We have previously demonstrated that other MEK inhibitors (PD098059, U0126, PD184161)
reduce ERK phosphorylation (MEK activity) and growth in human HCC cells.27, 29 PD0325901 is much more potent than these MEK inhibitors in HCC cells in vitro based on its median inhibitory concentration, which lies in the nanomolar range. In a recent study, Raf-1 small interfering RNA NVP-BGJ398 supplier (100 nM) caused a 50% decrease in phosphorylated ERK levels that was associated with a 50% decrease in HCC growth in vitro30 (and unpublished results). Similar results were obtained with ERK1,2 olignonucleotide anti-sense (300 nM) that decreased total ERK levels with a corresponding decrease in HCC cell growth.27 The effective dose and inhibitory effects of the small interfering RNA and anti-sense are comparable to that of
PD0325901 in HepG2 and Hep3B cells in vitro. Taken together, these results suggest that PD0325901 is a MEK inhibitor with absolute specificity. To investigate the efficacy of MEK inhibition in EPZ-6438 concentration a more clinically relevant model, TGF-α transgenic mice from which the TAMH line was derived were employed. These animals have a human TGF-α transgene that is specifically up-regulated in HCC tumors within the liver.31 The TGF-α transgenic mice are known to develop well-differentiated HCC in 70% of animals by 15 months of age.32 Indeed, studies of rat HCC show that preneoplastic regions in the liver grow at a threefold faster rate with up-regulation of TGF-α.33 Furthermore, because most human HCC tumors have an increased Non-specific serine/threonine protein kinase amount of TGF-α present, the TGF-α transgenic mouse is believed to be a valid model of HCC for the current study.34, 35 Because TGF-α is a potent activator of the MEK-ERK pathway, these animals are ideal for treatment with a MEK inhibitor.26 In TGF-α transgenic mice at 30 weeks of age, we previously demonstrated an eightfold increase in P-ERK expression within HCC hepatocytes compared with normal hepatocytes. In addition, the ability of PD0325901 to decrease P-ERK within normal
hepatocytes in the treatment arm correlated with its ability to prevent HCC formation in this model.36 In the current randomized study, the incidence of carcinoma in the diethylnitrosamine accelerated transgenic model was determined. A 3.5-fold decrease in tumor incidence was seen in animals given the MEK inhibitor. We wanted to further examine the mechanism of PD0325901 and determine whether it was preventing formation, halting progression, or causing regression of established tumors in this developmental model. To achieve this, MRI confirmation of the presence of tumors was performed, and then treatment was initiated. After serial examinations, a dramatic regression in tumor volume was observed (Fig. 4). Taken together, the animals that were examined by MRI showed a difference in tumor volume approaching a threefold decrease in the MEK-inhibitor treated mice compared with the vehicle-treated mice (Fig. 5).
Inhibitor development, because of its impact on patients’ morbidity and quality of life, is presently the most serious complication of haemophilia A treatment. The see more identification of several
genetic and non-genetic risk factors may be used for the stratification of inhibitor risk and the definition of prevention strategies, particularly for patients with a high-risk genetic profile. The most extensively studied genetic factor is the type of F8 mutation, i.e. large deletions, nonsense mutations and inversions, which are associated with a higher risk of inhibitor development. This is the basis for the increased risk in patients
with inhibitor family history; however, concordance family studies showed that factors other than F8 mutations are involved. An emerging role is investigated for polymorphisms of immune-regulatory genes that may increase (IL-10 and TNF-α) or reduce (CTLA-4) inhibitor risk and whose heterogeneous ethnic distribution may correlate to Ibrutinib the higher inhibitor risk in non-caucasian patients. A role for FVIII haplotypes, particularly in black haemophiliacs, has been recently proposed. Recent studies report an increased inhibitor risk for initial intensive treatments (surgery or severe bleeds requiring high-dose and/or prolonged treatment, presence of danger signals),
whereas regular prophylaxis (absence of danger signals) exerts a protective effect. A clinical PRKD3 score including the type of F8 mutation, family history of inhibitors and intensive treatment has been recently validated for predicting inhibitor risk. Because of the lack of useful data regarding the role of different types of FVIII concentrates, the stratification of risk in patients starting replacement treatment together with the careful evaluation of indications, doses and duration of treatment at first exposures and further efforts for overcoming barriers to early implementation of prophylaxis are encouraged, particularly for patients with a predictable high inhibitor risk. Approximately 30% of severe haemophilic patients generates antibodies (inhibitors) against therapeutically administered factor VIII (FVIII), typically during the first 20 exposure days (ED) . Inhibitor development remains the most serious and challenging complication of modern treatment of haemophilia A in developed countries , where safe FVIII concentrates are largely available and where prophylaxis is increasingly used to prevent arthropathy.
36, 37 We aimed to gain more insight into the biological significance of shedding of the TNFR1 ectodomain in these pathologies by studying the extent to which ectodomain shedding of the TNFR1 controls the initiation
and progression of NAFLD towards NASH and the development of insulin resistance. Using knockin mice expressing a mutated nonsheddable TNFR1,29 we demonstrated for the first time that ectodomain shedding of the TNFR1 is not an essential feedback mechanism in preventing the development of hepatic steatosis and insulin resistance. However, this mechanism of the TNFα-inflammatory loop is pivotal for protecting against the transition from “simple steatosis” towards NASH. We have shown that p55Δns/Δns mice on a normal R428 chow diet do not develop hepatic steatosis, www.selleckchem.com/products/DAPT-GSI-IX.html despite increased hepatic inflammation (Fig. 2E). Moreover, 12 weeks of HF feeding did
not exacerbate hepatic lipid levels, nor alter the zonal distribution or severity of microvesicular steatosis in p55Δns/Δns mice compared to controls (Fig. 2D-F), suggesting that shedding of TNFR1 does not prevent the development of hepatic steatosis. It was known that the shedding of TNFR1 ectodomains attenuates the inflammatory response induced by TNFα,23 but our data now show that persistent TNFR1 signaling is not involved in the initiation of NAFLD. Consistent with this, mice with a genetic deletion of TNFα or TNFR1 are not protected against developing obesity-induced hepatic steatosis.10, 13, 14 However, TNFα has been shown to be a potent lipid metabolism regulator38 and many studies in rodents have described a role for TNFα in the development of hepatic steatosis.8, 33 Most of these have studied the effects of TNFα within 24 hours of a high PI-1840 dose of human recombinant TNFα. Although administration of TNFα induces acute hepatitis, it does not mimic the chronic low-grade
inflammation associated with obesity. The inflammatory gene expression seen in livers from p55Δns/Δns mice was approximately 10- to 100-fold lower than that seen after a single injection with TNFα (Supporting Fig. 1); it thus led to a more physiologically relevant situation of chronic low-grade hepatic inflammation in our study. Although our data do not support a role for shedding of TNFR1 in the initiation of steatosis, we did see an advanced NASH-like phenotype in the livers of p55Δns/Δns mice fed an HFD compared to wildtype mice. This included the presence of inflammatory infiltrates, apoptotic hepatocytes, and large areas of hepatocellular necrosis surrounded by neutrophils and lymphocytes (Fig. 3A,B). Because our data indicated an important role for TNFR1 in the progression of NAFLD towards NASH, we investigated the effect of ectodomain shedding of TNFR1 on hepatic fibrosis, an advanced hallmark of NASH. P55Δns/Δns mice demonstrated increased levels of collagen staining, as detected by Masson’s Trichrome staining (Fig. 4E).
Blockade of HMGB1-RAGE interaction has been shown to effectively reduce liver damage upon acute injury.14, 15, 49 Unfortunately, Hmgb1−/− mice die at birth50 and, hitherto, no conditional Hmgb1−/− mouse has been established to test whether HMGB1 ablation in see more the Mdr2−/− mouse phenocopies the dKO liver phenotype. Furthermore, it will be of great interest to correlate the expression of RAGE and the abundance of its ligands, in particular HMGB1, with the severity of disease and its clinical outcome in different human liver disorders, and to prove the concept that pharmacological inhibition of RAGE
signaling represents a novel strategy for the prevention of HCC development during early stages of liver injury. We thank Tine Bauer, Angelika Krischke, and Sandra Prokosch for technical support, Prof. George Yeoh (University of Western Australia) and Janina Tirnitz-Parker for BMOL cells, and Valentina Factor (NIH) for the A6 antibody. Additional Supporting Information may be found in the online version of this article. “
“Fafi-Kremer S, Fofana I, Soulier E, Carolla P, Meuleman P, Leroux-Roels G, et al. Viral entry and escape from antibody-mediated neutralization http://www.selleckchem.com/products/chir-99021-ct99021-hcl.html influence hepatitis
C virus reinfection in liver transplantation. J Exp Med 2010;207:2019-2031. (Reprinted with permission.) End-stage liver disease caused by chronic hepatitis C virus (HCV) infection is a leading cause for liver transplantation (LT). Due to viral evasion from host immune responses and the absence of preventive antiviral strategies, reinfection of the graft is universal. The mechanisms by which the virus evades host immunity to reinfect the
liver graft are unknown. In a longitudinal analysis of six HCV-infected patients undergoing LT, we demonstrate that HCV variants reinfecting the liver graft were characterized by efficient entry and poor neutralization by antibodies present in pretransplant serum compared with variants not detected after transplantation. Monoclonal antibodies directed against HCV envelope glycoproteins or a cellular entry factor efficiently cross-neutralized infection of human hepatocytes by patient-derived viral isolates that were resistant to autologous host-neutralizing responses. These findings provide significant insights into the molecular mechanisms Ribonucleotide reductase of viral evasion during HCV reinfection and suggest that viral entry is a viable target for prevention of HCV reinfection of the liver graft. Recurrent hepatitis C after orthotopic liver transplantation (OLT) for hepatitis C–associated end-stage liver disease or hepatocellular carcinoma is a vexing clinical problem. Rapid reinfection of the liver graft by hepatitis C virus (HCV) particles in the blood is nearly universal, and the ensuing disease often runs an accelerated course quickly progressing to graft cirrhosis and retransplantation or death.1 In contrast to hepatitis B, no passive immunoprophylaxis is currently available.
The introduction of a new generation of products with different treatment profiles warrants the prospective collection of data in children to determine safety profiles for immunogenicity as well as for pharmacokinetics in prophylactic regimens. Another complication is the large interlaboratory variability of the inhibitor assay. Although the Nijmegen modification of the Bethesda assay was promising, recent results of external validation
studies still demonstrate up to 50% difference between the test results of a single sample [20, 21]. This has increased the demand for central testing, but equally important is that laboratory results need to be confirmed in a second SRT1720 datasheet independent sample and in the presence of reduced recovery before a diagnosis of an inhibitor can be made . The conflicting results presented in the Wight and
Paisley paper were a reason for the members of the European Pediatric Network (PedNet) to include all children diagnosed Pexidartinib cost in their centres into a prospective registry. The PedNet registry started in 2004 and is ongoing, collecting data on all reasons for exposure during the first 75 exposure days. It has now prospectively collected data on > 700 children with severe haemophilia A and B. By collecting data of complete age cohorts, patient outcomes and treatment regimens are more comparable. The number of exclusions is very small (96% of all eligible patients with severe haemophilia diagnosed in the centres were included); for the patients born between 2000 and 2009 only 4% of all data on the first 75 exposure days in severe haemophilia were missing (K. Fischer et al., personal communication). The development of an inhibitor is the result of complex interactions between the patient’s immune system and genetic and environmental factors . Much has been learned by combining treatment-related risk factors and genetic factors. Interesting work has been done to unravel the complex immune regulatory genes and their interplay . The reported
increase in inhibitor development needs critical consideration. We have demonstrated that from 2000 onward over 10% of all inhibitors are of low titre. There is an urgent need to investigate the clinical importance Uroporphyrinogen III synthase of these low-titre inhibitors; whether they need immune tolerance induction therapy or just disappear without causing problems. If it is true that the majority of these low-titre inhibitors are found only because of more frequent and more sensitive testing, having no relation to increased bleeding tendency, the definition of inhibitors might have to be reconsidered. As is well documented in many studies, different mutations in the factor VIII gene have different risk profiles for inhibitor development [3-5]. However, in a well-defined cohort of patients with severe haemophilia, almost 60% have large gene defects and therefore have comparable inhibitor risk.
ATP8B1 deficiency constitutes a potentially lethal form of intrahepatic cholestasis. We and others have previously identified many distinct mutations in ATP8B1.1, 3, 11, 17–19 Correlations between missense mutations and phenotypes of individual patients Dactolisib cost remained limited, because most mutations are confined to only few patients and because of the high variation in penetrance and clinical presentation. Furthermore, the molecular consequence of ATP8B1 mutations remained largely unexplored. Here, we combined protein expression and localization studies with homology modeling to demonstrate the effects of selected ATP8B1 mutations on the protein
level. These studies have high relevance for the patient population affected with ATP8B1 deficiency, because three of the selected mutations—p.G308V detected in Amish families, p.D554N in Greenland Inuit, and p.I661T in most European BRIC1 patients—are the most frequently identified mutations, together affecting the vast majority of
all patients characterized with ATP8B1 deficiency. Although our data do not fully explain the large variability in clinical presentation, they demonstrate that ATP8B1 deficiency presents as a protein folding disease, for a surprisingly large majority of the selected mutations. This conclusion is supported by several lines of evidence. First, with the exception of ATP8B1 L127P, all ATP8B1 mutants displayed significantly reduced protein ALK inhibitor expression, whereas mRNA expression was unaffected. Second, the recovery of ATP8B1 mutant expression upon MG132 and epoxomycin Calpain treatment indicates that increased proteasomal degradation is a common consequence of these ATP8B1
mutations. Third, incubation at reduced temperature has been demonstrated to restore proper folding of mutated proteins, and increased ATP8B1 mutant expression was observed when cells were cultured at 30°C. Fourth, most ATP8B1 mutants showed minimal plasma membrane localization. Instead, they were retained in the ER. Fifth, homology modeling predicted significant changes in the ATP8B1 structure due to the various mutations. In conclusion, in most cases, ATP8B1 deficiency is a consequence of protein misfolding, resulting in reduced expression at the plasma membrane. In vitro, a further reduction of ATP8B1 I661T protein abundance at the cell surface occurs when cells are cultured at 40°C. This may suggest that temporary decrease in ATP8B1 abundance at the plasma membrane could trigger the onset of a cholestatic episode in BRIC1 patients afflicted with the p.I661T mutation, because patients report that episodes are sometimes preceded by fever. Current treatment of ATP8B1 deficiency has major obstacles. Reduction of the (hydrophobic) bile salt pool using ursodeoxycholate or cholestyramine is only rarely effective.2 Surgical and/or endoscopic drainage of bile salts is more successful, but involves invasive procedures.
Brushing was performed in a toothbrushing machine (Pepsodent) with a soft brush and a suspension of toothpaste and distilled water for 300 minutes, representing 6 years of brushing. Weight was measured initially and after the trial period; roughness was measured after the trial period only. The results of roughness and weight loss were analyzed using ANOVA and Tukey
tests at 5%. Results: The negative control (2.82 ± 4.41 mg) showed the lowest weight loss. Experimental 1 (13.62 ± 4.29 mg) and Experimental 2 (15.4 ± 5.80 mg) were equal statistically, and Sorriso (23.22 ± 7.23 mg) and Corega (28.83 ± 6.34 mg) produced the greatest weight loss. Concerning roughness, the negative control group
(0.03 ± 0.01 μm) showed the lowest value. No significant differences were found between Corega (13.43 ± 1.65 μm), Experimental 1 (12.28 ± 0.85 μm), and PLX3397 in vivo Experimental 2 (10.68 ± 2.56 μm). The Sorriso toothpaste produced the greatest amount of surface roughness (19.15 ± 2.36 μm). Conclusion: Of the tested dentifrices, the experimental preparations proved to be the least abrasive and resulted in the lowest weight loss after brushing of the acrylic. Based on these findings, the use of these experimental dentifrices is advocated. Further evaluation based on the ability of these preparations to remove biofilms is learn more required. “
“Immediate load protocols for the edentulous mandible offer the patient 4-Aminobutyrate aminotransferase many advantages in terms of
decreased number of visits, improved early function, and reduction of surgical exposure; however, this treatment modality is not universally appropriate for all patients. The available evidence will assist the clinician in developing a customized and comprehensive informed consent. Patient selection and patient-mediated factors will dictate the suitability of not only a fixed or removable prosthesis, but also whether immediate loading would enhance the cost/benefit ratio. The indications, objective and subjective patient considerations, and design strategies are discussed for the immediate load scenario. “
“Purpose: Candida albicans is the predominant oral yeast associated with denture-induced stomatitis, and with an increasing population of denture wearers its incidence is increasing. Maintaining good oral and denture hygiene, through chemical and/or mechanical intervention, is essential to reducing this disease. The aim of this study, using a robust adherent C. albicans cell model system, was to evaluate and compare the efficacy of a novel denture cleanser to the efficacy of a commonly used dentifrice coupled with brushing. Materials and Methods: Four C.
05) showed that Kooliner was significantly affected by all disinfection
cycles (p < 0.05) when compared with baseline measurements. New Truliner resin was significantly affected by three and four cycles of microwave disinfection when compared with baseline measurements (p < 0.05). For Tokuyama Rebase II, Ufi Gel hard, and Lucitone 550, no significant dimensional changes were found. Conclusions: Microwave disinfection promoted shrinkage of Kooliner and New Truliner. The dimensional stability of Tokuyama Rebase II, Ufi Gel Hard, and Lucitone 550 was not affected by microwave disinfection. "
“Severe bilateral cleft-lip/palate patients are difficult to manage even if nasoalveolar molding therapy is advocated before surgical repair. A 5-day-old male infant with bilateral cleft-lip-palate was managed with the nasoalveolar molding technique. Periodic adjustments of the Selleckchem GSK1120212 appliance were continued every week to mold the nasoalveolar complex into the desired shape for the 5 months of infancy. The cleft width of 12 mm on the right and 14 mm on the left side was completely reduced, and the Ku-0059436 in vitro absent columella was lengthened to 6 mm with the active molding appliance. The horizontal bar of the nasal stent of the appliance was modified by adding an additional 1 mm layer of resilient liner on the tissue surface to achieve rapid columellar lengthening.
In severe bilateral cleft-lip/palate cases, simple modifications in the appliance can achieve rapid results. “
“Purpose: Previous studies considering retention of cast metal restorations to implant abutments incorporated some degree of frictional fit due to internal surface nodules and
roughness of the restoration. In comparison, CAD/CAM restorations have minimal surface irregularities, possibly impacting retention. There is insufficient knowledge of retentive force of CAD/CAM restorations to titanium abutments, and therefore the topic warrants further investigation. This in vitro study investigated the retention of all-ceramic CAD/CAM restorations to three different prefabricated implant abutments using five different cements. Materials and Methods: A total of 150 Astra Tech dental implant abutments were used, with each group of 50 being subdivided into five groups selleck chemical of 10. An optical impression of each size of abutment was made with the CEREC 3D intraoral camera. A full-coverage restoration was designed and milled with an enlarged, conical-shaped occlusal surface, which served to secure the restoration into a brass jig used with a universal testing machine. Five different cements were used with three different-sized abutments. Following cementation, the implant/abutment/restoration assemblies were stored for 24 hours at 37°C in 100% humidity. A pull-out test using a universal testing machine, set at a 0.5 mm/min crosshead speed, was used to evaluate retention of the individual restorations. The load required to remove each all-ceramic restoration was recorded.
His vital signs were within normal limit. He drug discovery had diffuse abdominal tenderness, especially in left upper quadrant and guarding. The laboratory findings were not significant. The CT showed 15 cm length intestinal wall
edematous enlargement at jejunum and high density area at mesentery around jejunum and ascites at Douglas cavum. He was radiologically diagnosed with small intestinal anisakiasis. It was resolved spontaneously in a few days. Conclusion: Discussion: Acute gastric anisakiasis can be easily diagnosed by the endoscopic visualization of Anisakis larvae along with mucosal edema, erythema, hemorrhage, and/or an ulcer. However, small intestinal anisakiasis Pexidartinib molecular weight is difficult to diagnose because we cannot endoscope it easily. The CT scan typically showed severe intestinal submucosal edema with ascites. The small intestinal anisakisis should be considered by the food history and the typical CT finding. If strongly suspected, small intestinal anisakaisis can be treated without surgery because the larvae will die within a few days and the symptoms will subside soon. Key Word(s): 1. Anisakiasis Presenting Author: OSAMU OGAWA Additional Authors: YUGO SUZUKI, AKIRA MATSUI, TOSHIFUMI MITANI, SHU HOTEYA, MITSURU
KAISE Corresponding Author: OSAMU OGAWA Affiliations: Toranomon Hospital, Toranomon Hospital, Toranomon Hospital, Toranomon Hospital, Toranomon Hospital Objective: Gastric adenocarcinoma of fundic grand type (GAFG) is neoplastic lesion mainly composed of highly differentiated columnar cells mimicking the fundic gland cells with nuclear atypia. It has been reported as a new, rare variant of gastric adenocarcinoma. Therefore, its endoscopic features are uncertain. The aim of the current study was to evaluate the endoscopic features of GAFG. Methods: From October 2012 to March 2013, three
consecutive patients with GAFG resected by endoscopic submucosal dissection (ESD) in our hospital were enrolled in this retrospective study. These specimens resected by ESD revealed well-differentiated adenocarcinoma mimicking fundic gland cells, which were positive for pepsinogen-1 GNAT2 (a marker of chief cells) and MUC6 (a marker of fundic gland cells). These findings were consistent with GAFG. To evaluate the endoscopic features of GAFG, they were examined for their location, background mucosa, shape, color, and size. Results: All three GAFGs were in the upper part of the stomach. In the background mucosa, all they had normal fundic gland mucosa without atrophic change. And all they had whitish submucosal tumor shape with dilated branching vessel, ranging in size from 5.0 to 6.0 mm (mean, 5.1 mm). Conclusion: Precise understanding of these endoscopic features must enhance efficacious detection of GAFG in endoscopic surveillance. Key Word(s): 1.
To target IFNγ to HSC, we modified IFNγ with PDGFβR-recognizing cyclic peptide (PPB) using different conjugation strategies as illustrated in Fig. 1E. PPB was directly conjugated to IFNγ (IFNγ-PPB) or by way of a 2 kDa hydrophilic hetero-bifunctional PEG linker (IFNγ-PEG-PPB). In addition, we synthesized IFNγ-PEG as a control. The synthesis details are illustrated in Supporting Fig. 1. The synthesized conjugates were characterized by western blot analyses with anti-IFNγ and anti-PPB antibodies (Supporting Fig. 2). Because chemical modifications of cytokines can
diminish their biological activity, click here we examined the activity of the IFNγ conjugates compared to unmodified IFNγ
in mouse RAW macrophages. These cells express the IFNγR but lack PDGFβR. IFNγ and its constructs IFNγ-PPB, IFNγ-PEG, and IFNγ-PEG-PPB all induced a similar dose-dependent increase in nitric oxide (NOx) release in RAW cells (Fig. 1F). There was no significant difference in dose-response slopes, demonstrating that all IFNγ conjugates retained full biological activity. IFNγ binds to its receptor, which is strictly species-specific, whereas PDGFβR binding is not. In order to discriminate between IFNγR- and PDGFβR-mediated http://www.selleckchem.com/products/Lapatinib-Ditosylate.html bindings, we used mouse NIH3T3 fibroblasts, primary rat HSC, and human LX2 hepatic stellate cells. The results Thiamine-diphosphate kinase confirmed the species specificity of IFNγ; mouse IFNγ and mouse derived IFNγ-PEG showed binding to mouse 3T3 fibroblasts (Fig. 2A) but not to rat HSC and human HSC (Fig. 2B; Supporting Fig. 3). However, PPB-modified mouse IFNγ conjugates showed high binding to mouse, rat, and human cells (Fig. 2A,B; Supporting Fig. 3), which was almost completely blocked with anti-PDGFβR IgG (Fig. 2B). This demonstrates the
specific binding of PPB-modified IFNγ constructs to PDGFβR, which is species-nonspecific. Subsequently, we investigated the antifibrotic effects of the constructs in mouse 3T3 fibroblasts and in human HSC after their activation with TGFβ. Both mouse IFNγ and IFNγ conjugates induced significant reduction in collagen expression in mouse cells (Fig. 2C,D). In addition, mouse IFNγ and IFNγ conjugates inhibited PDGF-induced cell proliferation in 3T3 fibroblasts as assessed by thymidine incorporation assays (Fig. 2E). Interestingly, in human LX2 cells, TGFβ1-induced collagen expression was strongly inhibited by treatment with the PDGFR-specific IFNγ constructs (Fig. 2C,D), whereas unmodified mouse IFNγ and IFNγ-PEG did not induce any effect in human cells due to species differences. These results clearly demonstrate that mouse IFNγ, which is inactive in human cells, can become biologically active in other species by directing it to the PDGFβR.