[205, 206] In fact, evaluation of cases of exacerbated hepatitis following cessation of NA therapy revealed significantly lower levels of HBcrAg (3.2 vs 4.9, P = 0.009) in the non-recurrence group compared

to the recurrence group,[207] indicating that HBcrAg is a potential marker for cessation of NA therapy. Similarly to HBcrAg, HBsAg is thought to be little affected by NA transcriptase inhibition, and the retreatment rate after cessation of NA therapy Selleck Alectinib was significantly lower for the group with low HBsAg levels (<1000 IU/mL) at the time of cessation (18% vs 63%, P = 0.049).[208] Based on the above results, the MHLW research group produced a report titled “Studies concerning efficacy of IFN therapy aimed at creation of treatment discontinuation standards and treatment discontinuation in NAs therapy for hepatitis B”, setting out policy regarding cessation of NA therapy.[209, 210] A summary is shown in Table 14. To determine the criteria for Proteases inhibitor therapy cessation, as shown below in Table 15, HBsAg and HBcrAg levels at therapy cessation were scored, the final score allocated to the following 3 categories of risk of relapse, and the success rate was predicted. Successful cessation was defined as “finally resulting in inactive carrier status, i.e. ALT ≤30 U/L and HBV DNA <4.0 log copies/mL”. Studies have shown that if this inactive carrier status is achieved,

there is no progression of liver disease, and risk of HCC also declines.[34, 211] Group for which cessation may be considered. However, even in the low risk group, recurrence of hepatitis can occur, so vigilance is required. Group for which cessation may be considered depending on circumstances. This group requires further evaluation concerning cessation criteria and methods. Continued treatment is recommended for this group. However, for patients aged <35, the cessation success rate is relatively high at 30∼40%. Recommendations The following 3 patient criteria must be met for cessation of NA therapy: (1) Both the treating physician

and the patient fully understand that after cessation of NA therapy, there is a high incidence of recurrence of hepatitis, possibly severe; (2) Follow-up is possible after treatment cessation, and appropriate treatment is possible even if hepatitis recurs, Coproporphyrinogen III oxidase (3) Even if recurrence of hepatitis occurs, it is unlikely to be severe if the degree of fibrosis is mild and the hepatic reserve is good. The 3 laboratory criteria for cessation of NA therapy are: (1) At least 2 years of administration of NAs; (2) undetectable serum HBV DNA levels (using real time PCR); (3) negative serum HBeAg at the time of treatment cessation. When the above criteria are met, it is possible to predict the risk of relapse from HBsAg and HBcrAg levels at the time of cessation of therapy. NA therapy should be continued in the high risk group.

[205, 206] In fact, evaluation of cases of exacerbated hepatitis following cessation of NA therapy revealed significantly lower levels of HBcrAg (3.2 vs 4.9, P = 0.009) in the non-recurrence group compared

to the recurrence group,[207] indicating that HBcrAg is a potential marker for cessation of NA therapy. Similarly to HBcrAg, HBsAg is thought to be little affected by NA transcriptase inhibition, and the retreatment rate after cessation of NA therapy Vincristine purchase was significantly lower for the group with low HBsAg levels (<1000 IU/mL) at the time of cessation (18% vs 63%, P = 0.049).[208] Based on the above results, the MHLW research group produced a report titled “Studies concerning efficacy of IFN therapy aimed at creation of treatment discontinuation standards and treatment discontinuation in NAs therapy for hepatitis B”, setting out policy regarding cessation of NA therapy.[209, 210] A summary is shown in Table 14. To determine the criteria for CH5424802 supplier therapy cessation, as shown below in Table 15, HBsAg and HBcrAg levels at therapy cessation were scored, the final score allocated to the following 3 categories of risk of relapse, and the success rate was predicted. Successful cessation was defined as “finally resulting in inactive carrier status, i.e. ALT ≤30 U/L and HBV DNA <4.0 log copies/mL”. Studies have shown that if this inactive carrier status is achieved,

there is no progression of liver disease, and risk of HCC also declines.[34, 211] Group for which cessation may be considered. However, even in the low risk group, recurrence of hepatitis can occur, so vigilance is required. Group for which cessation may be considered depending on circumstances. This group requires further evaluation concerning cessation criteria and methods. Continued treatment is recommended for this group. However, for patients aged <35, the cessation success rate is relatively high at 30∼40%. Recommendations The following 3 patient criteria must be met for cessation of NA therapy: (1) Both the treating physician

and the patient fully understand that after cessation of NA therapy, there is a high incidence of recurrence of hepatitis, possibly severe; (2) Follow-up is possible after treatment cessation, and appropriate treatment is possible even if hepatitis recurs, MYO10 (3) Even if recurrence of hepatitis occurs, it is unlikely to be severe if the degree of fibrosis is mild and the hepatic reserve is good. The 3 laboratory criteria for cessation of NA therapy are: (1) At least 2 years of administration of NAs; (2) undetectable serum HBV DNA levels (using real time PCR); (3) negative serum HBeAg at the time of treatment cessation. When the above criteria are met, it is possible to predict the risk of relapse from HBsAg and HBcrAg levels at the time of cessation of therapy. NA therapy should be continued in the high risk group.

5B). The plasma TG levels were not significantly different between the three groups, but serum cholesterol was significantly higher in HFHC mice (372.3 ± 21.9 mg/dL) compared with both HF mice (277.3 ± 50.5 mg/dL; P < 0.001) and chow-fed mice (127.5 ± 7.1 mg/dL; P < 0.001) (Fig. 5E). Plasma oxCoQ 9 levels in mice at 16 weeks were significantly higher in HFHC mice (0.06 ± 0.004 μg/mL)

compared with HF mice (0.03 ± 0.004 μg/mL) and chow-fed mice (0.02 ± 0.004 μg/mL; P < 0.0001) (Table 2 and Fig. 5D). The correlation Vemurafenib concentration of liver tissue collagen 1 mRNA relative expression and absolute plasma oxCoQ 9 levels had an R2 value of 0.51. Thus, the fructose-containing HFHC diet had the most hepatic ROS, hypercholesterolemia, and hepatic fibrosis. This was mirrored by the levels of plasma oxCoQ9, which differed significantly among all three

groups and correlated with the presence of fibrosis in this model. The rising rates Sirolimus molecular weight of NASH make addressing the underlying causes of this serious condition more pressing. Hepatic steatosis is common in obese patients, but only a subset of these patients develop NASH, emphasizing the contribution of genetic and potential environmental risk factors. Human NASH histopathology has been associated with steatosis, lobular and portal inflammation, hepatocyte ballooning, and fibrosis. Specifically, zone 3 predominant macrovesicular steatosis, ballooning, and perisinusoidal fibrosis is deemed consistent with adult or type 1 NASH. Type 2 or pediatric NASH histopathology has been reported to have panacinar or periportal (zone 1) steatosis, rare ballooning and portal tract expansion by chronic inflammation or fibrosis.37 Individuals who have NASH with fibrosis have progressive disease and greater morbidity and mortality including the potential for cirrhosis, liver failure, and liver transplantation.3 However, the specific biological determinants that lead to development of NASH with fibrosis are not

well-defined. Fructose consumption accounts for approximately 10.2% of all calories in our average diet in the United States.38 In comparison with other simple sugars such as glucose, use of fructose for hepatic metabolism is not restricted by the rate-limiting click here step of phosphofructokinase, thus avoiding the regulating action of insulin.39 Fructose intake is two- to three-fold higher in patients with NASH compared with body mass index–matched controls, and daily fructose ingestion has been associated with increased hepatic fibrosis.40, 41 These epidemiologic data prompted us to investigate the potential mechanistic role that fructose and other simple sugars may play in the development of NASH. The present study focused on the development of a dietary model of NASH. To this end, we compared HF mice with mice maintained on the same diet but also given ad libitum access to fructose in their drinking water (HFHC).

[12] Cell migration was assessed using cell-culture inserts (BD Biosciences, Bedford, MA), according to the manufacterer’s guidelines. Ibrutinib Details are provided in the Supporting Materials. The sphere formation assay was performed as previously described[13] (and in the Supporting Materials). Flow cytomety analysis was performed as previously described[13] and detected using a FACSCanto

II flow cytometer (BD Biosciences). Antibodies (Abs) and the procedure are described in the Supporting Materials. The 3′-UTR (untranslated region) sequence of PTEN and SMAD7 are predicted to interact with miR-216a, and miR-217 or a mutated sequence within the predicted target sites was synthesized and inserted into the XbaI and FseI sites of the pGL3 control luciferase reporter vector (Promega, Madison, WI) (Supporting Fig. 3). The luciferase reporter assay was performed as previously described[11, 13] (and in the Supporting Materials). All experiments on mice were approved by the SingHealth Institutional Animal Care and Use Committee. Details of animal studies are provided in the Supporting Materials. Experimental data are presented as the mean ± standard deviation (SD). All statistical analyses were performed using analysis of variance (ANOVA) or a two-tailed Student t test with either GraphPad

Prism 5 (GraphPad Software, Inc., La Jolla, CA) or Partek Genomics Suite software (Partek Incorporated, St. Louis, MO). Survival curves were

calculated using Kaplan-Meier’s method. Differences selleckchem were considered statistically significant when P values were less than 0.05. Because there have only been a few reports on the expression of miRNAs and their relation to early recurrent disease in patients with HCC, we conducted Molecular motor comprehensive miRNA profiling of liver biopsies from HCC patients with early and nonrecurrent diseases to identify miRNAs that are relevant to early recurrent disease. Early recurrence was defined as a recurrence within 2 years after a curative resection. miRNA expression profiles of 30 cases of liver biopsies, including 10 samples from HCC patients who had early recurrent disease over the 24-month observation period and 10 from patients who did not have early recurrent disease, were compared to 10 histologically normal tissue samples using the GeneChip miRNA 2.0 Array (Affymetrix, Inc., Santa Clara, CA). A panel of miRNAs with significant differential expression between early and nonrecurrent HCC and histologically normal liver tissue was derived through a series of ANOVA contrasts, and these miRNAs were selected for further validation and functional characterization. Among these miRNAs, expression of the miR-216a/217 cluster was significantly up-regulated in biopsies from HCC patients associated with early recurrent disease. Expression of miR-216a and miR-217 was >20- and >16-fold, respectively, when compared between HCC and normal liver tissue.

[12] Cell migration was assessed using cell-culture inserts (BD Biosciences, Bedford, MA), according to the manufacterer’s guidelines. see more Details are provided in the Supporting Materials. The sphere formation assay was performed as previously described[13] (and in the Supporting Materials). Flow cytomety analysis was performed as previously described[13] and detected using a FACSCanto

II flow cytometer (BD Biosciences). Antibodies (Abs) and the procedure are described in the Supporting Materials. The 3′-UTR (untranslated region) sequence of PTEN and SMAD7 are predicted to interact with miR-216a, and miR-217 or a mutated sequence within the predicted target sites was synthesized and inserted into the XbaI and FseI sites of the pGL3 control luciferase reporter vector (Promega, Madison, WI) (Supporting Fig. 3). The luciferase reporter assay was performed as previously described[11, 13] (and in the Supporting Materials). All experiments on mice were approved by the SingHealth Institutional Animal Care and Use Committee. Details of animal studies are provided in the Supporting Materials. Experimental data are presented as the mean ± standard deviation (SD). All statistical analyses were performed using analysis of variance (ANOVA) or a two-tailed Student t test with either GraphPad

Prism 5 (GraphPad Software, Inc., La Jolla, CA) or Partek Genomics Suite software (Partek Incorporated, St. Louis, MO). Survival curves were

calculated using Kaplan-Meier’s method. Differences Dabrafenib molecular weight were considered statistically significant when P values were less than 0.05. Because there have only been a few reports on the expression of miRNAs and their relation to early recurrent disease in patients with HCC, we conducted SSR128129E comprehensive miRNA profiling of liver biopsies from HCC patients with early and nonrecurrent diseases to identify miRNAs that are relevant to early recurrent disease. Early recurrence was defined as a recurrence within 2 years after a curative resection. miRNA expression profiles of 30 cases of liver biopsies, including 10 samples from HCC patients who had early recurrent disease over the 24-month observation period and 10 from patients who did not have early recurrent disease, were compared to 10 histologically normal tissue samples using the GeneChip miRNA 2.0 Array (Affymetrix, Inc., Santa Clara, CA). A panel of miRNAs with significant differential expression between early and nonrecurrent HCC and histologically normal liver tissue was derived through a series of ANOVA contrasts, and these miRNAs were selected for further validation and functional characterization. Among these miRNAs, expression of the miR-216a/217 cluster was significantly up-regulated in biopsies from HCC patients associated with early recurrent disease. Expression of miR-216a and miR-217 was >20- and >16-fold, respectively, when compared between HCC and normal liver tissue.

Based on these results it is recommended that all haemophilia centres have available a chromogenic or two-stage clotting assay and that this should be performed in subjects with normal APTT and one-stage FVIII activity in the presence of a personal or family history consistent with mild haemophilia A. Platelet

function testing is important for the diagnosis of many inherited and acquired bleeding disorders but it has lacked standardization [12]. Furthermore, heterogeneity in the biology and laboratory manifestations of platelet function disorders poses additional challenges check details to standardizing the diagnostic testing [12–15]. Recent surveys, including the largest worldwide survey of clinical laboratories by the International Society on Thrombosis and Haemostasis [16], have been helpful to identify which aspects of commonly performed platelet function tests, such as light transmission aggregation (LTA), show the greatest deviation in practice [17–19]. The lack of standardization in testing has led a number of organizations to

develop guidelines and recommendations, using expert opinion and/or systematic reviews of the literature [12,20–23]. Presently, many diagnostic laboratories need to update their practices in order to meet these new recommendations [22]. A number of organizations have led efforts to improve and standardize the laboratory assessment of platelet disorders [11,17–19,22,24]. http://www.selleckchem.com/products/lee011.html Some efforts have focused on defining common practices [16–19,24] and the heterogeneity in practice stimulated the development of published guidelines from organizations such as the International Society on Haemostasis and Thrombosis and the Clinical and Laboratory Standards Institute [22]. Presently, the most common type of diagnostic assay

used to investigate a known or suspected platelet function disorder is an assessment Amino acid of platelet aggregation function, often by LTA [12,16,17]. Although some laboratories perform ‘screening tests’ [such as the bleeding time and Platelet Function Analyzer-100® (Siemens/Dade-behing, Marburg, Germany) closure time] [19], neither the bleeding time nor the closure time has sufficient sensitivity to rule out common platelet function disorders [23,25]. LTA has considerable diagnostic utility when performed by rigorously standardized procedures [25,26] with validated reference intervals [27] on samples from individuals referred for bleeding problems. Laboratories need to consider the potential for false positives, as LTA abnormalities with two or more agonists are much more highly predictive of a bleeding disorder than a single agonist abnormality [25].

Based on these results it is recommended that all haemophilia centres have available a chromogenic or two-stage clotting assay and that this should be performed in subjects with normal APTT and one-stage FVIII activity in the presence of a personal or family history consistent with mild haemophilia A. Platelet

function testing is important for the diagnosis of many inherited and acquired bleeding disorders but it has lacked standardization [12]. Furthermore, heterogeneity in the biology and laboratory manifestations of platelet function disorders poses additional challenges Smoothened Agonist supplier to standardizing the diagnostic testing [12–15]. Recent surveys, including the largest worldwide survey of clinical laboratories by the International Society on Thrombosis and Haemostasis [16], have been helpful to identify which aspects of commonly performed platelet function tests, such as light transmission aggregation (LTA), show the greatest deviation in practice [17–19]. The lack of standardization in testing has led a number of organizations to

develop guidelines and recommendations, using expert opinion and/or systematic reviews of the literature [12,20–23]. Presently, many diagnostic laboratories need to update their practices in order to meet these new recommendations [22]. A number of organizations have led efforts to improve and standardize the laboratory assessment of platelet disorders [11,17–19,22,24]. KU-57788 in vitro Some efforts have focused on defining common practices [16–19,24] and the heterogeneity in practice stimulated the development of published guidelines from organizations such as the International Society on Haemostasis and Thrombosis and the Clinical and Laboratory Standards Institute [22]. Presently, the most common type of diagnostic assay

used to investigate a known or suspected platelet function disorder is an assessment C-X-C chemokine receptor type 7 (CXCR-7) of platelet aggregation function, often by LTA [12,16,17]. Although some laboratories perform ‘screening tests’ [such as the bleeding time and Platelet Function Analyzer-100® (Siemens/Dade-behing, Marburg, Germany) closure time] [19], neither the bleeding time nor the closure time has sufficient sensitivity to rule out common platelet function disorders [23,25]. LTA has considerable diagnostic utility when performed by rigorously standardized procedures [25,26] with validated reference intervals [27] on samples from individuals referred for bleeding problems. Laboratories need to consider the potential for false positives, as LTA abnormalities with two or more agonists are much more highly predictive of a bleeding disorder than a single agonist abnormality [25].

Continuous infusion (CI) is an alternative to the traditional intermittent bolus injections (BI). The rationale behind CI is to administer the factor Paclitaxel molecular weight concentrate at a rate corresponding to its elimination thus maintain steady factor levels. CI offers an improved hemostatic control and has a potential to reduce markedly factor consumption and thereby treatment costs, especially if the dose is adjusted according to the actual daily levels and clearance. The adjusted dose CI introduced in the early 1990s, during the last two decades has gained increased interest and acceptance among hemophilia treaters. Currently, CI is used particularly for the management of major bleeds and surgery, including the most demanding

surgical procedures such as total joint replacements. This chapter reviews actual issues of CI therapy in hemophilia. “
“Summary.  Replacement therapy using factor VIII (FVIII) elicits FVIII-specific antibodies (abs) in about 25% of the patients. A majority of such abs are directed towards specific FVIII regions in which major epitopes have been identified (C-terminal end of the C2 domain, the N-terminal

end of the A2 domain and C1 domain in cases of mild/moderate haemophilia A). We derived five human monoclonal abs (mabs) that react with Cisplatin high affinity to the FVIII C1, C2 or A2 domains respectively and are representative of most of the specific inhibitors observed in haemophilia A patients. We generated mouse anti-idiotypic mabs (anti-Ids) against the paratope of each of the inhibitors. We demonstrated that a combination of these anti-Ids (anti-anti-A2, -C1, -C2) had the ability to neutralize the inhibitory properties of human polyclonal abs in plasma. In 16 of the 18 plasmas

tested, the inhibiting FVIII activity was neutralized up to 100% by the anti-Ids mixture with restoration of full FVIII activity. These data allow us to conclude that polyclonal Fludarabine high-affinity FVIII inhibitors could be neutralized with an anti-Ids mixture and that only a limited number of anti-Ids were required for inhibitor neutralization in 90% of the patients. We also demonstrated that anti-Id Abs bound to anti-FVIII human B cell line produced the corresponding anti-FVIII Ab and that this binding was followed by surface capping of complexes. Data obtained in vitro at monoclonal and polyclonal level, confirmed by in vivo assays, and the preliminary results obtained at BCR level, indicate that anti-id mixture made of only a limited number of anti-Ids could be useful in the restoration of haemostasis in haemophilia patients with inhibitor. Administration of factor VIII (FVIII) to haemophilia A patients elicits an immune response that includes Abs inhibiting FVIII cofactor activity (referred to as inhibitors). The prevalence of inhibitors varies from one study to another, but a consensus value of ±25% is generally accepted.

Additional Supporting Information may be found in the online version of this article. “
“To retrospectively compare the short-term antitumor efficacy and safety of transcatheter arterial chemoembolization (TACE) with a cisplatin-iodized oil suspension (C-IS) and a miriplatin-iodized oil suspension (M-IS) for hepatocellular carcinoma (HCC). Of patients who underwent

TACE for unresectable HCC between January 2010 and PF-02341066 solubility dmso August 2011, 25 and 21 patients received C-IS and M-IS, respectively. The short-term therapeutic efficacy of both groups was evaluated by the treatment effect seen on dynamic enhanced computed tomography or magnetic resonance imaging of tumor nodules 3 months after treatment. Adverse events were evaluated to compare C-IS and M-IS. After TACE using C-IS and M-IS, 100% necrosis or tumor size reduction was achieved in 30 and 18 tumor nodules, respectively (81% vs 53%; P = 0.006). Objective responses were achieved in 30 nodules exposed to TACE using C-IS and 17 exposed to TACE using M-IS (81% vs 50%; P = 0.011). Disease control was achieved in 36 nodules exposed to C-IS and 27 exposed to M-IS (97% vs 79%; P = 0.017). The percentage of patients attaining a complete response, an objective response and disease control was significantly greater in the C-IS group than in the M-IS

group. No significant differences were found in the aspartate aminotransferase, alanine aminotransferase, total bilirubin and creatinine levels between the two Selleckchem Alectinib groups either before treatment or 1 month after treatment. The short-term antitumor effects of TACE Edoxaban with C-IS may be superior to those with M-IS in terms of the complete response, objective response and disease control rates. “
“STAT3-driven expression of small proline rich protein 2a (SPRR2a), which acts as an src homology 3 (SH3) domain ligand, induces biliary epithelial cell (BEC) epithelial-mesenchymal transition (EMT), which, in turn, promotes wound healing. SPRR2a also quenches

free radicals and protects against oxidative stress and DNA damage in nonneoplastic BEC. Sprr2a-induced EMT also increases local invasiveness of cholangiocarcinomas (CC), but prevents metastases. Understanding SPRR2a regulation of EMT has potential for therapeutic targeting in both benign and malignant liver disease. Molecular mechanisms responsible for SPRR2a-induced EMT were characterized, in vitro, and then evidence for utilization of these pathways was sought in human intrahepatic CC, in vivo, using multiplex labeling and software-assisted morphometric analysis. SPRR2a complexes with ZEB1 and CtBP on the microRNA (miR)-200c/141 promoter resulting in synergic suppression of miR-200c/141 transcription, which is required for maintenance of the BEC epithelial phenotype. SPRR2a induction promotes dephosphorylation and nuclear translocation of the SH3-domain containing protein GRB2 and an SH3-domain ligand in ZEB1 is required for SPRR2a-induced synergic suppression of miR-200c/141.

2A; Supporting Fig. 1F). Twenty-three of twenty-seven cases of HCCs (85%) showed a decreased Rnd3 expression, when compared to peritumor tissue. Mean tumor/nontumor ratio Dinaciclib mw was 0.68 ± 0.08 (P = 0.0005). Using IHC, Rnd3 expression in peritumor tissue varied from faint to intense and was predominantly localized to the cytoplasm of hepatocytes (Fig. 2B). In contrast, low or no expression was observed in tumor samples (Fig. 2B). Rnd3 protein expression was also determined in healthy primary human hepatocytes as well as in the tumor cell lines, Huh6, Huh7, SNU398, SNU475, Hep3B,

and HepG2. Results showed that Rnd3 expression was reduced in all tumor cell lines tested, as compared to primary hepatocytes (Fig. 2C). Because RND3 expression showed a strong correlation with the presence of satellite nodules in HCC, we analyzed the effect of changes in RND3 expression level on cell invasion in the

Hep3B HCC cell line. Lentiviral transduction led to a 6-fold overexpression of Rnd3, which was associated with a 4.5-fold reduction in cell ability to invade Matrigel (Fig. 3A). On the other hand, transient Rnd3 knockdown using two different siRNAs led to decreased expression of Rnd3 protein by 95% (S1) or 75% (S3) (Fig. 3B) and resulted in a significant increase in invasion (Fig. 3B). The increase was more drastic with S1 than S3, which is in agreement with their silencing efficacy. While performing invasion assays, we also analyzed cell growth and found that Rnd3 knockdown led to an inhibition of HCC cell growth (Fig. 3C). Thus, our results demonstrate that Rnd3 expression levels Ku-0059436 in vivo inversely regulate HCC cell-invasion and growth properties. Because of our initial observation that down-regulation of Rnd3 was associated with evidence of an invasive phenotype in patients, and to better characterize Rnd3 involvement in HCC progression, we focused our study on the invasion mechanism. Because loss of the cell-junction protein, E-cadherin, is associated cAMP with HCC cell invasiveness,23, 24 we evaluated E-cadherin

expression in Rnd3-depleted cells. Rnd3 silencing in Hep3B (Fig. 4A,B) using both siRNAs led to a significant decrease in E-cadherin mRNA expression, whereas a significant down-regulation of E-cadherin protein was only observed with S1. However, decrease of E-cadherin protein expression was significantly observed with both siRNAs in Huh7 cells (Supporting Fig. 2A). IF analyses confirmed that E-cadherin expression was strongly reduced at cell-cell contacts in Rnd3-silenced cells (Supporting Fig. 2B). These results suggest that Rnd3 depletion affected the integrity of adherens junctions. We then sought to analyze E-cadherin protein levels in the 27 HCCs previously used for measuring Rnd3 expression. E-cadherin expression was down-regulated in 16 of 27 HCCs, as compared to peritumor tissue (Supporting Fig. 3).