ATR2C ala2 from Arabidopsis-infecting downy mildew requires 4 TIR-NLR immune receptors for full recognition.

Arabidopsis Col-0 RPP2A and RPP2B confer recognition of Arabidopsis downy mildew (Hyaloperonospora arabidopsidis [Hpa]) isolate Cala2, but the identity of the recognized ATR2Cala2 effector was unknown. To reveal ATR2Cala2, an F2 population was generated from a cross between Hpa-Cala2 and Hpa-Noks1. We identified ATR2Cala2 as a non-canonical RxLR-type effector that carries a signal peptide, a dEER motif, and WY domains but no RxLR motif. Recognition of ATR2Cala2 and its effector function were verified by biolistic bombardment, ectopic expression and Hpa infection. ATR2Cala2 is recognized in accession Col-0 but not in Ler-0 in which RPP2A and RPP2B are absent. In ATR2Emoy2 and ATR2Noks1 alleles, a frameshift results in an early stop codon. RPP2A and RPP2B are essential for the recognition of ATR2Cala2. Stable and transient expression of ATR2Cala2 under 35S promoter in Arabidopsis and Nicotiana benthamiana enhances disease susceptibility. Two additional Col-0 TIR-NLR (TNL) genes (RPP2C and RPP2D) adjacent to RPP2A and RPP2B are quantitatively required for full resistance to Hpa-Cala2. We compared RPP2 haplotypes in multiple Arabidopsis accessions and showed that all four genes are present in all ATR2Cala2-recognizing accessions.

Factors Affecting Adherence to National Colorectal Cancer Screening: A 12-Year Longitudinal Study Using Multi-Institutional Pooled Data in Korea.

BACKGROUND: Consistent uptake of colorectal cancer (CRC) screening is important to reduce the incidence and mortality from advanced-stage CRC and increase the survival rate of the patients. We conducted a longitudinal study to determine the factors affecting CRC screening compliance in Korean adults using individual-level linked data from the Korean National Health and Nutrition Examination Survey, Korean National Health Insurance Service, and Korean Health Insurance Review and Assessment Service. METHODS: We selected 3,464 adults aged 50-79 years as the study population and followed them for 12 years (January 2007-December 2018). The outcome variable was the level of adherence to CRC screening, categorized as nonadherent, intermittently adherent, and consistently adherent. An ordinal logistic regression model was designed to determine the socioeconomic factors, family history of CRC, and medical conditions that could facilitate the consistent uptake of CRC screening. RESULTS: The results showed a significant and positive association between consistent uptake of CRC screening and the 100-150% income category (odds ratio [OR], 1.710; 95% confidence interval [CI], 1.401-2.088); clerical, sales and service job category (OR, 1.962; 95% CI, 1.582-2.433); residency at medium-sized cities (OR, 1.295; 95% CI, 1.094-1.532); high-school graduation (OR, 1.440; 95% CI, 1.210-1.713); married status (OR, 2.281; 95% CI, 1.946-2.674); use of employment-based national health insurance (OR, 1.820; 95% CI, 1.261-2.626); use of private insurance (OR, 2.259; 95% CI, 1.970-2.589); no disability (OR, 1.428; 95% CI, 1.175-1.737); family history of CRC (OR, 2.027; 95% CI, 1.514-2.714); and history of colorectal neoplasm (OR, 1.216; 95% CI; 1.039-1.422). CONCLUSION: The lack of regular participation in CRC screening programs in the Republic of Korea was found to be an issue that requires attention. Policies on CRC screening must place increased emphasis on strengthening educational and public relations initiatives.

The Effect of the Mixed Extract of Kalopanax pictus Nakai and Achyranthes japonica Nakai on the Improvement of Degenerative Osteoarthritis through Inflammation Inhibition in the Monosodium Iodoacetate-Induced Mouse Model.

Osteoarthritis is a chronic inflammatory disease, and, due to the lack of fundamental treatment, the main objective is to alleviate pain and prevent cartilage damage. Kalopanax pictus Nakai and Achyranthes japonica Nakai are herbal plants known for their excellent anti-inflammatory properties. The objective of this study is to confirm the potential of a mixture extract of Kalopanax pictus Nakai and Achyranthes japonica Nakai as a functional raw material for improving osteoarthritis through anti-inflammatory effects in macrophages and MIA-induced arthritis experimental animals. In macrophages inflamed by lipopolysaccharide (LPS), treatment of Kalopanax pictus Nakai and Achyranthes japonica Nakai mixture inhibits NF-κB and mitogen-activated protein kinase (MAPK) activities, thereby inhibiting inflammatory cytokine tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6), inflammatory factors PGE2, MMP-2, and MMP-9, and nitric oxide (NO) was reduced. In addition, in an animal model of arthritis induced by MIA (monosodium iodoacetate), administration of Kalopanax pictus Nakai and Achyranthes japonica Nakai mixture reduced blood levels of inflammatory cytokines TNF-α and IL-6, inflammatory factors prostaglandin E2(PGE2), matrix metalloproteinase-2(MMP-2), and NO. Through these anti-inflammatory effects, MIA-induced pain reduction (recovery of clinical index, increase in weight bearing, and increase in area and width of the foot), recovery of meniscus damage, loss of cartilage tissue or inflammatory cells in tissue infiltration reduction, and recovery of the proteglycan layer were confirmed. Therefore, it is considered that Kalopanax pictus Nakai and Achyranthes japonica Nakai mixture has the potential as a functional raw material that promotes joint health.

The Arabidopsis WRR4A and WRR4B paralogous NLR proteins both confer recognition of multiple Albugo candida effectors.

The oomycete Albugo candida causes white blister rust, an important disease of Brassica crops. Distinct races of A. candida are defined by their capacity to infect different host plant species. Each A. candida race encodes secreted proteins with a CX2 CX5 G ('CCG') motif that are polymorphic and show presence/absence variation, and are therefore candidate effectors. The White Rust Resistance 4 (WRR4) locus in Arabidopsis thaliana accession Col-0 contains three genes that encode intracellular nucleotide-binding domain leucine-rich repeat immune receptors. The Col-0 alleles of WRR4A and WRR4B confer resistance to multiple A. candida races, although both WRR4A and WRR4B can be overcome by the Col-0-virulent race 4 isolate AcEx1. Comparison of CCG candidate effectors in avirulent and virulent races, and transient co-expression of CCG effectors from four A. candida races in Nicotiana sp. or A. thaliana, revealed CCG effectors that trigger WRR4A- or WRR4B-dependent hypersensitive responses. We found eight WRR4A-recognised CCGs and four WRR4B-recognised CCGs, the first recognised proteins from A. candida for which the cognate immune receptors in A. thaliana are known. This multiple recognition capacity potentially explains the broad-spectrum resistance to several A. candida races conferred by WRR4 paralogues. We further show that of five tested CCGs, three confer enhanced disease susceptibility when expressed in planta, consistent with A. candida CCG proteins being effectors.

Plastid-mediated RNA interference: A potential strategy for efficient pest control.

RNA interference (RNAi) technology is a promising and effective approach for pest insect management. Owing to its sequence-guided working mechanism, RNAi has a high degree of species-selectivity, thus minimizing potential adverse effects on nontarget organisms. Recently, engineering plastid (chloroplast) genome, rather than the nuclear genome, to produce double-stranded RNAs has emerged as a powerful way to protect plants from multiple arthropod pests. Here, we review the recent progresses in the plastid-mediated RNAi (PM-RNAi) approach for pest control and the factors influencing its efficacy, and propose the strategies for further efficiency improvement. We also discuss the current challenges and the biosafety-related issues of PM-RNAi technology that need to be addressed for commercial production.

Positional effects of double-stranded RNAs targeting ß-Actin gene affect RNA interference efficiency in Colorado potato beetle.

Pesticide resistance in pests drives the development of RNA interference (RNAi)-based technology as a novel approach for pest control. To investigate the effects of the positional dependency of double-stranded RNAs (dsRNAs), we newly designed four different 200 bp dsRNAs targeting Colorado potato beetle (CPB) ß-Actin gene, termed as dsACT200-1 to dsACT200-4, to compare their insecticidal activity to CPB larvae together with our previously used 200 bp and 700 bp dsRNAs (dsACT200 and dsACT700), respectively (He et al., 2020a). Each of dsRNAs harbors different numbers of expected siRNAs predicted by sequence-based prediction platform, dsACT200 and dsACT200-2 have a relatively higher number of siRNA than other 200 bps dsRNAs. When CPB larvae were fed with in vitro synthesized dsRNA-painted potato leaves, all the tested dsRNAs showed significant effects to protect against CPB larvae. Combined with the survival rate of CPB larvae, ß-Actin gene expression level and the surviving CPB larvae weight, various positional dsRNAs from the same allele showed different plant protection activity against CPB larvae and partially correlated with the predicted siRNA numbers and distribution on the target sequence. This study suggests the specific allelic locus for rational dsRNA design triggering RNAi efficiency for target gene silencing is an essential factor in enhancing the insecticidal activity.

NFIB induces functional astrocytes from human pluripotent stem cell-derived neural precursor cells mimicking in vivo astrogliogenesis.

The ability to generate astrocytes from human pluripotent stem cells (hPSCs) offers a promising cellular model to study the development and physiology of human astrocytes. The extant methods for generating functional astrocytes required long culture periods and there remained much ambiguity on whether such paradigms follow the innate developmental program. In this report, we provided an efficient and rapid method for generating physiologically functional astrocytes from hPSCs. Overexpressing the nuclear factor IB in hPSC-derived neural precursor cells induced a highly enriched astrocyte population in 2 weeks. RNA sequencing and functional analyses demonstrated progressive transcriptomic and physiological changes in the cells, resembling in vivo astrocyte development. Further analyses substantiated previous results and established the MAPK pathway necessary for astrocyte differentiation. Hence, this differentiation paradigm provides a prospective in vitro model for human astrogliogenesis studies and the pathophysiology of neurological diseases concerning astrocytes.

Risk factors for rebleeding in Crohn's disease patients with acute severe lower gastrointestinal bleeding: With special reference to the role of anti-tumor necrosis factor therapy.

BACKGROUND AND AIM: Acute severe lower gastrointestinal bleeding (LGIB) in patients with Crohn's disease (CD) is uncommon; however, it is a potentially life-threatening complication, and its recurrence is common. We thus aimed to identify the predictors for rebleeding in CD patients with acute severe LGIB and particularly focused on whether anti-tumor necrosis factor (TNF) therapy lowers the risk of rebleeding compared with conventional medical therapy (CMT) or surgery. METHODS: The risk of rebleeding was analyzed in 131 CD patients with acute severe LGIB. Patients were classified into the CMT group (n = 99), anti-TNF therapy group (n = 22), and surgery group (n = 10). No patients in the surgery group received anti-TNF therapy. RESULTS: During the median follow-up of 98 months after the first episode of acute severe LGIB, rebleeding occurred in 50.5%, 18.2%, and 30.0% of the CMT group, anti-TNF therapy group, and surgery group, respectively (P = 0.015). The cumulative risks of rebleeding at 1 and 10 years were 20.0% and 64.7% in the CMT group, 13.6% and 18.4% in the anti-TNF therapy group, and 0% and 40.7% in the surgery group, respectively (P = 0.020). Multivariable Cox regression analysis showed that anti-TNF therapy was associated with a lower risk of rebleeding compared with CMT (hazard ratio, 0.303; 95% confidence interval, 0.108-0.849; P = 0.023). CONCLUSIONS: In CD patients with acute severe LGIB, anti-TNF therapy may reduce the risk of rebleeding compared with CMT. Although surgery is considered effective in preventing early rebleeding, concomitant anti-TNF therapy may be helpful in further lowering the long-term risk of rebleeding.

Chronic Viral Hepatitis Is Associated with Colorectal Neoplasia: A Systematic Review and Meta-Analysis.

BACKGROUND: Chronic viral hepatitis is associated with a wide range of extrahepatic diseases; however, evidence on a link between chronic viral hepatitis and colorectal neoplasia is still lacking. AIMS: To analyze the association between chronic viral hepatitis and prevalence of colorectal neoplasia. METHODS: A systematic review of articles published in the MEDLINE, EMBASE, and Cochrane Library between 2000 and 2020 was performed. Subgroup analyses based on the types of colorectal neoplasia and the etiology of chronic viral hepatitis were conducted. RESULTS: Twelve eligible studies with 48,428 hepatitis B virus (HBV) patients and 46,561 hepatitis C virus (HCV) patients were included. Chronic viral hepatitis was significantly associated with an increased risk of both colorectal adenoma (odds ratio [OR], 1.53; 95% confidence interval [CI], 1.16-2.02; I2 = 83%) and colorectal cancer (CRC) (OR, 1.32; 95% CI, 1.08-1.61; I2 = 94%). The etiology of chronic viral hepatitis was an independent factor related to heterogeneity for CRC subgroup analysis revealed an increased risk of CRC in both HBV (OR, 1.18; 95% CI, 1.09-1.27; I2 = 37%) and HCV (OR, 1.88; 95% CI, 1.78-1.97; I2 = 0%). HCV was associated with an increased risk of colorectal adenoma (OR, 1.48; 95% CI, 1.22-1.79; I2 = 0%); however, HBV was not associated with an increased risk of colorectal adenoma and had considerable heterogeneity (OR, 1.65; 95% CI, 0.88-3.09; I2 = 90%). CONCLUSION: Our meta-analysis showed that chronic viral hepatitis is associated with an increased risk of colorectal neoplasia. The strategy of stricter screening colonoscopy may benefit from patients with chronic viral hepatitis.

Maintenance and differentiation of human ES cells on polyvinylidene fluoride scaffolds immobilized with a vitronectin-derived peptide.

Polyvinylidene fluoride (PVDF) is biocompatible, easy to fabricate, and has piezoelectric properties; it has been used for many biomedical applications including stem cell engineering. However, long-term cultivation of human embryonic stem cells (hESCs) and their differentiation toward cardiac lineages on PVDF have not been investigated. Herein, PVDF nanoscaled membrane scaffolds were fabricated by electrospinning; a vitronectin-derived peptide-mussel adhesive protein fusion (VNm) was immobilized on the scaffolds. hESCs cultured on the VNm-coated PVDF scaffold (VNm-PVDF scaffold) were stably expanded for more than 10 passages while maintaining the expression of pluripotency markers and genomic integrity. Under cardiac differentiation conditions, hESCs on the VNm-PVDF scaffold generated more spontaneously beating colonies and showed the upregulation of cardiac-related genes, compared with those cultured on Matrigel and VNm alone. Thus, VNm-PVDF scaffolds may be suitable for the long-term culture of hESCs and their differentiation into cardiac cells, thus expanding their application in regenerative medicine.

Microneedle Array Patch (MAP) Consisting of Crosslinked Hyaluronic Acid Nanoparticles for Processability and Sustained Release.

AIMS: Crosslinked hyaluronic acid (X-linked HA) is not suitable for making microneedles because of the low fluidity of X-linked HA hydrogel. Microneedles were fabricated using X-linked HA nanoparticles (X-linked HA-NPs) to utilize the sustained drug delivery capability of X-linked HA-NPs and to obtain the processability advantages of X-linked HA. METHOD: The puncture performance of a microneedle array patch (MAP) made of crosslinked hyaluronic acid nanoparticles (X-linked HA-NP-MAP) was evaluated by insertion in vitro into porcine skin. After a predetermined attachment time, the remaining height of the X-linked HA-NP-MAP was measured to determine the dissolution rate. X-linked HA-NP-MAP and free HA-MAP containing Rhodamine B isothiocyanate-dextran were administered into the back skin of mice, and the relative fluorescent intensity in the back skin was measured over time. RESULTS: The puncture performance of the X-linked HA-NP-MAP was over 90%. The diameter of redispersed X-linked HA-NPs was same as that of the premolded X-linked HA-NPs. The dissolution rate was not different from that of free HA-MAP. In an in vivo experiment, X-linked HA-NP-MAP was administered into the mouse's back skin successfully and the relative fluorescent intensity of X-linked HA-NP-MAP lasted longer than that of HA-MAP. CONCLUSION: X-linked HA-NPs provide the biocompatibility, the processability of micromolding, sustained drug release, successful penetration into the skin, and relatively short insertion time for full disintegration of NPs in the skin. X-linked HA-NP-MAP can be used for various applications that require several days of sustained drug release.

Diverse NLR immune receptors activate defence via the RPW8-NLR NRG1.

Most land plant genomes carry genes that encode RPW8-NLR Resistance (R) proteins. Angiosperms carry two RPW8-NLR subclasses: ADR1 and NRG1. ADR1s act as 'helper' NLRs for multiple TIR- and CC-NLR R proteins in Arabidopsis. In angiosperm families, NRG1 co-occurs with TIR-NLR Resistance (R) genes. We tested whether NRG1 is required for signalling of multiple TIR-NLRs. Using CRISPR mutagenesis, we obtained an nrg1a-nrg1b double mutant in two Arabidopsis accessions, and an nrg1 mutant in Nicotiana benthamiana. These mutants are compromised in signalling of all TIR-NLRs tested, including WRR4A, WRR4B, RPP1, RPP2, RPP4 and the pairs RRS1/RPS4, RRS1B/RPS4B, CHS1/SOC3 and CHS3/CSA1. In Arabidopsis, NRG1 is required for the hypersensitive cell death response (HR) and full oomycete resistance, but not for salicylic acid induction or bacterial resistance. By contrast, nrg1 loss of function does not compromise the CC-NLR R proteins RPS5 and MLA. RPM1 and RPS2 (CC-NLRs) function is slightly compromised in an nrg1 mutant. Thus, NRG1 is required for full TIR-NLR function and contributes to the signalling of some CC-NLRs. Some NRG1-dependent R proteins also signal partially via the NRG1 sister clade, ADR1. We propose that some NLRs signal via NRG1 only, some via ADR1 only and some via both or neither.

Development of Botulinum Toxin A-Coated Microneedles for Treating Palmar Hyperhidrosis.

Hyperhidrosis is a disorder that is characterized by the production of excess amounts of sweat. The botulinum neurotoxin A (BoNT/A) has been used to treat hyperhidrosis through multiple intradermal injections at the site of the condition. However, because of BoNT/A toxicity, it is important to precisely deliver the proper dose of the toxin to the target site. In addition, the use of a conventional hypodermic needle for multiple injections in the palm makes the approach undesirable and painful. Here, we designed a BoNT/A-coated microneedle (BoNT-MN) array and tested its efficacy as a substitute pain-free method to treat hyperhidrosis. BoNT-MNs were prepared by coating polylactic acid microneedles with a BoNT/A formulation and were found to successfully penetrate into a thick skin in vitro. The coating formulations were then tested for their stability at 4, 25, and 37 °C for 24 h. BoNT-MNs were found to be much more stable than BoNT/A in a liquid state. Additionally, we carried out in vivo experiments by treating the right paws of mice with BoNT-MNs and found that the treatment induced a significant reduction in the sweating response in the mouse foot pad. Thus, BoNT/A treatment using microneedles is beneficial and may be used as a more efficient and less painful approach to treat hyperhidrosis.

Effect of Slurry Viscosity and Dispersant Agent on the Sintering of 3Y-TZP Ceramics Fabricated by Slip Casting.

Dental zirconia implants are typically manufactured by mechanical machining of a zirconia block into a tooth shape. Many cracks are created by this machining process on the surface of the green body, which induces deterioration of mechanical strength and reliability of the sintered zirconia implant. In this study, we fabricated a dense zirconia specimen by slip casting and sintering. The zirconia slurry for slip casting was prepared by mixing yttria-stabilized zirconia powder with an average particle size of 20 nm, distilled water, and dispersant. Slurry viscosity was controlled by varying pH, dispersant concentration, and solid content; the lower viscosity being achieved at pH 11 controlled by ammonium hydroxide. With a dispersant content of 0.4-0.8 wt%, the viscosity was reduced from 190 cP to 40 cP at pH 11. After casting and sintering at 1550 °C for 2 h, the sintered body reached a density of 5.70-6.01 g/cm³ and a grain size of 300-700 nm, depending on the slurry preparation conditions.

Effect of Solid Loading on the Sintered Properties of 3 mol% Yttria-Stabilized Tetragonal Zirconia Polycrystals (3Y-TZP) Ceramics via Slip Casting.

Dental zirconia implants fabricated by the mechanical machining and sintering of zirconia blocks have many surface cracks that lead to the deterioration of mechanical strength and the failure of the implant in the body. In this study, we attempted to manufacture an extremely dense and crack-free zirconia specimen by slip casting and pressureless sintering. After the preparation of zirconia slurry by control of its viscosity and by solid loading, highly dense zirconia specimens could be obtained by pressureless sintering at 1450 °C for 2 h. Slurry viscosity was controlled by adjusting the mixing ratio of 3Y-TZP powder, a dispersant, and a pH adjustment agent. Highly dense 3Y-TZP specimens with a relative density of 99% and small grain size of 200-400 nm could be obtained at a solid loading of 50-65 wt%. An optimally dense specimen was fabricated from zirconia slurry with 60 wt% solid loading that had the highest apparent density of 6.07 g/cm³ (99.5%).

Arabidopsis downy mildew effector HaRxL106 suppresses plant immunity by binding to RADICAL-INDUCED CELL DEATH1.

The oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) causes downy mildew disease on Arabidopsis. To colonize its host, Hpa translocates effector proteins that suppress plant immunity into infected host cells. Here, we investigate the relevance of the interaction between one of these effectors, HaRxL106, and Arabidopsis RADICAL-INDUCED CELL DEATH1 (RCD1). We use pathogen infection assays as well as molecular and biochemical analyses to test the hypothesis that HaRxL106 manipulates RCD1 to attenuate transcriptional activation of defense genes. We report that HaRxL106 suppresses transcriptional activation of salicylic acid (SA)-induced defense genes and alters plant growth responses to light. HaRxL106-mediated suppression of immunity is abolished in RCD1 loss-of-function mutants. We report that RCD1-type proteins are phosphorylated, and we identified Mut9-like kinases (MLKs), which function as phosphoregulatory nodes at the level of photoreceptors, as RCD1-interacting proteins. An mlk1,3,4 triple mutant exhibits stronger SA-induced defense marker gene expression compared with wild-type plants, suggesting that MLKs also affect transcriptional regulation of SA signaling. Based on the combined evidence, we hypothesize that nuclear RCD1/MLK complexes act as signaling nodes that integrate information from environmental cues and pathogen sensors, and that the Arabidopsis downy mildew pathogen targets RCD1 to prevent activation of plant immunity.

Evaluation of estrogenic potential by herbal formula, HPC 03 for in vitro and in vivo.

HPC 03 is herbal formula that consists of extracts from Angelica gigas, Cnidium officinale Makino and Cinnamomum cassia Presl. The present study evaluated the estrogenic potential of HPC 03 by using in vitro and in vivo models. The regulatory mechanisms of HPC 03 in estrogen-dependent MCF-7 cells were assessed. HPC 03 induced the proliferation of estrogen receptor-positive MCF-7 cells, and the proliferation was blocked by the addition of the estrogen antagonist tamoxifen. The estrogen receptorα/ß luciferase activities were significantly increased by HPC 03 treatment, which also increased the mRNA expression of the estrogen-responsive genes Psen2, Pgr and Ctsd Also, we evaluated the ameliorative effects of HPC 03 on menopausal symptoms in ovariectomized rats. HPC 03 treatment in OVX rats significantly affected the uterine weight, increased the expression of estrogen-responsive genes Pgr and Psen2 in uterus, increased bone mineral density loss in the femur and inhibited body weight increase. Serum E2, collagen type 1 and osteocalcin were significantly increased, while serum LH, FSH and ALP were decreased compared with OVX rats. HPC 03 may be a promising candidate for the treatment of menopause, but further research is necessary to determine whether the observed effects also occur in humans.

Synthesis of Nanoscale Fully Stabilized Zirconia Powders by Urea Hydrolysis, Sintering and Electrical Characterization.

Active nanoscale powders of cubic phase zirconia stabilized with yttria, gadolinia, and scandia were successfully prepared by urea hydrolysis. Synthetic cubic zirconia powders had homogeneous, nanoscale, and less-agglomeration characteristics. Dense pellets of grain size about 0.4 µm exhibited grain boundary blocking resistance compared to the high frequency bulk resistance. Gadolinium doped system exhibited highest ionic resistivity. Yttria stabilized zirconia by urea hydrolysis in this work showed smaller ionic resistivity than the sample prepared from the commercial powder.

Scopoletin Stimulates Melanogenesis via cAMP/PKA Pathway and Partially p38 Activation.

Scopoletin was recently shown to stimulate melanogenesis through cAMP-response element-binding protein (CREB) phosphorylation. In this study, we investigated the molecular events of melanogenesis-induced by scopoletin. After exposure to scopoletin, the protein levels of tyrosinase and tyrosianse related protein-1 (TRP-1) were significantly increased in B16F10 cells. The mRNA levels of tyrosinase and microphthalmia-associated transcription factor (MITF) were also enhanced by scopoletin. cAMP production and phosphorylation of p38 mitogen-activated protein kinase (MAPK) were increased by scopoletin treatment. Scopoletin-mediated increase of intracellular melanin and tyrosinase expression were significantly attenuated by protein kinase A (PKA) inhibitors (H-89 and KT5720), while a protein kinase C (PKC) inhibitor (Ro-32-0432) had no effect and a p38 MAPK inhibitor (SB203580) partially blocked the scopoletin-induced intracellular melanin and tyrosinase expression. Moreover, scopoletin synergistically with cell-permeable cAMP analog (dibutyryl cAMP) significantly induced tyrosinase activity and melanin content in B16F10 cells. The silencing of p38 MAPK by small interfering RNA (siRNA) decreased the scopoletin-induced tyrosinase expression in B16F10 cells. These results suggest that scopoletin could induce melanin synthesis through the cAMP/PKA pathway and partially p38 MAPK activation in B16F10 cells.

Pathogenesis-related protein 4b interacts with leucine-rich repeat protein 1 to suppress PR4b-triggered cell death and defense response in pepper.

To control defense and cell-death signaling, plants contain an abundance of pathogen recognition receptors such as leucine-rich repeat (LRR) proteins. Here we show that pepper (Capsicum annuum) LRR1 interacts with the pepper pathogenesis-related (PR) protein 4b, PR4b, in yeast and in planta. PR4b is synthesized in the endoplasmic reticulum, interacts with LRR1 in the plasma membrane, and is secreted to the apoplast via the plasma membrane. Binding of PR4b to LRR1 requires the chitin-binding domain of PR4b. Purified PR4b protein inhibits spore germination and mycelial growth of plant fungal pathogens. Transient expression of PR4b triggers hypersensitive cell death. This cell death is compromised by co-expression of LRR1 as a negative regulator in Nicotiana benthamiana leaves. LRR1/PR4b silencing in pepper and PR4b over-expression in Arabidopsis thaliana demonstrated that LRR1 and PR4b are necessary for defense responses to Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis (Hpa) infection. The mutant of the PR4b Arabidopsis ortholog, pr4, showed enhanced susceptibility to Hpa infection. Together, our results suggest that PR4b functions as a positive modulator of plant cell death and defense responses. However, the activity of PR4b is suppressed by interaction with LRR1.