Cannabis genus, i.e., the “phytocannabinoids”  can influence a wide array of biological processes, and the human body is able to produce endogenous analogs of these substances . These ligands, together with multiple receptors , and a complex enzyme and transporter apparatus involved in the synthesis and degradation of the ligands constitute the endocannabinoid system (ECS), a recently emerging regulator of several physiological processes. The ECS is widely expressed in the human body, including several members of the innate and adaptive immune system, where eCBs, as well as several pCBs were shown to deeply influence immune functions thereby regulating inflammation, autoimmunity, antitumor, as well as antipathogen immune responses, etc. Based on this knowledge, many in vitro and in vivo studies aimed at exploiting the putative therapeutic potential of cannabinoid signaling in inflammation-accompanied diseases  or in organ transplantation, and to dissect the complex immunological effects of medical and “recreational” marijuana consumption. Thus, the objective of the current article is (i) to summarize the most recent findings of the field; (ii) to highlight the putative therapeutic potential of targeting cannabinoid signaling; (iii) to identify open questions and key challenges; and (iv) to suggest promising future directions for cannabinoid-based drug development.It is well known that certain active ingredients of the plants of  On one hand, signaling bias can lie in the “choice” of the ligand in preferring  the β-arrestin pathway(s) over the G protein-coupled one or vice versa. On the other hand, in some cases coupling to Gαs, Gαq, or Gα12/13 proteins  was also observed, adding an extra layer of complexity to CB1 signaling   , A2A adereceptor , D2 dopareceptor , orexin-receptor , etc., wreceptor , or GPR5receptor . On top 1 and CB2 double KO mice exhibited stronger allergic inflammation than wild-type mice. Importantly, these alterations were shown to be mediated via the pathologically increased chemokine release of epidermal keratinocytes, suggesting that lack of homeostatic eCB signaling skewed keratinocytes toward a proinflammatory phenotype. On the other hand, FAAH−/− mice (having elevated eCB levels) showed reduced allergic responses , these processes include regulation of appetite, pain sensation, mood, and memory, whereas in the peripheral tissues, e.g., bone formation, spermatogenesis, sebum production, etc., and, maybe most importantly, immune functions , 29–37. esponses , 36–41].Cannabis sativa can lead to psychotropic effects. Moreover, mostly, but not exclusively because of its potent analgesic actions, it was considered to be beneficial in the management of several diseases (2 agonist sesquiterpene β-caryophyllene (BCP) (trans-Δ9-tetrahydrocannabinol (THC) and the non-psychotropic (−)-cannabidiol (CBD) are the best-studied ones, exerting a wide-variety of biological actions , multipne (BCP) , 45, 46 ne (BCP) , 47, theg., Ref. , 19]. Ofg., Ref. ] of CB1 MT, etc. , 49. MorMT, etc. , 51. TakMT, etc. .As we briefly mentioned above, ECS is generally considered to be one of the “gate-keepers” of the immune system, preventing the onset of pathological immune responses .Since clinical administration of medical marijuana as well as of purified/synthetic pCBs is nowadays under increasing scrutiny, in the next chapters we intend to summarize the most important data of the field, and, while also highlighting existing controversies and limitations, to point toward possible future directions of cannabinoid-based medicines Table .graft-versus-host disease (GVHD) after bone marrow transplantation are key clinical challenges. Indeed, in order to overcome these problems, a number of different promising therapeutic opportunities are currently investigated, including, e.g., transplantation of tolerogenic dendritic cells (DCs)  , modulatls (DCs)  or regulls (DCs) , as wellls (DCs) , etc. Duls (DCs) .2−/− mice compared to wild-type recipients. In these experiments, bone marrow-derived dendritic cells (BM-DCs) of CB2−/− mice exhibited enhanced secretion of the proinflammatory cytokines interleukin (IL)-1β, IL-6, and tumor necrosis factor, as well as transforming growth factor-β1 upon Toll-like receptor (TLR) activation by lipopolysaccharide (LPS) or CpG. In addition, secretion of the Th1/Th17-promoting IL-12 and IL-23 cytokines was also increased in CB2−/− BM-DCs, and CD4+ T cells of the KO mice showed an enhanced capacity to differentiate into interferon (IFN)-γ- or IL-17-producing effector cells, altogether suggesting that CB2 may be a potential therapeutic target in the clinical management of host-versus-graft reactions  reducing T cell proliferation and activation in draining lymph nodes of the recipient mice and (ii) decreasing early stage rejection-indicator cytokines, including IL-2 and IFN-γ. Moreover, by employing selective antagonists, as well as CB1 and CB2 KO mice, the authors also showed that these effects were mediated via inducing MDSCs and activation of CB1 receptors  normalized the impaired hematopoiesis seen during GVHD. The effects were thought to be CB1- and CB2-mediated ones, since both CB1 (AM251) and CB2 (SR144528) specific inverse agonists were able to partially prevent the effect of THC in normalizing splenomegaly. Among them the CB2-selective SR144528 appeared to be more efficient, and the combination of the two inverse agonists could completely abrogate THC’s beneficial effects arguing for that targeting CB1 and/or CB2 cannabinoid receptors may constitute a novel treatment modality against acute GVHD  showed that orally administered CBD (300 mg/day) is a safe and efficient way to improve the standard GVHD prophylaxis , which is accompanied by a reduced capability of CB2-mediated signaling to suppress T cell proliferation  polymorphism of the eCB synthesizing enzyme PTPN22  was found to be associated with increased risk of type 1 diabetes mellitus (T1DM), rheumatoid arthritis (RA), juvenile idiopathic arthritis, systemic lupus erythematosus, Graves disease, myasthenia gravis, generalized vitiligo, and granulomatosis with polyangiitis . Moreovferation , was assferation , and celferation . In lineferation , whereasunctions . Thus, titution QR, which  as well –81, but 1  and Mycobacterium tuberculosis (200 µg) in a 1:1 mix with incomplete Freund’s adjuvant in mice. VCE-003 inhibited the secretion of Th1/Th17 cytokines and chemokines in primary murine T cells and dampened the IL-17-induced, proinflammatory M1 polarization of macrophages in a CB2 and PPARγ-dependent manner -cannabigerol (CBG)] from day 6 postimmunization for 21 days was able to ameliorate the neurological defects and the severity of experimental autoimmune encephalomyelitis  induced by subcutaneous immunization with myelin oligodendrocyte glycoprotein  and other pCB-based formulations in alleviating symptoms  of MS are also well-described  was found to be effective in alleviating spasticity in 85% of Nabiximols (United States Adopted Name of Sativex®) non-responder patients  suppressing proinflammatory Th17-related transcription; (ii) promoting T cell exhaustion/tolerance; (iii) enhancing IFN-dependent antiproliferative program; (iv) hampering antigen presentation; and (v) inducing antioxidant milieu resolving inflammation   and the subsequently elevated “adenosine tone” -induced demyelinating disease  eceptors . It is nry model  and in hry model  too, andne tone” .Via activating CB1, CB2, or other cellular targets, both endo- and pCBs were already convincingly shown to exert complex , moer (CB2) , 139. Horesponse , 139. Inresponse . Thus, a2 appears to be the most promising candidate to adjust such immune responses, but recently other receptors  were also proven to be potent and relevant regulators. The available  data about the roles of these receptors in sepsis, and especially the possible therapeutic exploitation of GPR55-antagonism in such conditions, is extensively reviewed in Ref.  it was shown that the non-specific CB receptor agonist R(+)WIN55,212 significantly reduced cardiac inflammation. However, it also led to considerably increased parasitemia, therefore therapeutic value of such non-specific drugs remained questionable , CB2antagonism, as well as the CB2−/− genotype were protective, and led to enhanced survival and a diminished blood-brain barrier disruption (2−/− (but not CB1−/−) mice were resistant to LPS-driven suppression of serum progesterone levels and preterm birth (2 inverse agonists AM630 (10 mg/kg) or JTE907 (3 mg/kg) during immunization was found to improve antigen-specific immune responses in young and aged mice through the upregulation of immunomodulatory genes in secondary lymphoid tissues  infection of rhesus macaques  antigen-specific immune responses, which occurred through both CB1/CB2-dependent and -independent mechanisms  reduced host immune response, and prevented cognitive impairments . Chronicmacaques , and didmacaques . Likewisammation . Moreovechanisms , and finpatients . Thus, ipatients .Hepatitis B Virus, 10 µM CBD inhibited HCV replication by 86.4% in vitro -1/2 signaling. However, it should also be noted that long-term cannabis exposure in two patients resulted in reversal of this effect  body of evidence, there are still significant gaps in our understanding with respect to the cannabinoid signaling, and its optimal therapeutic exploitation, inviting obviously a number of specific complementary 1 [overviewed in Ref. , as it became evidenced by the infamous, brain-penetrating CB1 inverse agonist rimonabant , which was applied as a potent anorexigenic agent for a few years in Europe, but was then retracted from the market  under international regulatory control, and among which several synthetic cannabinoids are now present at the black market cannabinoid signaling, and restoration of the homeostatic eCB tone of the tissues augur to be very promising future directions in the management of several pathological inflammation-accompanied diseases. Moreover, in depth analysis of the (quite complex) mechanism-of-action of the most promising pCBs is likely to shed light to previously unknown immune regulatory mechanisms and can therefore pave new “high”-ways toward developing completely novel classes of therapeutic agents to manage a wide-variety of diseases.All authors wrote, edited, and approved the final version of this manuscript.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.


Angiopoietin-like proteins (ANGPTLs) 3, 4, and 8 reportedly contribute to progression of metabolic disease, a risk factor for cardiovascular disease (CVD). The purpose of this study was to investigate whether circulating ANGPTL levels are associated with CVD risk after adjustment for potential confounding factors.We conducted a single center, cross-sectional study of 988 Japanese subjects undergoing routine health checks. Serum ANGPTL3, 4, and 8 levels were measured using an enzyme-linked immunosorbent assay. Using multiple regression analysis we evaluated potential association of circulating ANGPTL3, 4, and 8 levels with general medical status including age, sex, smoking, drinking, obesity, hypertension, impaired glycometabolism, dyslipidemia, hyperuricemia, hepatic impairment, chronic kidney disease, anemia, cardiac abnormality, and inflammation.Circulating ANGPTL3 levels were relatively high in health-related categories of hepatic impairment and inflammation. Circulating ANGPTL4 levels were also significantly high in impaired glycometabolism or hepatic impairment but decreased in inflammation. Finally, increased ANGPTL8 levels were observed in obesity, impaired glycometabolism and dyslipidemia. Particularly, increased levels of circulating ANGPTL8 were positively correlated with circulating triglycerides and LDL-cholesterol levels and inversely correlated with circulating HDL-cholesterol levels.Circulating ANGPTL3, 4, and 8 levels reflect some risk factors for CVD development. Cardiovascular disease (CVD) is a serious health concern worldwide , 2. To pANGPTL3 and ANGPTL4 decrease circulating TG concentrations, whereas loss-of-function mutations in human ANGPTL8 have no significant effect on circulating TG levels , and a human ANGPTL4 ELISA kit designed to detect both full length and cleaved protein using antibodies against the N-terminal of ANGPTL4 (IBL). Antibody specificity was confirmed, and none cross-reacted with other ANGPTLs. In particular, antibodies supplied in ANGPTL3 and 4 kits showed little (<0.1%) cross-reactivity with ANGPTL8.We developed a sandwich ELISA to detect human ANGPTL8 using two antibodies: a rabbit polyclonal antibody (PoAb-169) and a mouse monoclonal antibody (MoAb-21C1). PoAb-169 was developed using a synthesized peptide (169-191aa) of human ANGPTL8, and MoAb-21C1 was developed using recombinant human ANGPTL8. PoAb-169 served as the capture antibody and was used to precoat a 96-well microtiter plate. MoAb-21C conjugated to horseradish peroxidase-conjugated (HRP) served as the detection antibody. Recombinant human ANGPTL8 served as the standard, and human serum samples were prepared as test samples. Specificity of antibodies was confirmed, and none cross-reacted with ANGPTL3 (≤0.1%) or ANGPTL4 (≤0.1%).2SO4 was added to stop the reaction, and absorbance at 450 nm was measured. Human ANGPTL8 levels in test samples were calculated based on evaluation of recombinant human ANGPTL8 standards.Serially-diluted standards and twenty-fold diluted test samples were added to antibody-precoated plates in duplicate and incubated 60 minutes at 37°C. Plates were then washed with PBS containing 0.05% Tween20 (PBST), and HRP-conjugated MoAb-21C1 mouse IgG Fab’ was added and incubated 30 minutes at 4°C. Plates were washed with PBST, and then tetramethylbenzidine (TMB) solution was added for 30 minutes at room temperature. An equal amount of 1 N HANGPTL3, ANGPTL4, ANGPTL8, HbA1c, Glucose, TG, AST, ALT, GGT, and hs-CRP were transformed to natural-log values for statistical analysis, as distributions of these variables were skewed. A generalized linear model was used in multiple regression. All statistical analysis was performed using JMP Pro 13.0.0 software . All P values were two-tailed, and P<0.05 was taken as statistically significant.Tables Next, to evaluate potential association of circulating ANGPTLs levels with health-related categories, we conducted multiple regression analysis. Calculation of regression coefficients (β) and P values revealed that circulating ANGPTL3 levels are relatively high in categories such as aging, hepatic impairment, or inflammation . By cont1C and glucose levels in the category of impaired glycometabolism, and with AST, ALT and GGT in the category of hepatic impairment (We next asked examined potential associations between circulating ANGPTLs levels and clinical variables shown to be statistically significant in pairment . By contpairment .Molecular cooperation of ANGPTL3 and ANGPTL8 is reportedly critical for ANGPTL8-induced LPL inhibition and subsequent increases in TG levels , 24. TheIn the current study, we asked whether associations exist between circulating ANGPTL3, ANGPTL4, and ANGPTL8 levels and general medical indexes, including CVD risk, in 988 Japanese persons aged 27–84 years who had undergone a routine medical check-up. We found that increased levels of circulating ANGPTL3 were positively associated with liver dysfunction and inflammation, elevated ANGPTL4 levels were positively associated with impaired glycometabolism and liver dysfunction and inversely correlated with inflammation, and increased ANGPTL8 levels were associated with obesity, impaired glycometabolism, and dyslipidemia. Thus, circulating ANGPTL3, 4, and 8 levels reflect some CVD risk factors.et al. reported that serum ANGPTL3 levels increase in patients with dengue hemorrhagic fever, a viral inflammatory disease [et al. previously reported that ANGPTL3 induces angiogenesis through αvβ3 integrin signaling, suggesting that ANGPTL3 may promote inflammation [Our study indicated that circulating ANGPTL3 levels are positively correlated with inflammation, as estimated by hs-CRP values. Although inflammation plays crucial roles in progression of metabolic diseases such as obesity or impaired glycometabolism , a posit disease , supportammation .High circulating ANGPTL3 levels were also positively correlated with hepatic impairment, as estimated by AST and GGT levels, but not ALT levels. ANGPTL3 expression is restricted to hepatocytes, and ANGPTL3 is secreted from only those cells, suggesting that ANGPTL3 is a “hepatokine” . AST andANGPTL3 mutations show decreased serum ANGPTL3 protein levels, as well as decreased TG, LDL-cholesterol, and HDL-cholesterol, as well as lower incidence of coronary artery disease [An observational study reports that human subjects harboring loss-of-function  disease . These f disease . Therefo disease , 14, 15,1C in the category of impaired glycometabolism, a finding consistent with a previous report [et al. previously reported that mice overexpressing ANGPTL4 in adipose tissue show predisposition to liver steatosis, potentially reflecting an association of ANGPTL4 with liver dysfunction including steatosis [We also found that circulating ANGPTL4 levels are positively correlated with serum glucose and HbAs report . We alsos report , 31. Ourteatosis .We also found that serum ANGPTL4 levels are inversely correlated with inflammation. A previous report showed that ANGPTL4 has anti-inflammatory activity in mice by suppressing expression of inflammatory genes expressed in macrophages . It is nANGPTL4 mutations promote decreases in levels of circulating TGs and increases in HDL-cholesterol [In human subjects, loss-of-function lesterol , suggestlesterol . Since tet al. reported that mutations that suppress ANGPTL8 activity are associated with decreased levels of circulating LDL- and HDL-cholesterol levels, but not with circulating TG levels, although transgenic mice overexpressing Angptl8 in liver exhibited hypertriglycemia [ANGPTL8 is an atypical ANGPTL with an N-terminal coiled-coil domain but lacking a C-terminal fibrinogen-like domain . Howeverglycemia . InteresANGPTL8 expression increases during differentiation of primary cultured human or mouse pre-adipocytes to adipocytes [Our study also reveals a positive correlation of circulating human ANGPTL8 levels with obesity, consistent with a previous study of human subjects . In thatipocytes . Taken tSome reports suggest that circulating ANGPTL8 levels are positively correlated with circulating levels of glucose or HbA1c , 38. HowReports also suggest that circulating levels of ANGPTL3, 4, and 8 reflect various health concerns including CVD risk, although these reports vary , 30, 35.Our current study has some limitations. First, circulating ANGPTL levels reported here differ from those reported in other reports , 26, 30.In summary, the current study reveals that levels of circulating ANGPTL3, 4, and 8 reflect some risk factors of CVD development. In particular, we found that circulating human ANGPTL8 levels were positively correlated with obesity and dyslipidemia, as estimated by increased TG and LDL-cholesterol levels and decreased HDL-cholesterol levels, suggesting that circulating ANGPTL8 concentrations predict future CVD development. However, the cross-sectional study design used here limits our interpretation of whether there is an association between ANGPTL8 levels and future CVD events. Further prospective cohort studies will be required to answer this question.


Pulse wave velocity was improved following acute supplementation only . These results support the findings of previous reviews that anthocyanin rich foods or extracts may indeed improve vascular health, particularly with respect to vascular reactivity measured by FMD. More research is required to determine the optimal dosage, and the long-term effects of consumption.Anthocyanins are of interest due to their anti-oxidative and vasodilatory properties. Earlier reviews have shown that berries and other anthocyanin rich foods or extracts can improve vascular health, however the effect of anthocyanins on vascular function has not yet been reviewed. To address this gap in the literature, we conducted a systematic review and meta-analysis of randomised-controlled trials examining anthocyanin-rich foods or extracts on measures of vascular reactivity and/or stiffness in adults. Data from 24 studies were pooled as standardized mean difference (SMD) with 95% confidence intervals (CI). Anthocyanin consumption significantly improved flow-mediated dilation (FMD) following acute (SMD: 3.92%, 95% CI: 1.47, 6.38,  The onset of cardiovascular disease (CVD) is often accompanied by a steady decline in vascular function. A number of biomarkers and methodologies exist to assess vascular health and function. Common biochemical markers include serum concentrations of inflammatory markers , adhesion molecules , lipids and lipoproteins  and triglycerides), oxidised LDL-C, and clotting factors  . VasculaAnthocyanins are polyphenols found in the flavonoid family. They are the red, blue or purple pigments observed in many fruits, vegetables and flowers. In addition to providing vibrant colour, anthocyanins are of interest because of their known bioactivity including potent antioxidant capacity  and potep < 0.00001), LDL-C (p = 0.003), fasting glucose (p = 0.004), haemoglobin A1c (p = 0.04) and tumour necrosis factor-α (p = 0.04) in both healthy and metabolically compromised participants. Similarly, a systematic review of 12 RCTs reported that consumption of purified anthocyanins and anthocyanin rich extracts  improved blood pressure and LDL-C in post-myocardial infarction and hyperlipidemic individuals, respectively [Numerous intervention studies have evaluated the effect of isolated anthocyanins, anthocyanin-rich foods or their extracts on CVD risk factors, including some markers for vascular health. In a recent meta-analysis of 22 randomised-controlled trials (RCTs), Huang and colleagues  demonstrectively . HoweverThis systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines . The stuThe eligibility criteria used in the review are summarised in Randomised-controlled trials that compared anthocyanin-rich foods, anthocyanin-rich extracts or purified anthocyanins against a placebo or control.Adult participants aged ≥18 years old.All sources , doses and durations of anthocyanin intake were considered. Studies included were acute, short-term interventions consisting of a single dose, or chronic, long-term intervention consisting of days, weeks or months of a daily dose. Studies were required to report a quantitative or quantifiable anthocyanin dose; where these data were not reported, information for the investigational product or food was sourced from other published material. If content was not quantified and the product or food did not have a reference value available from other peer-reviewed literature, the study was excluded from analysis. Studies were excluded if they combined anthocyanin-rich food or extracts with other substances, or did not compare anthocyanin-rich food or extract alone to a placebo. Studies evaluating whether anthocyanin-rich foods or extracts could mitigate postprandial effects or other vascular insults  were also excluded.Any comparators (including a placebo or control) that did not contain anthocyanins were eligible for inclusion.Functional measure of vascular health, including assessments of arterial stiffness via PWV, digital volume pulse (DVP), PWA, PAT and Finapres, and vascular reactivity via PAT, LDI and FMD. Studies were excluded if the outcomes comprised only of blood pressure or biochemical markers of vascular function, as these measures have been examined in previous reviews ,17.No restrictions regarding publication dates were imposed, however, studies were only eligible if they were published in English or a translation was accessible. Only study outcomes published as full papers in peer reviewed scientific journals were included.Researchers (LF and AMH) searched the following databases: Embase (1980), Medline (1946), Cochrane (1993), CINAHL  and Scopus (1960) for relevant articles from inception until 13 June 2017. Further literature searches included manually checking the reference lists of all pertinent articles and reviews for studies missed by the database searches.Search terms and MeSH/keyword terms were collated into 2 key concepts: anthocyanin rich foods or extracts and vascular function. A review article evaluating high anthocyanin containing foods was used to assist with the development of the anthocyanin concept key words . RelevanThe following terms were searched in all databases:  and ).Once duplicates were removed, the titles and abstracts of articles were independently screened for eligibility by two investigators (LF and AMH). If consensus was reached, ineligible articles were excluded and eligible articles were moved to the next stage for a full-text review. Articles without sufficient information in the title and abstract were also moved to full text review. All disagreements were resolved by discussion among the research team until a consensus was reached.A data extraction template was developed and pilot tested by 2 authors (LF and AMH) on 5 randomly selected studies. The template was refined according to pilot testing, and any disagreements were resolved by consensus. All data were independently extracted by two investigators (LF and EF), checked for consistency and resolved by consensus.Extracted data included study design, country, sample size, participant characteristics , amount of food/extract and anthocyanins consumed, placebo or control, duration of the intervention, and vascular function outcome of interest.Assessors (LFJ and AMH) independently evaluated the quality of included studies using the 3-category Jadad scoring system . The quad) for individual studies were determined. Effect sizes were calculated as the difference between treatment and control post-test means, divided by their pooled standard deviations [A descriptive analysis was completed and effect sizes .Additionally, meta-analyses were performed using Stata 10.0 . Chronic and acute study data were assessed separately, and in each analysis, data were grouped by vascular function outcome . Any outcomes involving fewer than two studies were excluded from the meta-analysis. Data from studies that involved multiple anthocyanin doses were averaged. Where acute studies examined multiple time points, only the dose with the peak effect was selected for inclusion in the meta-analysis. Where there were multiple measures from the same outcome , these data were combined. Effect sizes were expressed as standardised mean difference (SMD), otherwise known as Cohen’s R) = 0.5:If not provided by the author, the mean difference (change between baseline and post-test results) was calculated for both intervention and control groups in each single study. The standard deviation of combined means was calculated using the following formula, assuming a correlation coefficient  [n = 8) [n = 3; Korea, n = 3) [Studies were predominantly conducted in the United Kingdom (n = 9) ,41,42,47 [n = 8) ,38,43,45, n = 3) ,30,35,48, n = 3) ,25,40, a, n = 3) , Greece , n = 3)  and Isra, n = 3) . Of the , n = 3) ,43,45,48, n = 3) ,47,48. n = 19) involved participants in the middle to older age group (mean age ≥ 40 years) [n = 13) [n = 13) [n = 13) [n = 5) [n = 2) [n = 4) [n = 4) [n = 1) [The number of participants that completed each study ranged from 10 to 146. Seven studies had exclusively male participants ,41,42,430 years) ,46,48. A[n = 13) ,41,42,43[n = 13) ,46,48, w[n = 13) ,44,47. P [n = 5) ,33,38,46 [n = 4) ,27,40, a [n = 4) ,28,30,45 [n = 1) .Anthocyanins were in the form of a fruit extract in all studies except the two reported by Zhu and colleagues , where pn = 9), PWV (n = 10), DVP-SI (n = 2) and total peripheral resistance (TPR) assessed via Finapres (n = 1). Outcomes measures for vascular reactivity included FMD (n = 13), PAT-Reactive Hyperemia Index  and LDI (n = 2).Various measures of vascular function were examined in the eligible studies . The reln = 23) had a Jadad score of 3 or greater. All studies were randomised and most featured double-blinding (n = 20). Most studies provided an account of participants (n = 23). Points were most frequently not attained for describing the method of randomisation (only 12 studies provided a sufficient description) and/or describing the method of blinding (provided by 17 studies). n = 9) and healthy populations (n = 9). Vascular function did not deteriorate significantly in any study. Of the 29 studies from 26 publications included in the systematic review, 18 reported significant improvements in at least one measure of vascular function, whilst 11 reported no improvement. These benefits were observed equally in non-healthy  [n = 3) [p = 0.000; I2 = 17.9%; p = 0.48; I2 = 74.3%; p = 0.061; I2 = 77.4%; Effects of acute anthocyanin supplementation on vascular reactivity assessed via FMD and RHI were reported in four and two studies, respectively. Compared to placebo-control, acute anthocyanin supplementation significantly improved FMD  ,41 and A [n = 3) ,33,41. Pp = 0.000, I2 = 62.5%). Compared to control, chronic anthocyanin supplementation did not significantly improve RHI  reported improvements in vascular function ,45,47,48p = 0.364; I2 = 28.9%; p = 0.088; I2 = 52.6%; p = 0.060; I2 = 39.8%; Less consistent effects were observed for arterial stiffness, with less than half of the studies (6 of 15) reporting improvements in PWV (3 of 8 studies) ,32,44 orp = 0.01).Visual inspection of the funnel plots showed symmetry for all outcomes aside from acute studies examining FMD. Begg’s adjusted rank correlation tests revealed non-significant Kendall Tau(s) for all measurements. Egger’s regression asymmetry tests yielded non-significant intercepts for all outcomes, except acute studies examining FMD  suggest that, overall, vascular stiffness may be improved by anthocyanin consumption, subject to confirmation with more data.These findings are in line with previous reviews that have found a relationship between anthocyanin intake and improvements in vascular function assessed by blood pressure and biochemical markers ,17, suppMost improvements were observed in vascular reactivity assessed via FMD, which may relate to the reliability of this measure. FMD is considered to be the highest standard of assessing endothelial function in subjects that are in the early, reversible stages of endothelial dysfunction . There aR2 ≤ 0.30) [Kay et al.  suggests ≤ 0.30) . This suThe majority of chronic studies included in this review were between 1 and 16 weeks, with one study of six-month duration . It is pAnthocyanin efficacy may be influenced by the chemical composition of extracts, or the process used to manufacture or store them. Anthocyanins are highly unstable and easily degraded by factors such as pH, temperature, light, oxygen exposure, solvents, enzymes, other flavonoids, and proteins, hence it is possible that anthocyanin content may have decreased prior to consumption ,59. Moren < 70) and were heterogeneous with regard to anthocyanin dose, duration and population characteristics. We attempted to reduce the impact of heterogeneity on estimated effect sizes by implementing a random-effects model of analysis. Additionally, some studies were incomplete, failing to report certain population characteristics or outcome data, and necessitating the estimation of anthocyanin intakes based on other publications. Finally, vascular function was assessed using a range of techniques, with varying degrees of accuracy. The Jadad tool used to evaluate study quality did not consider the reliability or validity of these techniques.There are several other limitations worth mentioning. While the funnel plots did not show asymmetry, Eggers test indicates that there is potential for publication bias in acute studies involving FMD measurements. However, the small number of published papers included in this review makes it challenging to effectively evaluate publication bias. The articles included in this review were limited to those published in English, which may have introduced language bias. Studies where the anthocyanin content was not provided or able to be determined were also excluded, limiting the scope of our evaluation. Most notably, we included a small number of studies, many of which had small sample sizes , not all studies gave an account of participants or adequately described their method of randomisation and blinding. This suggests the possibility of reporting bias, and therefore the results of lower quality studies should be considered with caution. Other systematic reviews have implemented a revised Jadad scale which may be a superior tool given that it assesses additional factors not addressed by the original scale, such as quality of exclusion criteria, the intervention/controls used, and data reporting ,65. HoweOur meta-analysis showed that consumption of anthocyanins from foods or extracts significantly improved vascular health. Indeed, acute or chronic anthocyanin consumption resulted in a significant overall increase in measures of vascular reactivity, and significant improvements when assessed specifically by FMD, but not RHI. This analysis included evidence from eight high quality studies (>4/5 Jadad score). Additionally, we found that acute supplementation significantly reduced arterial stiffness as measured by PWV. However, no significant benefits were observed for overall measures of arterial stiffness or AI individually in either acute or chronic studies. Randomised-controlled trials looking at the effect of anthocyanins on vascular reactivity and stiffness have not previously been reviewed; therefore, this meta-analysis is an appropriate addition to the current literature. More research is needed to understand the role of anthocyanins in vascular health, the long-term consequences, and the recommended intake of anthocyanins to assist the reduction of CVD morbidity and mortality. Thus, recommended directions for future research include: (1) dose-dependent studies in specific populations  to assist developing dose recommendations; and (2) intervention studies examining a variety of vascular regions, including cerebrovascular vessels.


LOC100520753 (CD68), LCN2, EMR1, S100A9, NCF1 (p47phox), and LEP, were mainly involved in inflammatory response, oxidation-reduction process, and lipid metabolic process, while downregulated genes in CM pigs, i.e., ABHD5, SPP1, and GAS6, were focused on cell differentiation and cell adhesion. Taken together, our study demonstrates that testosterone deficiency alters the expression of numerous genes involved in key biological processes of VAT accumulation under HFC diet and provides a novel genome-wide view on the role of androgen on VAT deposition under HFC diet, thus improving our understanding of the molecular mechanisms involved in VAT changes induced by testosterone deficiency.Testosterone deficiency causes fat deposition, particularly in visceral fat, and its replacement might reverse fat accumulation, however, the underlying mechanisms of such processes under diet-induced adiposity are largely unknown. To gain insights into the genome-wide role of androgen on visceral adipose tissue (VAT), RNA-Seq was used to investigate testosterone deficiency induced changes of VAT in miniature pigs fed a high-fat and high-cholesterol (HFC) diet among intact male pigs (IM), castrated male pigs (CM), and castrated male pigs with testosterone replacement (CMT) treatments. The results showed that testosterone deficiency significantly increased VAT deposition and serum leptin concentrations. Moreover, a total of 1732 differentially expressed genes (DEGs) were identified between any two groups. Compared with gene expression profiles in IM and CMT pigs, upregulated genes in CM pigs, i.e.,  Visceral adiposity represents excess visceral fat tissue (VAT) deposition in body, which is associated with several critical human diseases such as type 2 diabetes, metabolic syndrome and cardiovascular disease, suggesting visceral adiposity is a high disease risk factor in the life. As we know, excessive nutrients intake from the daily diet, such as high-fat and high-cholesterol (HFC) diet, significantly increases VAT accumulation ,2. On thConversely, to avoid visceral adiposity induced by testosterone deficiency, a feasible strategy is testosterone replacement, which resulted in a significant decrease in body mass index, fat mass, and regional fat distribution in hypogonadal and aging men ,7. FurthIn general, pig model has been widely used for studying the changes induced by testosterone deficiency because it was shown to have similar characteristics concerning obesity and metabolic syndrome to human ,15,16. TCastration significantly enhanced VAT fat, while testosterone treatment significantly decreased VAT fat, with no significant difference between IM and CMT pig groups A. By conA total of approximately 26.81, 24.65, and 23.87 million high quality clean reads were obtained from IM, CM, and CMT pigs, respectively. More than 81% of reads could be mapped to the pig reference genome 10.2. Furthermore, more than 77% of these mapped reads aligned with unique genes . FinallyFrom 16,836 expressed mRNAs, 15,806, 15,811 and 15,785 expressed genes were detected in VAT of IM, CM, and CMT pig group, respectively. Among these genes, 14,814 genes were expressed in all three groups; 314, 280, and 344 genes were expressed in each pair of groups , while 334, 403, and 347 genes were exclusively found in IM, CM, and CMT pigs, respectively .Based on our criteria of defining DEGs, 747 (312 upregulated and 435 downregulated), 826 (398 upregulated and 428 downregulated), and 926 (366 upregulated and 560 downregulated) DEGs were identified between groups of CM versus IM, CMT versus CM, and CMT versus IM, respectively . TotallyTo obtain the biological functions of DEGs in VAT of IM, CM, and CMT pigs, Gene Ontology (GO) enrichment analysis was further performed. The results showed that significantly upregulated GO terms between CM and IM pigs were mainly enriched in inflammatory response, innate immune response, and oxidation-reduction, while downregulated DEGs were mainly involved in extracellular matrix organization, regulation of transcription from RNA polymerase II promoter, and cell adhesion . FurtherTo further refine the expression modes of DEGs in all groups, after excluding 39 DEGs with 0 value in each group, 1693 out of 1732 DEGs were used for series-cluster analysis, which were categorized into eight clusters A. Two ofNCF1, S100A9, LEP, LOC100520753 (CD68), EMR1, and LCN2) related with inflammatory response, oxidation-reduction process and lipid metabolic process from profile 5 and four genes  related of cell differentiation and small molecule metabolic process from profile 2, were selected for qRT-PCR verification. Consistent with RNA-Seq data, the expression of six genes from profile 5 were increased in CM pigs but decreased in CMT pigs  with diabetogenic or HFD diet, the fat mass was found to be significantly lower than that in APOE+/+ or wild-type mice, indicating that APOE-deficient might be resistant to adiposity and APOE was positively related with diet-induced adiposity [APOE under testosterone deficiency in our data might amplify castration-induced VAT deposition under HFC diet. Actually, expression levels of other well-known lipoproteins for modulating triglyceride metabolism, such as APOA5 and APOC3 [APOC3 were not detected in IM pig groups , EMR1, LCN2, S100A8, S100A9, and S100A12  and Yamaoka et al (2013) [S100A8, S100A9, and S100A12 were positively correlated with VAT adiposity in human and overexpression of S100A8 and S100A9 could increase adipose inflammation. Most recently, upregulated S100A8 expression levels were shown to contribute to the very early stage of pathogenesis of obesity and induce local inflammation [As expected, testosterone deficiency with HFC induced the expression of genes related with inflammatory response. The marked genes, such as  S100A12 , were upficiency ; CD68+ cin women ; percentin women ; and theice only , showingividuals  and werepatients . Therefol (2013) ,40, who ammation . ObviousNCF1 (p47phox) and NOX2 (GP91-PHOX)) and down-regulation of antioxidant-related genes   . It is n or rats ,44, suggHFD mice ,46 as weNCF1 pphox and ABHD5 for cell differentiation, SPP1 for both cell differentiation and cell adhesion, GAS6 for cell adhesion, and DIO3 and PCK1 for small molecule metabolic process  were obtained from the Institute of Animal Sciences, Hainan Academy of Agricultural Sciences . The animals received a standard diet without cholesterol during a 7-week “pretreatment period” for acclimation to the environment. At week 7, the pigs were divided into three groups as follows: intact male pigs fed a HFC diet (IM), castrated male pigs fed a HFC diet (CM), and castrated pigs with testosterone replacement fed a HFC diet (CMT). Among them, IM pigs received only skin incisions which were closed by suture, CM pigs were gonadectomized through two incisions in the perineum which were closed by suture, and CMT pigs were given testosterone treatment after castration by intramuscular weekly injection with testosterone propionate  dissolved in corn oil. All surgical operations were performed under anesthesia. The HFC diet was fed for 12 weeks starting from week 8. The HFC diet was comprised of 73% normal swine diet, 15% lard, 10% egg yolk power, 1.5% cholesterol, and 0.5% sodium cholate. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Zhejiang Chinese Medical University .g at 4 °C for 15 min and then stored at −80 °C for serum measurement. VAT of each animal was derived from retroperitoneal adipose tissue and weighed, followed by freezing storage in liquid nitrogen for RNA extraction. VAT fat was measured as a ratio of VAT weight refers to the proportion of body weight. Serum testosterone concentrations were measured using an RIA kit , and serum leptin levels were measured using a commercially available kit  and analyzed by an Automatic Biochemistry Analyzer . The intra-assay and inter-assay coefficients of variation were below 10% and 15% for testosterone, respectively, and the inter-assay coefficients of variation were below 6% for leptin. The assay sensitivities were 0.02 ng/mL and 0.1 ng/L for testosterone and leptin, respectively.At the end of the experimental period, animals were weighted and killed by exsanguination under sodium pentobarbital anesthesia. Blood samples were collected and centrifuged at 3000× n = 4 per group) were pooled with equal quantities, respectively, and were used for cDNA library construction and Ion-proton sequencing. Briefly, cDNA libraries for single-end sequencing were prepared using the Ion Total RNA-Seq Kit v2.0  according to the manufacturer’s instructions. After diluting and mixing of samples, the mixture was processed on OneTouch 2 instrument and was enriched on OneTouch 2 ES station . Finally, the enriched mixture samples were loaded on to 1 P1v2 Proton Chip and sequenced on Proton Sequencers according to the Ion PI Sequencing 200 Kit v2.0 .Total RNA was extracted using TRIzol reagent  according to the manufacturer’s protocol. RNA integrities and qualities were assessed by Bioanalyzer 2100  and agarose gel electrophoresis. The RNA integrity number (RIN) of all samples was more than 7.0. Then, RNA samples from IM, CM, and CMT groups  correction, and UQRPKM values greater than or equal to 1 in any two groups.Raw sequencing data were filtered by removing the adaptor sequences, reads with >5% ambiguous bases, and low-quality reads with qualities <13. These clean data were then mapped to the current pig reference genome (Sscrofa 10.2) using the MapSplice program . UQRPKM Hierarchical clustering analysis was performed using Cluster 3.0 and was visualized using the Java Treeview . The expression profiles of DEGs were determined by series-cluster analysis based on the Short Time-Series Expression Miner (STEM) method .http://www.geneontology.org/). Fisher’s exact test was applied to identify the significant GO categories. Terms with p values less than 0.05 and the number of matching genes in term (DifGene) more than 5% of all used genes (AllDifGene) were selected as significant or enriched terms.Gene ontology (GO) analysis of DEGs using GO annotation from the Gene Ontology (Ct−ΔΔ method was used to determine the expression level differences [Ten genes (six from profile 5 and four from profile 2) were analyzed by quantitative real-time RT-PCR (qRT-PCR) for validating the data from RNA sequencing. The primers of each gene are listed in ferences .p values of less than 0.05 and 0.01 were considered as significant and highly significant, respectively.Statistical analyses were performed using SPSS v17.0 software  and the results showed as means ± SEMs. Statistical differences between groups were examined using ANOVA followed Bonferroni multiple comparison, and The datasets have been submitted to the Gene Expression Omnibus (GEO) database of NCBI (accession number GSE77544).In summary, a genome-wide view of transcriptome changes of VAT with HFC induced by testosterone deficiency in miniature pig model was investigated here. Our data discovered that testosterone deficiency in pigs fed a HFC diet mainly increased the changes of genes associated with inflammatory process, oxidation-reduction process, and lipid metabolic process, while reduced other genes mainly involved in cell differentiation and cell adhesion. Furthermore, this study provides novel insights for comprehensively understanding the mechanisms of the VAT changes induced by testosterone deficiency, revealing possible clues for treatment or abatement of visceral adiposity under testosterone deficiency.


CCR and CCR-like genes, collectively called OsCCRs. To elucidate the functions of OsCCRs, their phylogenetic relationships, expression patterns at the transcription levels and biochemical characteristics were thoroughly analyzed. Of the 33 OsCCRs, 24 of them encoded polypeptides of lengths similar to those of previously identified plant CCRs. The other nine OsCCRs had much shorter peptide lengths. Phylogenetic tree and sequence similarities suggested OsCCR4, 5, 17, 18, 19, 20, and 21 as likely candidates for functional CCRs in rice. To elucidate biochemical functions, OsCCR1, 5, 17, 19, 20, 21, and 26 were heterologously expressed in Escherichia coli and the resulting recombinant OsCCRs were purified to apparent homogeneity. Activity assays of the recombinant OsCCRs with hydroxycinnamoyl-CoAs revealed that OsCCR17, 19, 20, and 21 were biochemically active CCRs, in which the NAD(P)-binding and NADP-specificity motifs as well as the CCR signature motif were fully conserved. The kinetic parameters of enzyme reactions revealed that feruloyl-CoA, a precursor for the guaiacyl (G)-unit of lignin, is the most preferred substrate of OsCCR20 and 21. This result is consistent with a high content (about 70%) of G-units in rice lignins. Phylogenetic analysis revealed that OsCCR19 and 20 were grouped with other plant CCRs involved in developmental lignification, whereas OsCCR17 and 21 were closely related to stress-responsible CCRs identified from other plant species. In agreement with the phylogenetic analysis, expression analysis demonstrated that OsCCR20 was constitutively expressed throughout the developmental stages of rice, showing particularly high expression levels in actively lignifying tissues, such as roots and stems. These results suggest that OsCCR20 is primarily involved in developmental deposition of lignins in secondary cell walls. As expected, the expressions of OsCCR17 and 21 were induced in response to biotic and abiotic stresses, such as Magnaporthe grisea and Xanthomonas oryzae pv. oryzae (Xoo) infections, UV-irradiation and high salinity, suggesting that these genes play a role in defense-related processes in rice.Cinnamoyl-CoA reductase (CCR) is the first committed enzyme in the monolignol pathway for lignin biosynthesis and catalyzes the conversion of hydroxycinnamoyl-CoAs into hydroxycinnamaldehydes. In the rice genome, 33 genes are annotated as  Plants are constantly confronted by both biotic and abiotic stresses, leading to significant reductions in their productivity -, G- and syringyl (S)-units derived from monolignols. Lignin is predominantly deposited in the secondary cell walls of xylem and fiber cells and makes the cell walls rigid and impervious  and 10 in Eucalyptus grandis , poplar (P. euramericana), maize (Zea mays), switchgrass (Panicum virgatum), and wheat (Triticum aestivum)  database, we found 33 genes annotated as O. sativa L. spp. Japonica cv. Dongjin) were germinated on Murashige and Skoog (MS) medium  in a growth chamber with a 12 h photoperiod and temperature of 28°C. Ten-day old seedlings were transferred to soil and grown in a greenhouse at 28°C during the day and 20°C at night. Stem and leaf samples were collected from 10-week-old rice plants, and panicle samples were collected from 14-week-old rice plants. Root and shoot samples were collected from 10-day old rice seedlings.Sterilized seeds of wild-type rice plants  and reduced β-nicotinamide adenine dinucleotide phosphate (NADPH) for hydroxycinnamoyl-CoA production were purchased from Sigma-Aldrich . Reagents for buffers, media and other solutions were obtained from Sigma-Aldrich and Duchefa.http://rice.plantbiology.msu.edu/, Kawahara et al., https://www.ncbi.nlm.nih.gov/) database, respectively. Multiple amino acid sequence alignment was performed with Clustal-W  conditions are provided in Supplementary Table OsCCR gene was cut out with the appropriate restriction enzymes and inserted into the pET28a(+) vector . The resulting OsCCR/pET28a(+) constructs were individually transformed into E. coli BL21(DE3) cells for heterologous expression of OsCCRs.Total RNA was isolated from 8-week-old rice leaves with RNAiso . The first cDNA was synthesized using the total RNA and SuPrimeScript RT premix with an oligo dT primer . Cloning primers for E. coli transformants harboring the OsCCR/pET28a(+) construct were grown at 37°C until an OD600 of ~0.6 in LB medium containing kanamycin (25 μg/mL) was achieved. At that point, 0.1 mM isopropyl β-D-thiogalactopyranoside (IPTG) was added in the culture for induction. After additional incubation at 18 or 25°C for 16 h, the cells were harvested by centrifugation . Cell pellets were resuspended in phosphate-buffered saline  supplemented with lysozyme (1 mg/mL) and phenylmethylsulfonyl fluoride (1 mM). The resuspended cells were sonicated on ice, and the crude protein extracts were obtained by centrifugation . The crude protein samples were mixed with Ni-NTA Agarose beads  and incubated at 4°C for 2 h with agitation. The mixtures were packed into a chromatography column and washed three times with a five-column volume of 20 mM imidazole in Tris buffer . The recombinant OsCCRs were eluted with 50–100 mM imidazole in Tris buffer. The eluted proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).The Arabidopsis 4-coumarate:CoA ligase 1 (At4CL1) was cloned from A. thaliana cDNA, and the resulting gene was inserted into pET28a(+) vector , 2 mg CoA, and 6.9 mg ATP were dissolved into a total volume of 10 mL of 50 mM Tris-HCl pH 7.5 buffer containing 2.5 mM MgCl2. The reaction was initiated by the addition of 0.25 mg purified At4CL1. After a 5 h incubation at room temperature with agitation, 6.9 mg ATP, 2 mg CoA, and 0.25 mg purified At4CL1 enzyme were added to the reaction mixture, and the incubation was continued at room temperature for 12 h. Ammonium acetate (0.4 g) was added to the mixture to halt the reaction. Hydroxycinnamoyl-CoA esters were purified using Sep-Pak® Vac tC18 cartridge  preconditioned with consecutive washes of MeOH, H2O, and 4% ammonium acetate solution (five column-volumes each). The reaction mixture was loaded on the preconditioned cartridge, and the column was rinsed with 4% ammonium acetate solution. The hydroxycinnamoyl-CoA esters were eluted with H2O. Fractions containing the hydroxycinnamoyl-CoA esters were identified by UV/Vis spectra recorded using a V-550 UV/Vis-spectrophotometer , and the purified products were lyophilized for storage.For the OsCCR activity assay, hydroxycinnamoyl-CoAs were synthesized by the method described by Beuerle and Pichersky . Arabido366 were monitored for 10 min by a Cary 300 Bio UV/Vis-spectrophotometer . For determination of KM and Vmax, the substrates were used at concentrations of 5–50 μM. KM and Vmax were determined by extrapolation from Lineweaver-Burk plots. The enzyme assays were carried out in quadruplicate and the result represented the mean ± standard deviation.OsCCR activity was measured according to the methods of Lüderitz and Grisebach . The reaWild-type Dongjin rice plants were grown in a greenhouse for 8 weeks after germination. UV-C treatment of rice plants were performed using the methods described by Park et al. . UV-treaOsCCR expression.To treat salt stress, rice seedlings were hydroponically grown on MS medium (Duchefa), and 10 day-old rice seedlings were treated with 250 mM NaCl. After 1, 3, 6, 12, and 24 h salt treatments, rice seedlings were collected for the analysis of OsCCR genes in various rice developmental stages as well as under biotic  and abiotic stresses  were downloaded from the Genevestigator plant biology database (https://genevestigator.com/gv/doc/intro_plant.jsp) .The public transcriptomic analysis data of UBQ5) gene (Os01g22490) was used as a reference gene, which expresses stably in rice  were represented with the letters a and b. All statistical analysis was carried out using SPSS statistics.Total RNA extraction from rice samples and cDNA synthesis were accomplished using the methods described above. Quantitative real-time PCR (qRT-PCR) was performed using a Prime Q-Mastermix  on a Rotor-Gene Q instrument system (Qiagen). For normalization of transcript levels, rice ubiquitin5 /plant dihydroflavonol reductase (DFR) superfamily  to known functional CCR genes, which encode 319–379 amino acids  followed that of Kawasaki et al. . Most previously studied functional CCRs, such as AtCCR1, EuCCR (E. gunnii CCR), ZmCCR1 (Z. mays CCR1), and SbCCR1 (Sorghum bicolor CCR1), are composed of five exons, as in the exon-intron structure Pattern 2 , SbCCR1, ZmCCR1, LpCCR (Lolium perenne CCR), HvCCR (Hordeum vulgare CCR) and TaCCR1 (T. aestivum CCR1). These CCRs have been suggested as monocot functional CCRs involved in developmental lignification  and alfalfa (Medicago truncatula), and divided these proteins into three classes (Classes I–III). Class I was subdivided into three clades (clades 1–3), with the clade 1 containing functional CCRs. Most rice CCRs belonged to Class I, with OsCCR1, 4, 5, 10, 11, 12, 17, 18, 19, 20, 21, and 24 falling into the clade 1 of Class I -binding motif at the N-terminus, and the catalytic motif for CCR activity  from wild type rice plants. The cDNAs of OsCCR5, 17, 19, 20, and 21 were successfully cloned from rice leaves. Despite many attempts, the cDNAs of OsCCR4 and 18 could not be cloned from rice, which was likely a result of very low expression levels of these genes throughout all developmental stages  -dependent short-chain dehydrogenase/reductases (SDRs)  Figure  (Pan et ) Figure . Chao et) Figure  suggesteE. coli culture was too small for kinetic analysis. The KM-values of OsCCR20 for p-coumaroyl-, feruloyl-, and sinapoyl-CoA were 24.08, 15.71, and 23.34 μM, respectively  was about five-fold higher than those for p-coumaroyl- and sinapoyl-CoAs , indicating that it has a greater catalytic efficiency toward feruloyl-CoA than toward the other substrates  than toward p-coumaroyl- or sinapoyl-CoAs . This result indicates that among three hydroxycinnamoyl-CoA substrates, both OsCCR20 and 21 have substrate preferences for feruloyl-CoA. The substrate preferences of both OsCCR20 and 21, with the strongest preference being toward feruloyl-CoA, is consistent with the lignin composition of rice. Gui et al.  displayed a high level of expression throughout all developmental stages including germination, seedling, tillering, stem elongation, booting, heading, flowering, milk, and dough stages , drought , and high salinity  -dependent SDRs  comprised of 4, 5, and 6 exons, respectively. In Pattern 2, the fourth exon is about two times longer than other exons. The length of the fourth exon in Pattern 2 is similar to the combined lengths of the fourth and fifth exons of Pattern 3 , with the length of the fourth exon equaling that seen in Pattern 2   showed that all OsCCRs, except OsCCR27, have no signal sequence. The expression of CCRs in other groups are induced under various stress conditions  -binding and NADP-specific motifs as well as the CCR signature motif. The recombinant OsCCR17, 19, 20, and 21 showed enzyme activity toward hydroxycinnamoyl-CoA substrates, indicating that these OsCCRs are biochemically functional CCRs in rice. Phylogenetic analysis revealed that OsCCR19 and 20 were closely related to other plant CCRs involved in developmental lignification. In silico transcriptomic analysis and qRT-PCR consistently demonstrated that OsCCR20 were constitutively expressed throughout all developmental stages of rice, with especially high expression levels in actively lignifying tissues such as roots, stems and panicles. These results suggest that OsCCR20 are primarily involved in the developmental deposition of lignins in secondary cell walls. Meanwhile, the expressions of OsCCR17 and 21 were induced in response to biotic and abiotic stresses, such as M. grisea and Xoo infections, UV-irradiation and high salinity. OsCCR17 and 21 were also grouped with stress-responsible CCRs identified from other plant species. Therefore, we suggest that OsCCR17 and 21 play a role in defense-related processes of rice under biotic and abiotic stress conditions.Expression patterns and biochemical properties of the rice M-HC and S-WL: conceived and designed the experiments; HLP and M-HC: performed the experiments and conducted bioinformatics analyses; MK: performed the experiments. M-HC, HLP, SHB, and S-WL: analyzed the data, and wrote the manuscript.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.


SREBP cleavage-activating protein (SCAP) is a key protein in the regulation of lipid metabolism and a potential target for treatment of dyslipidemia. SCAP is required for activation of the transcription factors SREBP-1 and -2. SREBPs regulate the expression of genes involved in fatty acid and cholesterol biosynthesis, and LDL-C clearance through the regulation of LDL receptor (LDLR) and PCSK9 expression. To further test the potential of SCAP as a novel target for treatment of dyslipidemia, we used siRNAs to inhibit hepatic SCAP expression and assess the effect on PCSK9, LDLR, and lipids in mice and rhesus monkeys. In mice, robust liver Scap mRNA knockdown (KD) was achieved, accompanied by dose-dependent reduction in SREBP-regulated gene expression, de novo lipogenesis, and plasma PCSK9 and lipids. In rhesus monkeys, over 90% SCAP mRNA KD was achieved resulting in approximately 75, 50, and 50% reduction of plasma PCSK9, TG, and LDL-C, respectively. Inhibition of SCAP function was demonstrated by reduced expression of SREBP-regulated genes and de novo lipogenesis. In conclusion, siRNA-mediated inhibition of SCAP resulted in a significant reduction in circulating PCSK9 and LDL-C in rodent and primate models supporting SCAP as a novel target for the treatment of dyslipidemia. SREBP cleavage-activating protein (SCAP) is a well-characterized key regulator of lipid metabolism , 2. Its Deletion of liver Scap in mouse models leads to a reduction in the expression of SREBP-regulated genes, de novo free fatty acid and cholesterol synthesis, liver TG levels and plasma lipid levels , 8. OverBecause of the complex role of SCAP in regulation of lipid metabolism, it is important to demonstrate the translatability of the rodent findings into a higher species with lipid metabolism pathways more closely resembling that of humans. Specifically, because inhibition of SCAP negatively regulates both LDLR and PCSK9 mRNA expression, and PCSK9 is a negative regulator of LDLR, it is critical to determine the effect of targeting SCAP on the balance of LDLR and PCSK9 and how this ultimately affects LDL-C levels in such a model. Rhesus monkeys have been extensively used as a model for human lipid metabolism –14. HereFor mouse siRNA identification, 42 sequences were designed against mouse Scap transcript NM_001103162. For nonhuman primate (NHP) siRNA identification, 84 sequences were designed to have 100% human/rhesus homology using human SCAP transcript NM_012235 and rhesus SCAP transcript XM_001100342. Oligos were synthesized and screened in mouse Hela 1-6 cells  and rhesus LLC-MK2 cells (ATCC) as previously described . The seqFor in vivo studies, lead siRNA oligos were formulated in lipid nanoparticles (LNP) as previously reported , 17. BriAll procedures described below were approved by the Merck Research Laboratories Institutional Animal Care and Use Committee and carried out in accordance with the Guide for the Care and Use of Laboratory Animals. Male C57BL/6 mice were obtained from Taconic Farms . Mice were maintained in a 12/12 h light-dark cycle with free access to food and water in group housing conditions in a temperature-controlled environment (22°C). All mice were maintained on regular rodent chow .2 and liver samples were collected. Scap mRNA levels were measured as previously described (2O)  at 20 ml/kg intraperitoneally. Four hours post D2O injection, mice were euthanized with CO2 and EDTA plasma was collected via cardiac puncture and was reserved for lipids, circulating PCSK9, and fatty acid/cholesterol synthesis analyses. Livers were collected either in RNAlater or snap-frozen in liquid nitrogen and stored at −80°C for gene expression/protein analysis.C57BL/6 mice (n = 4) received a single intravenous dose of 1 mg/kg of Scap siRNA-LNP, nt siRNA-LNP (nt control-1), or vehicle control. Animals were dosed via tail vein injections. On days 7, 14, 21, and 28 after siRNA dosing, the mice were euthanized with COescribed . For theTaqMan quantitative (q)PCR was done on an ABI 7900 real-time PCR system using commercially available TaqMan probes and primers . Data analysis was performed as described previously .13C18]oleic acid (Cambridge Isotope Labs) in 8% fatty acid-free BSA. Serial blood samples were obtained via tail nick at t = 0 (pre-tracer) and 15, 30, 45, 60, and 90 min post-tracer administration. The concentrations of the M0, M18, and M36 isotopologues of TG 52:2 were determined by LC-MS/MS as previously described  was fit to a mono-exponential decay function to determine a value for the fractional synthetic rate (k). The rate of production was then determined as multiplied by the average steady-state concentration of TG 52:2 from 15 to 90 min . On day 10, food was removed in the morning for 4 h following which all mice were administered an intravenous dose of 50 mg/kg (10 ml/kg) of  at room temperature for 1 h. Band intensities were quantified on an Odyssey infrared imaging system (Li-Cor).PCSK9 was measured using an in-house-developed ELISA assay . Briefly1) siRNA sequence selection study; 2) siRNA KD duration study; and 3) siRNA dose-titration study. For all three studies, animals were dosed intravenously based on body surface area with a single-rate controlled timed 2 min infusion. Blood samples were taken as alert procedure and percutaneous liver biopsies as sedated procedure. In each study, serum analysis for clinical chemistry, hematology, and coagulation parameters was performed for each blood collection time point, and the level of hepatic SCAP expression at each biopsy time point.All NHP studies were conducted at New Iberia Research Center (NIRC) with the approval by NIRC’s and Merck’s Institutional Animal Care and Use Committees. The NIRC facility is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. Sexually mature male and female lean rhesus macaque monkeys weighing approximately 6–8 kg at the time of the study were randomized into groups based on prestudy body weight and serum lipid levels. Three rhesus experiments were performed: 2  dose of LNP-encapsulated siRNAs (n = 4 per group). Blood samples were taken on days −4 and 0 (prior to dosing) and on days 3, 5, 6, 7, 9, 14, 19, 21, and 28 following LNP injections. Percutaneous liver biopsies were performed on days −4 prior, 7, 14, and 28. Total cholesterol, LDL-C, and HDL-C were all measured directly from plasma using the Siemens Dimension assays for Clinical Chemistry Analyzer: CHOL, ALDL, and AHDL, respectively . Plasma PCSK9 levels were measured as described , 3, 7, 10, 14, 16, 17, 18, 21, 25, 28, 35, and 48 following LNP injection. In order to assess the effect of hepatic SCAP KD on palmitate and cholesterol synthesis, animals were administered D2O  on day 16. Blood samples for the determination of palmitate and cholesterol synthesis were collected as fasted bleeds at −0.5 h and +4 h, +24 h, +48 h, and +5 days relative to D2O dosing. Percutaneous liver biopsies were performed on days −7, 7, 14, and 28. Serum analysis and liver TaqMan RT-PCR were done as described above.For the follow up siRNA KD duration study, group size was increased to 10 per group for SCAP (NHP SCAP-1) and nt control (nt control-2) siRNA-LNP. PCSK9 siRNA was used as a positive control in the study (n = 4). Animals received a 26.7 mg/m2 (n = 4), 13.35 mg/m2 (n = 8), and 6.68 mg/m2 (n = 8). The dose levels were determined based on body surface area and approximately corresponded to 2, 1, and 0.5 mg/kg, respectively. The nt control-2 was dosed at 13.35 mg/m2 (n = 8). Blood samples were taken on days −2 and 0 (prior to dosing) and days 2, 3, 7, 8, 9, 10, 12, 14, 16, 17, 18, 19, 21, 23, and 26 following LNP injection. Percutaneous liver biopsy procedures were performed on day −2 (prior) and days 3, 12, and 26 post dosing. Serum analysis and liver TaqMan RT-PCR were done as described above.The siRNA dose titration study was done using three dose levels of SCAP siRNA oligo: 26.7 mg/mDeuterium enrichment in plasma water (precursor labeling) was determined by GC-MS  and enriA 384-well format custom-designed rhesus PCR array was developed in collaboration with SABiosciences-Qiagen. Genes representing the major lipid and glucose metabolism pathways as well as genes involved in apoptosis, autophagy, and inflammatory pathways were selected based on publicly available canonical pathway databases . A total of 372 individual genes and five housekeeping genes were assayed. Rhesus Genomic DNA Contamination primer control, reverse transcription control (duplicate), and positive PCR control (duplicate) were included on the array as part of the SABiosciences-Qiagen standard array setup. The array is available from SABiosciences-Qiagen, catalog number CAPQ09560. Real-time PCR was performed as previously described . ACTB, Pt-test was used with statistical significance determined using the Holm-Sidak method with α = 0.05. Bar graphs represent the mean ± SEM, whereas the box and whiskers graphs represent the minimum, the 25th percentile, the median, the 75th percentile, and the maximum. All mouse data were analyzed using GraphPad Prism version 7.00 for Windows .Mouse data was analyzed by one-way ANOVA with Dunnett’s post test, except For the longitudinal NHP studies, the data come in the format of repeated measures, for which it has become standard to quantify the mean profiles of treatments and both variation between-subject and variation within-subject over time, using a linear mixed-effects model : Y = Timhttps://cran.r-project.org/web/packages/gmodels/index.html). To compare the treatment effects across different biomarkers, we further characterize the treatment effect using effect size (ES), where the mean effect is divided by the SD of the population. Unlike P values or T statistics, ESs are independent of the sample sizes. ESs are also normalized by their standard deviations, therefore, we can compare and rank the sensitivities among biomarkers based on their ESs.To test the drug effects, we compare the mean change-from-baseline of a treated group versus that of the vehicle group using a linear contrast (combination) of Time × Treatment levels, where the mean effect and the its standard error can be estimated using an R-package “gmodels”, which was developed by Warnes et al. . In order to assess the magnitude and duration of KD of Scap mRNA in the liver, animals were dosed with a single dose of m-Scap-1 siRNA-LNP and liver samples collected at different time points. As shown in supplemental Fig. S1A, greater than 90% mRNA KD was observed following a single dose of m-Scap-1 siRNA. This degree of silencing was maintained over a period of 3 weeks and mRNA levels were reduced by 75% at day 28 post dosing. In agreement with the earlier reports for this LNP formulation , siRNA-LA siRNA-LNP dose-titration study was performed in C57BL/6 mice (n = 4 per group) to select doses covering a range of liver Scap mRNA KD. As shown in supplemental Fig. S1B, m-Scap-1 siRNA-LNP treatment with doses ranging from 0.0625 to 1 mg/kg caused a significant and dose-dependent reduction in liver Scap mRNA. The KD of liver Scap mRNA also resulted in a dose-dependent reduction of the SREBP-2 target gene, HMG-CoA reductase (Hmgcr), as well as the SREBP-1c target gene, Fasn, indicating SCAP loss of function . The effects on Scap, Hmgcr, and Fasn mRNA reached a maximum at the 0.5 mg/kg dose, and doses of 0.03125, 0.0625, 0.125, and 0.5 mg/kg were selected for further studies.The dose-dependent effect of m-Scap-1 siRNA-LNP on Scap mRNA and SREBP activity was further explored in a follow-up study (n = 8 per group). The siRNA-LNP reagents did not cause any obvious toxic effects on liver, as measured by AST/ALT levels, at any of the doses tested and there was no effect on body weight in any of the treatment groups (data not shown). Scap mRNA was reduced by a maximum of 95% for the 0.5 mg/kg dose on day 10 post dosing .2O to measure palmitate and cholesterol synthesis. Consistent with the SCAP’s role in modulating SREBP-1c and SREBP-2, Scap mRNA KD caused a reduction in both palmitate and cholesterol synthesis. The effects were dose dependent with a greater percent reduction of de novo fatty acid synthesis relative to cholesterol synthesis . Dose-dependent reductions were also observed for plasma total cholesterol, LDL-C, VLDL-C, and HDL-C . Interesoduction . TreatmeThe statin class of drugs inhibits HMGCR, the rate-limiting enzyme in cholesterol biosynthesis, thereby reducing cholesterol synthesis and free cholesterol in the cell. In the liver, the reduction in free cholesterol leads to a compensatory upregulation of SREBP activity, including upregulation of LDLR and PCSK9 expression levels. In humans, a net increase in LDLR activity and LDL-C lowering is the result of statin treatment . To testsiRNA lead qualification was done in lean rhesus monkeys with two independent SCAP siRNA leads. Animals (n = 4) were dosed with a single dose of SCAP, PCSK9, or nt control (nt control-2) siRNAs and mRNA levels assessed from liver biopsies obtained 4 days prior to dosing and on days 7, 14, and 28 following siRNA administration . Both SCAP siRNAs showed over 80% mRNA KD in the liver at day 7. SCAP KD was associated with significant reduction in plasma PCSK9 levels . Maximum plasma PCSK9 reduction was close to 75% for the first 9 days following siRNA dosing. Based on these results, NHP SCAP-1 siRNA was selected as the lead for the follow up studies.2O tracer on day 16 post siRNA-LNP dosing. Animals were dosed with a single dose of SCAP, PCSK9, and nt control (nt control-2) siRNA and the expression of SCAP, PCSK9, and LDLR mRNA was measured in liver biopsies 7 days before and 7, 14, 28, and 35 days post siRNA-LNP dosing. SCAP mRNA was reduced by 94% on day 7 relative to a predose liver biopsy. The expression of SCAP gradually increased over time as shown in To further characterize the effect of hepatic SCAP KD on SREBP activity, including de novo lipogenesis, a follow up study was performed with a larger group size and addition of the D2 of the SCAP siRNA-LNP reagent. The nt siRNA-LNP was dosed at 26.7 mg/m2 as a control. Liver SCAP mRNA was dose-dependently reduced by the escalating doses of SCAP siRNA at all time points measured  . Other wThe SCAP siRNA studies reported herein were carried out in lean normolipidemic mouse and rhesus monkey models that were fed standard relatively low-fat diets. Horton and colleagues investigated the effects of liver SCAP loss of function in ob/ob mice, mice fed a high-fat diet, and hamsters fed a high-sucrose diet and found effects on plasma lipids and lipid metabolism similar to what is reported here . SpecifiThe requirement of SCAP for the activation of lipid biosynthesis could be a potential driver of toxicity in conjunction with systemic SCAP inhibition, e.g., using a small molecule inhibitor approach. Indeed, conditional knockout of SCAP in Swann cells causes hypomyelination  and condIn summary, we have demonstrated for the first time that SCAP siRNA-mediated KD in a NHP model leads to a reduction in plasma PCSK9, TG, and LDL-C suggesting a cardioprotective outcome of SCAP inhibition in humans. This opens up potential targeting of SCAP in the liver for the treatment of dyslipidemia and potentially other metabolic diseases that are driven by increased SREBP activation such as fatty liver disease.


Bunyaviridae that is endemic in East Asia. However, the genetic and evolutionary characteristics shared between tick- and human-derived Korean SFTSV strains are still limited.Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne viral disease caused by the SFTS virus (SFTSV) from Haemaphysalis longicornis)-derived Korean SFTSV strain (designated as KAGWT) and compare this virus with recent human SFTSV isolates to identify the genetic variations and relationships among SFTSV strains. The genome of the KAGWT strain is consistent with the described genome of other members of the genus Phlebovirus with 6,368 nucleotides (nt), 3,378 nt, and 1,746 nt in the Large (L), Medium (M) and Small (S) segments, respectively. Compared with other completely sequenced human-derived Korean SFTSV strains, the KAGWT strain had highest sequence identities at the nucleotide and deduced amino acid level in each segment with the KAGWH3 strain which was isolated from SFTS patient within the same region, although there is one unique amino acid substitution in the Gn protein (A66S). Phylogenetic analyses of complete genome sequences revealed that at least four different genotypes of SFTSV are co-circulating in South Korea, and that the tick- and human-derived Korean SFTSV strains (genotype B) are closely related to one another. Although we could not detect reassortant, which are commonly observed in segmented viruses, further large-scale surveillance and detailed genomic analysis studies are needed to better understand the molecular epidemiology, genetic diversity, and evolution of SFTSV.In this study we identify, for the first time, the genome sequence of a tick  is an emerging tick-borne viral disease caused by the SFTS virus (SFTSV). During entomological surveillance of SFTSV infection in Korean ticks collected from SFTS outbreak areas, we isolated a single SFTSV strain which we designated KAGWT. In addition, we isolated three SFTSVs from human patients with typical SFTS symptoms. In this study, we report the genomic sequences of each of these isolates and compare the genetic and evolutionary characteristics between tick- and human-derived Korean SFTSV isolates. Genetic and phylogenetic analyses of these sequences revealed that the tick-derived Korean SFTSV strain is clustered into genotype B, the most prevalent genotype in South Korea, and was closely related to other SFTSV in the same group. Furthermore, our results show that at least four different genotypes of SFTSV strains are co-circulating in South Korea. Phlebovirus in the family Bunyaviridae [Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne viral disease characterized by fever, gastrointestinal symptoms, leukopenia, and thrombocytopenia. It was first reported in China in 2010 . The cauaviridae , 6, batsaviridae  and tickaviridae , respectPhlebovirus, SFTSV contains a tripartite RNA genome consisting of three single-stranded RNA segments of negative polarity, designated large (L), medium (M), and small (S). The L, M, and S segments encode the RNA-dependent RNA polymerase (RdRp), the viral envelope glycoproteins (Gn and Gc) and both a nucleoprotein (NP) and a nonstructural protein (NSs) in an ambisense orientation, respectively [Like other members of the genus ectively , 9.Haemaphysalis longicornis, Rhipicephalus microplus, H. flava, H. concinna, Amblyomma testudinarium, and Ixodes nipponensis [Although human-to-human transmission of SFTSV through contact with blood and/or body secretions of patients has been reported –12, the ponensis –18. Furtponensis –22, and ponensis .H. longicornis nymphs collected from the Samcheok-si, Gangwon Province [Since the first reported fatal case in 2012 in Gangwon Province in South Korea . MoreoveProvince . In addiChungbuk National University Hospital received written consent for sample collection from each patient with SFTSV infection. All participants were adults and this study was approved by the institutional review board (IRB) of Chungbuk National University Hospital (IRB no. 2015-08-009-001).H. longicornis ticks collected from the Samcheok-si, Gangwon Province in South Korea as described previously [The KAGWT strain was isolated from eviously . The CB12. Cell culture supernatant was collected after seven days and stored at -80°C as the working virus stock for whole genome sequencing.For virus propagation, the virus was passaged five times on confluent monolayers of Vero E6 cells  in Dulbecco’s Modified Eagle Medium  containing 8% fetal bovine serum  with penicillin (100IU/mL) and streptomycin  placed in 37°C incubator supplemented with 5% COViral RNA was extracted from 140 μL of the viral stock using QIAamp Viral RNA Mini Kits  according to the manufacturer’s instructions. Single strand cDNA was synthesized using viral RNA and primers specific for each segment using a cDNA synthesis kit  according to the manufacturer’s instructions.For the whole genome sequencing, one to six overlapping PCR fragments covering the SFTSV genome containing full-length L, M, and S segments were amplified by PCR from cDNA using SP-Taq DNA polymerase . PCR for each segment was performed at 95°C for 5 minutes, followed by 45 cycles of amplification consisting of 95°C for 30 seconds, annealing at 53 or 60°C for 30 seconds according to the primer sets for each segment, and 72°C for 2 minutes, with a final extension at 72°C for 5 minutes. The non-coding 5′ and 3′ ends of the viral genome were determined by rapid amplification of cDNA ends (RACE) method as described previously . PCR proGenetic and phylogenetic analyses were conducted by aligning published full-length sequences of SFTSV obtained from China, Japan and Korea isolates, which are available in GenBank, together with the closely related SFTSV sequences obtained from the basic local alignment search tool results. A total of 41 full-length sequences of the L, M, and S segments, including the strains isolated in this study , were inMultiple sequence alignments were performed using the Clustal W algorithm in DNASTAR version 5.0.6 or MEGA version 6.0 . The aliPhylogenetic analyses were performed based on the full-length L, M, and S segments of SFTSVs. Phylogenetic trees were constructed with MEGA version 6.0 software using the Maximum Likelihood (ML) method based on the Kimura 2-parameter model. The reliability of the ML tree was evaluated by the bootstrap test with 1,000 replications.The full-length L, M, and S segment sequences of tick- and human-derived strains determined in this study have been deposited in GenBank under the following accession numbers: KAGWT (KY273136 to KY273138), CB1 , CB2 , and CB3  strains.Phlebovirus [The genomes of tick- and human-derived Korean SFTSV strains analyzed in this study were consistent with 6,368 nucleotide (nt), 3,378 nt and 1,746 nt present in the L, M and S segments respectively, consistent with what has been reported for other members of the genus ebovirus . The L sAlignment and pairwise comparisons of each segment between the tick-derived KAGWT strain and other fully sequenced human-derived Korean SFTSV strains showed nucleotide (and deduced amino acid) sequence homology ranging from 95.9 (99.3) to 99.9 (100), 94.0 (98.4) to 99.8 (99.8), and 94.9 (98.3) to 99.9 (100)% for L, M and S segments, respectively . This hiIn particular, the KAGWT strain showed the highest sequence identities at the nucleotide and deduced amino acid levels with the KAGWH3 strain isolated in 2014 from the serum of a patient from Gangwon Province, South Korea who experienced typical SFTS symptoms . These rCompared with the other fully sequenced SFTSV strains, KAGWT showed higher identity with the genotype B SFTSV strains than with the other genotypes at each segment as shown in In comparison with the other fully sequenced SFTSV strains, CB2 was identified as the first genotype A SFTSV strain out of 14 full-length sequenced Korean SFTSV isolates. Genetic comparison results showed that the CB2 strain had nucleotide sequence identity ranging from 97.9% (L), 97.9 to 98.0% (M), 98% (NP) and 97.2% (NSs), respectively with the other genotype A SFTSV strains circulating in China. However, the CB2 strain showed relatively low nucleotide sequence identity ranging from 96.1 to 96.7% (L genes), 93.5 to 96.2% (M genes), 95.3 to 97.6% (NP genes) and 93.9 to 96.8% (NSs genes), respectively with other SFTSV strains .To investigate the genetic evolutionary origins and relationship between the tick- and human-derived Korean SFTSV strains, phylogenetic analyses were conducted with full-length complete sequences of previous SFTSV isolates from China, Japan, and South Korea. Phylogenetic analyses of complete genome sequences  indicated that the tick-derived Korean SFTSV strain (KAGWT) was clustered with the genotype B SFTSV strains circulating in humans in China, Japan, and South Korea Figs –4, althoFurthermore, the CB1 and CB3 human isolates also clustered with genotype B viruses although they are separated into a different sub-node from tick-driven Korean SFTSV strains (KAGWT) and are closely related with recent Korean SFTSV human isolates, KADGH and KACNH3. In addition, the CB2 human isolate was clustered together with recent China genotype A viruses.Overall the phylogenetic trees revealed that Korean SFTSV strains can be classified with high branch support into four distinct genotypes  out of the six genotypes described previously . To invePhlebovirus, SFTSV that is highly endemic in China, Japan, and South Korea [SFTS is an emerging tick-borne infectious disease caused by a novel th Korea , 2, 4.Phlebovirus genus [In this study, we determined the whole genome sequences of the first tick and patient-derived Korean SFTSV strains and compared them with other available whole genomic SFTSV sequences. To the best of our knowledge, this is the first report of the whole genomic sequence of an SFTSV strain isolated from ticks and of a genotype A SFTSV strain collected from South Korea. Comparison of the whole genome sequence of tick- and human-derived strains revealed that all SFTSVs consist of three segments with 6,368 nt in the L segment, 3,378 nt in the M segment, and 1,746 nt in the S segment. Thus, the genome organization of Korean strains are consistent with the known genome organization of SFTSV belonging to the us genus . Complemus genus , 30. AllH. longicornis nymphs in 2013 [Genetic and phylogenetic analysis revealed that the tick-driven KAGWT strain isolated from  in 2013  has high in 2013 , and furet al. [Phlebovirus, genetic reassortment is known to be an important process resulting in the sequence diversity necessary for viral evolution [Although several studies about SFTSV classification have been reported , 31–34, et al.  which devolution . Previouvolution , 37, Rifvolution , and SFTvolution , 31–34. volution , 31–34, In conclusion, we report here the first whole genomic sequence of the tick-derived SFTSV KAGWT strain isolated from the ticks in the Gangwon province of South Korea and the comparison of the genetic characteristics of this virus with recent human SFTSV isolates. Genetic and phylogenetic analyses with full-length genome sequences revealed that tick- and human-derived Korean SFTSV strains are closely related to one another and that at least four different genotypes of SFTSV are co-circulating in South Korea. These results provide insight into the genetic origins of human of SFTSV strains as well as shed light on the molecular epidemiology, genetic diversity, and evolution of SFTSV. Furthermore, these associations will have important implications for the design of diagnostic procedures and vaccines against SFTSV.S1 TableNucleotide identities (%) are shown above the diagonal and the deduced amino acid identities (%) are shown below the diagonal. 1, CB2 strain; 2, HL/Injected strain; 3, HL/Egg/G2 strain; 4, HL/Larvae/G2 strain; 5, HL/Nymph/G2 strain; 6, HL/Adult/G2 strain; 7, KAGWT strain; 8, KAGWH3 strain; 9, KAGBH5 strain; 10, KAGBH6 strain; 11, KACNH3 strain; 12, KADGH strain; 13, CB1 strain; 14, CB3 strain; 15, KAGNH strain; 16, KAGNH4 strain; 17, KAJNH2 strain; 18, YG1 strain; 19, SPL003A strain; 20, SPL010A strain; 21, SPL035A strain; 22, Zhao strain; 23, ZJZHSH-FDE/China/06/2012 strain; 24, ZJZHSH-LWL/China/08/2014 strain; 25, ZJZHSH-WRF/China/08/2014 strain; 26, SPL087A strain; 27, AHL/China/2011 strain; 28, KASJH strain; 29, HB154/China/2011 strain; 30, HB155/China/2011 strain; 31, HB156/China/2011 strain; 32, SD24 strain; 33, HB29 strain; 34, JS4 strain; 35, SD4 strain; 36, Gangwon/Korea/2012 strain; 37, SDLZtick12/2010 strain; 38; JS2012-tick01 strain; 39, AH12 strain; 40, QD7 strain; 41, AH-YTY/China/05/2012 strain.(DOCX)Click here for additional data file.


JAK2V617F homozygous mutation was associated with a larger spleen size. In other data, CALR mutations in MF were signigicantly associated with longer larger splenomegaly-free survivals than others. In addition, MF patients with ≥1 mutations in AZXL1, EZH1 or IDH1/2 had significantly low spleen reduction response in ruxolitinib treatment. Developments of JAK inhibitors, such as ruxolitinib, pacritinib, momelotinib, and febratinib enabled the effective management in MF patients. Especially, significant spleen reduction responses of the drugs were demonstrated in several randomized clinical studies, although those could not eradicate allele burdens of MF.Myelofibrosis (MF) is a clinical manifestation of chronic BCR-ABL1-negative chronic myeloproliferative neoplasms. Splenomegaly is one of the major clinical manifestations of MF and is directly linked to splenic extramedullary hematopoiesis (EMH). EMH is associated with abnormal trafficking patterns of clonal hematopoietic cells due to the dysregulated bone marrow (BM) microenvironment leading to progressive splenomegaly. Several recent data have emphasized the role of several cytokines for splenic EMH. Alteration of CXCL12/CXCR4 pathway could also lead to splenic EMH by migrated clonal hematopoietic cells from BM to the spleen. Moreover, low Gata1 expression was found to be significantly associated with the EMH. Several gene mutations were found to be associated with significant splenomegaly in MF. In recent data,  Myelofibrosis (MF) is a clinical manifestation of chronic BCR-ABL1-negative chronic myeloproliferative neoplasms (MPNs), which is similar to essential thrombocythemia (ET) and polycythemia vera (PV), characterized by progressive bone marrow (BM) fibrosis, extramedullary hematopoiesis (EMH), progressive splenomegaly, and leukoerythroblastosis in the blood ,3.JAK2) V617F mutation is present in 60% of the patients with PMF or post-ET MF and 95% of those with post-PV MF [MPL) have been found in 10–25% of patients with PMF and post-ET MF [CALR) have been observed in JAK2- and MPL-negative patients with PMF (75%) and post-ET MF (50–60%) [The Janus kinase 2  and HSCs due to the dysregulation of the BM microenvironment. Progressive splenomegaly is significantly associated with debilitating symptoms, such as early satiety, deteriorative abdominal pain, portal hypertension, decreased physical activity, and the progression of cytopenia due to splenic sequestration in MF . BecauseThe symptoms of splenomegaly are associated with spleen size. In one study, palpable splenomegaly was observed in 80% of the asymptomatic patients, and about 10% of the patients with MF showed severe symptomatic splenomegaly when diagnosed with MF .The JAK tyrosine kinase family consists of JAK1, JAK2, JAK3, and tyrosine kinase 2 (TYK2), which is associated with the cytoplasmic juxtamembrane region of cytokine receptors, including the MPL, erythropoietin receptor (EPOR), and granulocyte colony-stimulating factor receptor (GCSF-R) . ActivatJAK2 V617F mutation is present in exon 14 on chromosome 9p24. When JAK2-V617F binds to cytokine receptors, such as MPL, EPOR, and GCSF-R, restricted signaling occurs via the STAT 3/5, phosphatidylinositol-3-kinase (PI3K)/AKT, and the RAS/mitogen-activated protein kinase (MAPK) pathways, resulting in the augmentation in gene expression and increase in all three myeloid lineages [The MPL is a thrombopoietin receptor and key to the growth and survival of megakaryocytes [MPL  cause its spontaneous activation, leading to cytokine-independent activation of the downstream JAK-STAT pathway. MPL W515K is involved in stem cell-derived events with both myeloid and lymphoid progenitors [ryocytes . Somaticgenitors .CALR mutants were found to activate MPL and the downstream JAK-STAT signaling pathway, as CALR mutants are abnormal chaperones and traffic with MPL to the cell surface [Calreticulin (CALR) is a multifunctional calcium-binding protein and not a signaling molecule; it is located primarily in the endoplasmic reticulum . In rece surface ,15.JAK2, MPL, and CALR are now recognized as driver mutations responsible for the MPN phenotype. Moreover, other discovered subclonal mutations in ASXL1, EZH2, CBL, IDH1/2, TP53, and SRSF2 are now known to be associated with disease progression in MF [Overall, somatic mutations of on in MF ,17.MF is characterized by abnormal trafficking of HSCs and hematopoietic progenitor cells (HPCs), leading to their migration from the BM and the engraftment to EMH sites . ExpansiThe C-X-C motif chemokine ligand 12 (CXCL12) is produced by mesenchymal stromal cells and osteoblasts in the BM and is known to play a critical role in the maintenance and development of HSCs in the BM ,25. ReceCXCL12 binds to the G-protein-coupled receptor, C-X-C chemokine receptor type 4 (CXCR4), on hematopoietic cells and other cells . Then, CJAK2 is involved in CXCL12/CXCR4-mediated cell transfer and engraftment [JAK2 activates MPL-mutant MO7e cells that promote CXCR4 signaling [JAK2 activation and CXCL12/CXCR4 signaling increased CXCL12-dependent migration and the downstream activation of the STAT, PI3K/AKT, and RAS/MAPK pathways (JAK2 inhibition by ruxolitinib or AZD1480 (JAK1/JAK2 inhibitor) reversed the enhanced migration response. This data demonstrate that oncogenic JAK2 activation as a driver mutation spontaneously activates the CXCL12/CXCR4 pathway and encourage EMH, resulting in progressive splenomegaly. A previous study demonstrated that raftment . Recentlignaling . In the pathways . MoreoveGATA1 expression in a different megakaryocyte cell line [In addition, Gata1 is a member of the GATA family of transcription factors that are indispensable for the appropriate maturation of hematopoietic cells of many lineages, including erythroid cells and megakaryocytes . MF is aell line . In the ell line . TherefoJAK2 V617F mutation has been associated with high blood cell counts, history of thrombosis or pruritus, and the low requirement of transfusion for follow-up duration. Barosi et al. investigated the usefulness of the JAK2 V617F mutation in explaining the phenotype variations [JAK2 V617F homozygous mutation showed higher proliferative profiles, such as larger spleen size (p = 0.003) and higher white blood cell count (p = 0.003) than those with wild-type or heterogeneous mutations . Rumi et al. evaluated the clinical impacts of the driver mutations of with PMF . In the JAK2, CALR, MPL, or triple-negative mutation status. However, patients with ≥1 mutation in ASXL1, EZH1, or IDH1/2 and those with ≥3 mutations of any type were significantly less likely to have a spleen response than those with no mutations in ASXL1, EZH1, or IDH1/2 (p = 0.01), and those with ≤2 mutations (p = 0.001).Recently, Patel et al. investigated whether genetic profiles impact the clinical outcomes for patients with MF  that are treated with ruxolitinib . In theiJAK2 V617F mutation has led to the development of JAK inhibitors for the management of MF. Ruxolitinib is the first FDA-approved selective JAK1/JAK2 inhibitor and has moderate activity against TYK2 and JAK3. In the phase 1/2 trial, it was well tolerated, with thrombocytopenia as the dose-limiting toxicity [p < 0.001; p < 0.001) [The discovery of toxicity . Clinicatoxicity ,43. In tJAK2 (wild and mutant type) inhibitor and also targets fms-like tyrosine kinase 3 (FLT3), colony-stimulating factor 1 receptor, and inerleukin-1 receptor-associated kinase 1 without meaningful inhibition of JAK1. It has an advantage as a relatively non-myelosuppressive JAK2 inhibitor [n = 220) and BAT (n = 107) in MF patients. At 24 weeks of the study, SVR of ≥35% on MRI was achieved in 19% of the pacritinib group versus 5% of the BAT group  and BAT (n = 100) involving MF patients. At 24 weeks, the SVR of ≥35% in the pooled pacritinib group was 18%, but in the BAT group, it was only 3% (p = 0.001) [Pacritinib is an oral selective nhibitor . In two = 0.001) . In the JAK1/JAK2 inhibitor with the feature of alleviating anemia due to reduced production of hepcidin in the liver. SMPLIFY-I, a phase-3 head-to-head clinical trial of momelotinib versus ruxolitinib involving JAK inhibitor-naïve MF patients, demonstrated non-inferiority in SVR response at 24 weeks (26.9% in the momelotinib group versus 29% in the ruxolitinib group (p = 0.011) [p = 0.90; Momelotinib is a potent and selective = 0.011) . In SIMP= 0.011) . In thisJAK2 inhibitor, targets FLT3, and is a rearranged during transfection (RET) kinase [JAK2 V617F, MPL W515K, and JAK2 exon 12 mutations and is more selective for JAK2 than for JAK1 and JAK3. In phase 3 of the randomized JAKARTA trial, two dose groups of fedratinib  were compared with the placebo group (n = 96) [p < 0.0001; Fedratinib is a selective and potent ) kinase . It inhiAlthough reduction of increased spleen size could be possible by development of the JAK inhibitors, non-responders remain present. For the patients with refractory symptomatic splenomegaly and/or portal hypertension, splenectomy might be a treatment option, but should only be considered if the qualifying patient has an adequate life expectancy . SimilarJAK2 activation in MPL-mutant cells was found to support the CXCL12/CXCR4 pathway and activate downstream JAK-STAT, PI3K/AKT, and RAS/MAPK activation pathways, leading to the expansion of malignant clones, EMH, and splenomegaly in MF patients [Splenomegaly is a cardinal feature and is associated with splenic EMH in MF. In previous studies, several cytokines, such as SCF, TNF-α, PDGF, TGF-β, bFGF, PF4, and IL-8 contributed to the expansion of malignant clones from the BM to the spleen as an EMH site, leading to the development of splenomegaly in MF patients. Recently, patients . Moreovepatients ,35,36,37JAK inhibitors for the treatment of MF. Several JAK2 inhibitors have been found to effectively reduce spleen volume in MF in several randomized clinical trials [JAK2 mutant HPCs derived in the spleen are supposed to be sensitive to JAK inhibition. However, JAK2 inhibitors did not eradicate JAK2 V617F allele burden because MF HSCs survive. Recent data demonstrated that the eradication of JAK2 V617F allele burden by JAK2 inhibition is not successful because several coexisting additional genetic mutations, such as TET2, ASXL1, and IDH1/2 mutations might participate in clonal expansion of BM HSCs [The understanding of driver mutations has preceded the development of l trials ,47,48,50 BM HSCs .Despite the clinical relevance, increased spleen size has not been proven as a significant prognostic factor in major prognostic models, including International Prognostic Scoring System (IPSS), Dynamic IPSS, Dynamic IPSS plus, MF Secondary to PV, and ET-Prognostic Model and Mutation-Enhanced IPSS 70 in patients with MF ,55,56,57Splenomegaly is associated with several clinical symptoms, genetic mutations, prognostic factors, and EMH in MF. However, the clinical value of the spleen appears to be underestimated in MF. Further well-designed clinical studies are needed for evaluating the significance of splenomegaly in MF.