochemistry markers. In such situations it may be that procedures can be adjusted to balance diagnostic priorities with preservation of tissue for molecular analysis. For example, immunohistochemistry investigations may be ordered in sequential rounds and slides may be precut upfront. This may result in longer turnaround times in certain patients but may help preserve tissue for molecular analysis.RET fusions, suggests a lack of efficacy of these agents in this setting  from Junshi Pharmaceuticals; and personal fees from Abbvie Pharmaceuticals, ARMO Biosciences, Biodesix, Bolt Biotherapeutics, Bristol-Myers Squibb, EMD Serrano, Genentech/Roche, Genmab, Heat Biologics, Halozyme, IMAB Biopharma, Immunocore, Loxo Oncology, Midas Health Analytics, Mirati Therapeutics, Nektar, Novartis, Pfizer, Sanofi, Seattle Genetics, Shire PLC, Spectrum Pharmaceuticals, Symphogen, Takeda, Tesaro, Tocagen, outside the submitted work. DA reports personal fees in relation to consultancies from Bayer Oncology, Genentech, AbbVie, and Bristol Myers Squibb, outside the submitted work. JS reports personal fees from Loxo Oncology in relation to advisory board attendance, during the conduct of the study. AT reports personal fees from Loxo Oncology in relation to advisory board attendance, during the conduct of the study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


More recent systems and methods have produced nanofiber yarns (NFY) for ease of use in textiles. This paper presents a method of NFY manufacture using a simplified dry electrospinning system to produce self-assembling functional NFY capable of conducting electrical charge. The polymer is a mixture of cellulose nanocrystals (CNC), polyvinyl acrylate (PVA) and poly polystyrene sulfonate (PEDOT:PSS). When treated with ethylene glycol (EG) to enhance conductivity, fibers touching the collector plate align to the applied electrostatic field and grow by twisting additional nanofiber polymers injected by the jet into the NFY bundle. The longer the electrospinning continues, the longer and more uniformly twisted the NFY becomes. This process has the added benefit of reducing the electric field required for NFY production from >2.43 kV cm Electronic textiles  refer to electronic systems embedded in clothing. Previously, these specially engineered textiles were designed mainly for extreme weather conditions ,2,3. MorTo produce electronic textiles, existing spinning systems for yarn fabrication will require modification or replacement. Structurally, yarn is made of a long continuous length of interlocked fibers. Conventional mechanical fiber-spinning technologies cannot produce fiber diameters smaller than a few micrometers, with most current commercial fibers featuring significantly larger diameters for cost efficiency. However, several traditional techniques, such as bicomponent spinning, melt-blowing and flash spinning, have been shown to produce sub-micrometer or nanometer scale fibers in the nonwoven mats . Other mElectrospinning has been widely explored to produce fibers with micrometer to nanometer diameters from polymers, composites, ceramics, and metals in the form of solution and melt. Electrospun yarns have been used in membranes, filtration, tissue scaffolds, and wound dressings with more recent applications in the mechanical, electrical, optical, and biomedical fields ,10. PrevLike conventional electrospinning systems, the proposed method uses electrostatic forces to produce fine nanofibers. This method differs by further using electrostatic repulsion in conductive fibers to align the fibers with the applied field while keeping the tips separated. Combined with the mechanical whipping motion of electrospinning, the process attracts newly spun nanofibers to attach to existing strands, twisting the charged nanofibers into bundles around the longitudinal axis to form the yarn .The process of yarn formation occurs in three stages. In stage 1, the conductive nanofibers are spun out of the charged droplet of the spinneret by the applied electric field between the spinneret and the collector. The nanofibers break off from the jet and attach to the collector electrode, aligning longitudinally in the direction of electrostatic force, while continuing to swirl from the naturally occurring whipping motion. The conductive properties of the standing fibers result in charge concentrations at their tips, attracting newly-spun fibers from the oppositely-charged jet, lengthening the standing fibers a. When i−1. Testing was conducted at various distances (7.0 to 8.5 cm) between the spinneret and aluminum foil covered collecting plate. All experiments were conducted at 25 °C room temperature and 70% humidity. The setup of the proposed electrospinning system is nearly identical to the conventional configuration, except for the use of a flat metal electrode. In this procedure, a 27-gauge needle is connected to a high-voltage power supply  capable of generating DC voltages up to 50 kV. During testing, the applied DC voltage ranges from 15 to 19 kV depending on the composition of the nanofiber solution. The solution is continuously supplied using a syringe pump  at a rate of 5 uL minTo explain the electrostatic fiber repelling and twisting motion aided by the electric field, a Finite Element Model (FEM) was created using COMSOL software. The model uses three simple assumptions: First, that a conductive nanofiber that settles on the charged collector plate concentrates charge at the tip as it aligns to the direction of the applied electric field. Second, that fibers spun out of the droplet will have an opposite charge to the collector nanofibers and be attracted to the collector fiber tips. Third, any additional nanofibers that join into a bundle will also concentrate charge at their tips.The model is based on a basic electrospinning system with one nozzle and a planar collection plate. The nozzle was set to 15 kV and two representative fiber tips (the two ‘black rice’ shapes) were set as the alternate grounds from the ground plate b. To be As only the assembly of NFY is of interest, the model only considers stages 2 and 3. In stage 2, the longitudinal electric field concentrates charge at the two fiber tips, attracting (and repelling) the tips vertically while the charge at the tips provides a transverse repulsion force. However, at the start of fiber growth c the proIn summary, while more nanofibers are gradually depositing/growing on the fiber tips, there is a visible variation of the electrostatic field distribution due to the charge concentration at the fiber tips providing a counterforce to the charge of the plate. The growth of the nanofiber yarn isolates the electric fields of the tips from the field from the ground plate shown in . FurtherIn the fabrication of the conductive yarn structure, the most important consideration is the electric field that aligns the nanofibers on the collection plate, allowing the twisting behavior. The conductivity of the polymer solution is also critical, as this impacts the induced surface charge for the nanofibers. Our experiments focused on these parameters.The three main ingredients are cellulose nanocrystal (CNC), poly polystyrene sulfonate (PEDOT:PSS), and polyvinyl alcohol (PVA). The CNC slurry (purchased from the University of Maine) was diluted to 5.7%. The PEDOT:PSS is Clevios™ PH1000 from Heraeus . The ethylene glycol (EG) (99.8%) and PVA were purchased from Sigma Aldrich. The PVA and PEDOT:PSS were selected for conductivity and mechanical integrity. Only a viscoelastic material that can undergo strong deformations while being cohesive enough to support the stresses developed during deformation can be drawn into nanofibers. The polymer solution for the spinning experiment was prepared by dissolving the PVA in PEDOT:PSS at 150 °C on a hotplate for 3 h before adding the dimethyl sulfoxide (DMSO) or EG.The conjugated chain of the π bond on PEDOT offers an electrical path for the transport of electrons, resulting in fascinating optical and electrical properties. Some polar solvents (such as dimethyl sulfoxide (DMSO), EG, and glycerol) have been found to enhance the conductivity of PEDOT:PSS by more than an order of magnitude ,29,30,31In our experiment, we explored EG and DMSO as polar solvents. −1. Instead, randomly oriented nanofibers are generated (−1 for either EG or DMSO. From experiments D to J (−1) for yarn formation than DMSO (>2.42 kV cm−1). Note that the E field must be below 2.8 kV cm−1 to prevent corona discharge between the needle and its surroundings.Different voltages and gap distances were examined for yarn formation. From the test results A, B and C , the solenerated a. For Mas D to J , we obse1) is around 28°, which can be observed in the SEM picture. This increases to a 38° twisting angle (Ɵ2) with 20 min of spinning.The influence of electrospinning time on yarn density was also investigated. Two runs were conducted using Material B, one electrospun for 10 min and the other for 20 min. The results are shown in Electrospinning is a promising method for producing nanofibers on an industrial scale. Fabricating electrospun nanofibers into assembled structures such as yarn increases their utility and creates the potential for new innovative applications of e-textile sensors and systems, such as incorporation with a flexible humidity and airflow sensors ,34.Previously proposed methods for assembling nanofibers into yarn included continuous, twisted, and hybrid approaches. However, these tend to involve complicated additions and modifications to existing electrospinning systems. In this paper, we presented a simple yarn fabrication method utilizing conductive polymer materials with no modification to the electrospinning system. We demonstrated the concept of how the yarn forms from nanofiber tips on the ground plate due to the longitudinal and transverse electric fields, and how electrostatic repulsion and mechanical swirling from the electrospinning system form the fibers into bundles using an electrical field FEM. We have successfully demonstrated the concept and conducted a series of studies on how to improve the overall yarn formation by the introduction of a solvent treatment to our base PVA/PEDOT:PSS solution and how different solvents (EG and DMSO) lower the electric field intensity required for yarn formation. We also studied the yarn density as a function of electrospinning time. Additional studies are still required to address some remaining challenges with this technique. We intend to address and optimize the design’s mechanical strength and electrical conductivity for a future publication. A US patent has been filed prior to the submission of this paper (106F0280-IE(JAP106054-US)).


Kaposi’s sarcoma (KS) remains the most frequent malignancy in persons living with HIV (PWH) in Latin America. We examined KS trends and outcomes from Latin American clinical sites in the era of increased access to antiretroviral therapy (ART).Cohorts in Brazil, Peru, Mexico, Honduras, Argentina and Chile contributed clinical data of PWH ≥16 years old from 2000 to 2017, excluding patients with KS diagnosed before clinic enrolment. We compared KS incidence over time using multivariable incidence rate ratios. Predictors of KS before/at or after ART initiation and of mortality after KS were examined using Cox regression.Of 25 981 PWH, 481 had incident KS, including 200 ART‐naïve and 281 ART‐treated patients. From 2000 to 2017, the incidence of KS decreased from 55.1 to 3.0 per 1000 person‐years. In models adjusting for CD4 and other factors, the relative risk for KS decreased from 2000 to 2008. Since 2010, the adjusted risk of KS increased in the periods before and ≤90 days after ART initiation but decreased >90 days after ART. In addition to low CD4 and male‐to‐male sex, KS risk after ART was associated with age and history of other AIDS‐defining illnesses. Mortality after KS (approximately 25% after five years) was not associated with either year of KS diagnosis nor timing of diagnosis relative to ART initiation.KS incidence in Latin America has remained stable in recent years and risk is highest before and shortly after ART initiation. Early diagnosis of HIV and ART initiation remain critical priorities in the region. In the Globally, KS has been strongly associated with severe immunodeficiency, male sex (particularly men who have sex with men [MSM]), and regional prevalence of human herpes virus 8 (HHV‐8) , 5. AmonThis study examines the epidemiology, risk factors and outcomes of incident KS in the current ART era among PWH in Latin America using a multinational cohort.22.1This study utilized data from two multi‐cohort, observational studies of adults living with HIV in Latin America: the Caribbean, Central and South America network for HIV Epidemiology (CCASAnet) and Coorte Brasil. CCASAnet includes clinical sites from seven Latin American countries and is a member of the International Epidemiologic Databases to Evaluate AIDS (IeDEA) consortium . CCASAnePWH ≥ 16 years of age enrolled in a CCASAnet or Coorte Brasil site between January 2000 and December 2017 were included in this study. KS can occur in children and adults with HIV. This study focused on adults and used age criteria consistent with other global studies of cancer epidemiology in PWH . We excl2.2We calculated KS incidence per calendar year and adjusted incidence rate ratios using multivariable general estimating equations including calendar year, clinical site, age at cohort entry, sex/sexual route of HIV infection , CD4 cell count at cohort entry (square root transformed) and prevalent AIDS‐defining illness (excluding KS) at cohort entry. Calendar year, age and CD4 cell count at enrolment were fit with restricted cubic splines (four knots) to relax assumptions of linearity . To exam2.3We used Cox proportional hazard models to examine patient characteristics associated with KS before and after ART initiation among ART‐naïve patients. In these analyses, observation time was again divided into before ART initiation and after ART initiation. In the first analysis, age, history of other AIDS‐defining illness, sex/sexual risk factor for HIV transmission, HIV RNA (logarithmic transformed) and CD4 cell count (square root transformed) at clinic entry were evaluated in univariate and multivariable Cox models for KS risk from date of clinic entry to date of ART initiation. Observation time was censored at first occurrence of date of KS diagnosis, ART initiation, death or loss to follow‐up. In the second analysis, the above patient characteristics and ART regimen class associated with KS risk following ART initiation were evaluated from the date of ART initiation to KS diagnosis, death or loss to follow‐up. All Cox models were stratified by country. Age, HIV RNA and CD4 cell count were fit using restricted cubic splines with three knots. Missing data  were multiply imputed using chained equations with ten imputations. Imputation models included all covariates in the main model as well as the interaction of the outcome (Kaposi’s sarcoma) and the person‐time of follow‐up.2.4Lastly, we evaluated whether the timing of KS diagnosis relative to ART initiation was associated with mortality. We included all patients with incident KS. Patients with ART treatment before clinic enrolment were considered to have KS after ART. Patients who never started ART were considered to have KS before ART. We examined whether patient characteristics at KS diagnosis  and timing of KS diagnosis (before or after ART initiation) were associated with mortality risk using Kaplan–Meier curves and multivariable Cox proportional hazard models stratified by country. Age and CD4 cell count (square root transformed) were fit using restricted cubic splines with three knots. Missing data  were multiply imputed using chained equations with 10 imputation replications. Imputation models included all covariates in the main model as well as the interaction of the outcome (death) and the person‐time of follow‐up.http://biostat.mc.vanderbilt.edu/ArchivedAnalyses.All analyses were completed using R version 3.5.3 (2019‐03‐11). Statistical code for all analyses is available at 33.1In total, 27 425 PWH were included in CCASAnet and Coorte Brasil cohorts between 2000 and 2017. Of those, 257 (0.9%) were excluded for KS diagnoses before clinic enrolment. Our analysis included a total of 25 981 PWH who contributed 133 233 person‐years of follow‐up. Of these, 78% were ART‐naive at clinic entry. There were 481 diagnoses of KS at or after clinic entry, including 107 (22%) diagnosed on the date of entry, and 145 (30%) diagnosed within 30 days following entry. Two hundred patients were diagnosed with KS before ART initiation, 125 were diagnosed within the first 90 days after ART initiation, and 156 were diagnosed more than 90 days following ART initiation. Among those who developed KS before ART, the median time from clinic entry to KS was five days (interquartile range [IQR]: 0 to 28 days). Among those who developed KS after ART, the median time from ART initiation to KS was 92 days (IQR: 28 days to 1.09 years).As shown in Table 3.2The incidence of KS decreased from 55.1 in 2000 to 3.0 per 1000 person‐years in 2017. This decrease largely occurred from 2000 to 2005. Incidence remained relatively stable between 3 and 5 per 1000 person‐years from 2005 to 2017 Figure . We exam3.3We examined characteristics associated with KS risk using Cox proportional hazard models among patients who were ART‐naïve upon clinic entry n = 20 229). Among the 371 KS diagnoses, 200 occurred before ART initiation and 171 occurred after ART initiation. Table 0 229. Am3.4Lastly, we evaluated mortality risk following KS diagnosis relative to occur before or after ART initiation. We included all 481 KS diagnoses of which 200 (42%) occurred before ART initiation and 281 (58%) occurred at any point after ART initiation. Patient characteristics at the time of KS diagnosis are shown in Table 4In this large, multinational cohort of PWH in Latin America, we observed both stable and dynamic trends in KS epidemiology, risk factors and outcomes in the current ART era. While the incidence of KS has decreased in the region since 2000, most of this occurred in the early years and the overall risk of KS has remained fairly stable since 2005 to 2008. After accounting for other factors, the risk of KS before and shortly after ART initiation appears to have increased in recent years, whereas the incidence >90 days after ART initiation has decreased, perhaps a result of improved long‐term adherence and HIV outcomes. Immunodeficiency and MSM HIV transmission risk factor were consistently associated with KS risk in the region. Lastly, the risk of mortality following KS (approximately 25% within five years after KS diagnosis) was statistically similar for those diagnosed before or after ART initiation and was unchanged over time, showing that ongoing work is needed to improve long‐term outcomes of PWH with KS in the region.The incidence of KS dramatically decreased in PWH in Latin America in the early 2000s. In many high‐income countries, rates of KS fell significantly in the early 1990s, though KS remains an important cause of cancer morbidity in the US and other countries , 14, 15.We observed differences in patient risk factors for KS relative to ART initiation. Before and after ART initiation, sex/sexual risk factor for HIV transmission and immune deficiency were strongly associated with KS. After ART initiation, age and history of other AIDS‐defining illnesses were also associated with risk. Numerous studies have shown higher rates of KS among MSM and those with low CD4 count , 24. HowWe observed a high risk of death following KS diagnosis, but no difference between PWH diagnosed with KS before versus after ART initiation. Despite improved survival among PWH, the history of KS has been associated with long‐term risk of mortality , 9. In gThis study has important limitations to consider. Our study was strengthened by careful validation of KS diagnoses. However, the lack of information regarding KS stage  and accompanying symptoms or progression  precluded evaluating disease‐specific characteristics and outcomes. Furthermore, without corresponding KS staging, our KS treatment data could not be reliably used in analyses. Many CCASAnet sites are tertiary referral clinics and the patients may not be representative of all PWH in their respective cities and countries. Two sites in Brazil  are referral centres for KS, specifically. We excluded patients with KS diagnoses prior to clinic entry and adjusted for site to reduce this bias. Furthermore, our results are only reflective of KS epidemiology of PWH without diagnosed KS at the time of clinic entry and exclude patients whose KS prompted HIV diagnosis and entry into care. This also meant we excluded patients with a second episode of KS after entry into care, who may have different outcomes. Our study was limited by lack of HHV‐8 seroprevalence data, and thus, we cannot describe how HHV‐8 epidemiology contribute to our results. Of note, studies from Brazil and Peru suggest that HHV‐8 prevalence is higher among PWH and MSM , 31. Our5In conclusion, we observed dynamic changes in KS epidemiology among clinical cohorts from Latin America. With the increasing availability of ART, the incidence of KS has dramatically decreased since 2000, even after controlling for CD4 cell count at clinic entry and other patient factors. However, we observed stable rates of KS from approximately 2008 to 2017, highlighting the ongoing need to reduce KS. Currently, our best tools for reducing KS are increasing early HIV diagnosis and treatment. The risk of KS remains highest before and shortly after ART initiation and is highest among MSM and patients with severe immunodeficiency. Mortality following KS diagnosis was not associated with the year of diagnosis in adjusted models. Further research in KS treatment interventions, long‐term morbidity, and specific causes of death of PWH with KS are needed to improve patient outcomes.All authors of this study declare they have no competing interests.All authors have read and approved the final manuscript. J.L.C., C.C.M., B.E.S., B.G., B.R.C., P.F.B.Z. and V.F. designed the research study. B.G., E.G., V.F., D.P., P.F.B.Z., B.C.R., M.M.E., R.A.S., S.B.T., S.R.P., M.L.R.I, P.R.A., U.T., C.B., E.L., J.N., C.P.C. and A.G. contributed essential research contributions to the collection and validation of high‐quality observational data. J.L.C., C.C.M., B.E.S., A.K., C.A.J., B.R.C., P.F.B.Z. and V.F. wrote the manuscript.Figure S1. Incidence of KS by year, 2000 to 2017.Click here for additional data file.Figure S2. Adjusted log hazard ratio for KS before ART by (a) CD4 cell count and (b) age. Model (a) adjusted relative to an individual aged 35 years from Brazil, MSM, median log10 HIV RNA and no other AIDS‐defining illness before enrolment. Model (b) adjusted relative to an individual from Brazil, MSM, CD4 cell count of 200 cells/μL, median log10 HIV RNA and no other AIDS‐defining illness before enrolment.Click here for additional data file.Figure S3. Adjusted log hazard ratio for KS after ART by (a) CD4 cell count and (b) age. Model (a) adjusted relative to an individual aged 35 years from Brazil, MSM, median log10 HIV RNA and no other AIDS‐defining illness before enrolment. Model (b) adjusted relative to an individual from Brazil, MSM, CD4 cell count of 200 cells/μL, median log10 HIV RNA and no other AIDS‐defining illness before enrolment.Click here for additional data file.Figure S4. Adjusted log hazard ratio for mortality after KS by (a) CD4 cell count and (b) age. Model (a) adjusted relative to an individual aged 35 years from Brazil, MSM, median log10 HIV RNA, KS diagnosis in 2011 and KS after ART initiation. Model (b) adjusted relative to an individual from Brazil, MSM, CD4 cell count of 100 cells/μL, median log10 HIV RNA, KS diagnosis in 2011 and KS after ART initiation.Click here for additional data file.Table S1. Descriptive characteristics at all patients with KS diagnosis following clinic entryClick here for additional data file.


Vibrio vulnificus is a well-known opportunistic pathogen causing food-borne illnesses by ingestion of contaminated seafood. A new strain of V. vulnificus FORC_016 was isolated from a patient’s blood sample in South Korea. The genome consists of two circular DNA chromosomes: chromosome I  and chromosome II . In addition, chromosome I has a super integron (SI) containing 209 ORFs, which is probably associated with various additional functions including antibiotic resistance and pathogenicity. Pan-genome analysis with other V. vulnificus genomes revealed that core genome regions contain most of the important virulence factors. However, accessory genome regions are located in the SI region and contain unique genes regarding cell wall biosynthesis and generation of host cell protecting capsule, suggesting possible resistance ability against environmental stresses. Comparative RNA-Seq analysis of samples between contact and no contact to the crab conditions showed that expressions of amino acid/peptide and carbohydrate transport and utilization genes were down-regulated, but expressions of cell division and growth-related genes were up-regulated, suggesting that the crab may be a nutrition reservoir for rapid propagation of V. vulnificus. Therefore, consumption of the contaminated fresh crab would provide a large number of V. vulnificus to humans, which may be more dangerous. Consequently, biocontrol of V. vulnificus may be critical to ensure the safety in seafood consumption. Vibrio vulnificus is an opportunistic human pathogen that causes fever, nausea, septic shock, and its average mortality rate is over 50% , cytolysin (vvhA), and repeats-in-toxins (RTX) are major virulence factors. For most bacterial infections, attachment and colonization of host surfaces are important steps in the early phase of infections. Pili are proteinaceous fibers that stick out from the cell surface of bacteria, which often mediate the initial attachment to the host surface. Likewise, V. vulnificus adherence has been demonstrated to correlate with increased piliation  of V. vulnificus abolished capsule expression, resulting in decreased mortality , a primary recombination site (attI), and multiple target-specific recombination sites (attC)  . The SI s (attC) . To furtV. vulnificus is one of the major pathogens causing seafood outbreaks, its genome information and deduced functions are not fully understood yet. To date (April 2020), 22 complete genome sequences of V. vulnificus are available in the GenBank database. Among them, only three reports, with the complete genome sequences of strains MO6/24-O, FORC_017, and VV2014DJH, were published, showing general genome features with pathogenicity and various virulence factors predicted by bioinformatics . The cytotoxicity of FORC_016 was confirmed using the lactate dehydrogenase (LDH) release assays  were assigned to be functional and 591 ORFs were hypothetical proteins. In addition, chromosome II consists of 1,837,945 bp with a GC content of 47.00% containing 1,572 ORFs, 13 tRNA genes, and 3 rRNA genes. Among them, 1,223 ORFs (77.80%) were assigned to be functional and 349 ORFs were hypothetical proteins. Furthermore, 2,711 ORFs on chromosome I and 1,453 ORFs on chromosome II were assigned into Clusters of Orthologous Groups (COGs) of proteins functional categories using the webMGA program  on chromosome I, which is a super integron region, suggesting that it may have been derived from a different origin , antibiotic resistance (β-lactamase and related efflux pump), and iron uptake (TonB-dependent heme receptor and vibriobactin genes for ferric iron uptake), associated with the additional functions to compete with other bacteria. Therefore, the combination of these two chromosomes may cover all required basic and additional functions for dominant growth and survival of the host strain.FORC_016 has two chromosomes and each chromosome has its own virulence factors . ChromosVibrio strains revealed that V. vulnificus FORC_016 was closely related to other V. vulnificus strains  analysis was conducted. The ANI analysis showed that FORC_016 belongs to the clinical type including other clinical V. vulnificus such as MO6-24/O, a clinical isolate from a patient with septicemia, suggesting that FORC_016 may be a potential pathogen causing foodborne outbreak  in the pan-genome map  may be associated with cell membrane biosynthesis , cytolysin (vvhA), RTX toxin (rtxABCD), and vibriolysin (vvp) (in (vvp) . The genin (vvp) . Furtherin (vvp) . They weV. vulnificus strains, CPS cluster I may be strain-specific (The genome of FORC_016 has antibiotic resistance-associated genes encoding a cyclic AMP receptor protein (FORC16_2625), β-lactamase (FORC16_3220), and a resistance-nodulation-division (RND) efflux system (FORC16_3925) . Cyclic specific . ComparaV. vulnificus in the host are highly associated with iron concentrations in infected individuals , suggesting the iron uptake ability of FORC_016    (Supplem16_3705) . TherefoluxS) encoding autoinducer (FORC16_2386), suggesting its QS signaling activity for toxin or biofilm formation (V. vulnificus is a major toxin that elicits the secondary wound damage for septicemia (vvhA), RTX toxin (rtxABCD), and hemolysin (tlh), suggesting its pathogenesis in human infection  signaling is widely used to communicate with each other and to regulate the virulence genes under given environmental conditions . FORC_01ormation . Vibriolpticemia . FORC_01pticemia . In addinfection . TherefoV. vulnificus FORC_016 reference genome. To elucidate the difference of RNA expression profiles between two conditions of FORC_016 with or without crab, their RNA expression profiles were compared after 1 or 4 h incubation  and nitrite (NO2–) to ammonia as well as their transport genes were down-regulated. Ammonia is an essential element for amino acid biosynthesis . It was identified using 16S rRNA gene sequencing with a 27F/1492R universal primer set . They were aerobically grown in modified Luria-Bertani medium supplemented with 2% (w/v) NaCl (LBS) for 12 h at 30°C. Agar medium was prepared with supplementation of 1.5% Bacto Agar   .A600 of 0.8) were harvested and washed with sterilized phosphate-buffered saline (PBS). Then, PBS-resuspended cells (4 × 109 CFU/250 ml) were subinoculated in either Vibrio fischeri minimal medium containing 32.6 mM sterilized glycerol (VFMG) with or without a tap water-prewashed fresh crab as test and control samples, respectively. These cultures were incubated for 1 or 4 h at 30°C. To remove the crab cells, the cultures were double-filtered using a syringe containing sterilized gauze and subsequent vacuum filter containing Whatman No.1 filter paper . After filtration, RNAprotect Bacteria Reagent  was added to the filtered solutions containing bacterial cells for RNA stability. Total RNAs were extracted and purified using a miRNeasy Mini Kit (Qiagen), according to the manufacturer’s procedure. The quality of total RNAs was determined using an Agilent 2100 Bioanalyzer and Agilent RNA 6000 Nano reagents .Genomic DNA was extracted and purified using a DNeasy Blood & Tissue Kit , according to the manufacturer’s protocol. Concentration of the purified genomic DNA was determined using a NanoVue spectrophotometer . For RNA extraction, the bacterial cells grown to log phase (V. vulnificus FORC_016 and MO6-24/O at a various multiplicity of infection (MOI) for 2 h as previously described  released from damaged cells as an indicator of cell damage, was measured using a Cytotoxicity Detection Kit  with the manufacturer’s protocol. The INT-407 human intestinal epithelial cells (ATCC CCL-6) were incubated and tested with escribed .V. vulnificus 16S rRNA genes were conducted using MEGA6 software with the neighbor-joining method and 1,000 bootstrap replicates  and a PacBio RS II platform  according to their standard protocols. For construction of complete genome sequence, the qualified DNA reads were assembled using CLC Genomics Workbench ver. 7.0.4  and PacBio SMRT Analysis ver. 2.0 software (Pacific Biosciences). The open reading frames (ORFs) and tRNA/rRNA of the assembled genome were predicted using a Rapid Annotation using Subsystem Technology (RAST) server , and theplicates . Average program . Pan-genV. vulnificus FORC_016, and the relative transcript abundance was measured with the concept of reads per kilobase of transcript per million mapped sequence reads (RPKM)  to remove rRNAs and converted to cDNA using an iScript cDNA Synthesis Kit . The cDNA library was evaluated using Bioanalyzer 2100 with DNA 1000 reagents . The cDNA sequencing was performed using an Illumina HiSeq 2500 sequencer. The cDNA sequence reads were mapped to complete genome sequences of s (RPKM)  using CLs (RPKM) . The recFor quantification of specific mRNA transcripts, quantitative real-time PCR (qRT-PCR) amplification was performed using the Chromo 4 real-time PCR detection system (Bio-Rad) with SYBR Green I and a pair of specific primers listed in Vibrio vulnificus FORC_016 grown to A600 of 0.8 was harvested and washed, and subsequently inoculated in VFMG, VFMG containing crab, or LBS. The growth of V. vulnificus was measured by counting the colony forming unit (CFU)/ml from serial dilutions by 1X PBS plated onto LBS agar. The incubation times were 0 h, 1 h, and 4 h. The plates were incubated in 30°C for 12 h and the colonies were counted. The experiments were run with biological duplicates and for each biological sample, technical triplicates were used.V. vulnificus FORC_016 were deposited in the GenBank database of the National Center for Biotechnology Information (NCBI)4 under accession numbers CP011775 and CP011776. Total mRNA sequences for transcriptome analysis in V. vulnificus FORC_016 were deposited in the NCBI Sequence Read Archive (SRA)5 under accession number PRJNA532503.Complete genome sequences of The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/SC and J-HL designed the research. SK, HC, and J-GK performed the research. SK, HC, J-GK, and J-HL analyzed the data. SK, HC, SC, and J-HL wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.