Reciprocal natural hybridization between Lycoris aurea and Lycoris radiata (Amaryllidaceae) identified by morphological, karyotypic and chloroplast genomic data.

BACKGROUND: Hybridization is considered as an important model of speciation, but the evolutionary process of natural hybridization is still poorly characterized in Lycoris. To reveal the phylogenetic relationship of two new putative natural hybrids in Lycoris, morphological, karyotypic and chloroplast genomic data of four Lycoris species were analyzed in this study. RESULTS: Two putative natural hybrids (2n = 18 = 4 m + 5t + 6st + 3 T) possessed obvious heterozygosity features of L. radiata (2n = 22 = 10t + 12st) and L. aurea (2n = 14 = 8 m + 6 T) in morphology (e.g. leaf shape and flower color), karyotype (e.g. chromosome numbers, CPD/DAPI bands, 45S rDNA-FISH signals etc.) and chloroplast genomes. Among four Lycoris species, the composition and structure features of chloroplast genomes between L. radiata and the putative natural hybrid 1 (L. hunanensis), while L. aurea and the hybrid 2, were completely the same or highly similar, respectively. However, the features of the cp genomes between L. radiata and the hybrid 2, while L. aurea and the hybrid 1, including IR-LSC/SSC boundaries, SSRs, SNPs, and SNVs etc., were significantly different, respectively. Combining the karyotypes and cp genomes analysis, we affirmed that the natural hybrid 1 originated from the natural hybridization of L. radiata (♀) × L. aurea (♂), while the natural hybrid 2 from the hybridization of L. radiata (♂) × L. aurea (♀). CONCLUSION: The strong evidences for natural hybridization between L. radiata (2n = 22) and L. aurea (2n = 14) were found based on morphological, karyotypic and chloroplast genomic data. Their reciprocal hybridization gave rise to two new taxa (2n = 18) of Lycoris. This study revealed the origin of two new species of Lycoris and strongly supported the role of natural hybridization that facilitated lineage diversification in this genus.

Bispecific Aptamer-Based Recognition-then-Conjugation Strategy for PD1/PDL1 Axis Blockade and Enhanced Immunotherapy.

Cytotoxic T cells initiate antitumor effects mainly through direct interactions with tumor cells. As a counter to this, tumor cells can put the brakes on such T-cell activity via specific linkage between programmed death ligand 1 (PDL1) and its receptor programmed cell death protein 1 (PD1). Bispecific inhibitors that enabled synchronous blockade of PD1 and PDL1, thereby releasing the brakes on T-cell antitumor activity, should significantly improve the efficacy of immune checkpoint blockade (ICB) therapy. In this work, we identified a DNA aptamer, Ap3, that could specifically recognize PDL1 on tumor cells and competed with the binding of PD1. By integrating Ap3 with an anti-PD1 aptamer, the bispecific aptamer Ap3-7c was constructed, and it showed promise for improving the T-cell immune response. We further designed a dibenzocyclooctyne (DBCO)-labeled bispecific aptamer, D-Ap3-7c, allowing covalent conjugation of aptamers onto PD1 and PDL1 after specific cell recognition. Our in vivo studies showed that this recognition-then-conjugation strategy could induce a potent immunological effect against tumors. This work is expected to provide clues for antitumor immunotherapy.

Co-Expression of Pig IL-2 and Fusion Bovine Cathelicidin Gene by Recombinant Plasmids in Yeast and Their Promotion of Mouse Antibacterial Defense.

In order to develop an effective and safe immunomodulator to enhance the antimicrobial bioactivity and immunity of animals against infectious bacterial diseases, a recombinant plasmid pGAPZαA-IL2-B co-expressing pig interleukin-2 (PIL-2) and fused bovine cathelicidin (FBC) genes were constructed using the 2A self-cleavage technique. After being expressed in Pichia pastoris strain SMD1168, the recombinant yeast was administered orally to 5-week-old female ICR mice. The control mice were similarly dosed with P. pastoris with a blank plasmid or FBC recombinant plasmid alone. At 28 days post-treatment, the mice were challenged intraperitoneally with virulent strains of either E. coli or S. aureus. Compared with the control groups, the mice that received recombinant yeast co-expressing PIL-2/FBC manifested significant increases in the number of leukocytes, CD4+ and CD8+ T cells, IgG, and the gene expressions of TLRs(TLR1,4,6,9), antimicrobial peptides(CRP4 and CRAMP) and cytokines (IL-2, 4, 6, 7, 12, 15, 23, IFN-γ, and TNF-α) in the blood. Furthermore, the treated mice displayed significantly higher survival than the other two control groups after the challenge. These results suggest that the antimicrobial activity and immunity of animals can be effectively enhanced by the in vivo co-expression of IL-2 and the FBS gene, which can facilitate the development of new immunopotentiation molecules to overcome the infection of antibiotic-resistant bacteria.

Combination predicting model of traffic congestion index in weekdays based on LightGBM-GRU.

Tree-based and deep learning methods can automatically generate useful features. Not only can it enhance the original feature representation, but it can also learn to generate new features. This paper develops a strategy based on Light Gradient Boosting Machine (LightGBM or LGB) and Gated Recurrent Unit (GRU) to generate features to improve the expression ability of limited features. Moreover, a SARIMA-GRU prediction model considering the weekly periodicity is introduced. First, LightGBM is used to learn features and enhance the original features representation; secondly, GRU neural network is used to generate features; finally, the result ensemble is used as the input for prediction. Moreover, the SARIMA-GRU model is constructed for predicting. The GRU prediction consequences are revised by the SARIMA model that a better prediction can be obtained. The experiment was carried out with the data collected by Ride-hailing in Chengdu, and four predicted indicators and two performance indexes are utilized to evaluate the model. The results validate that the model proposed has significant improvements in the accuracy and performance of each component.

Elucidation and Structural Modeling of CD71 as a Molecular Target for Cell-Specific Aptamer Binding.

Pancreatic cancer is a highly lethal malignancy associated with tissues of the pancreas. Early diagnosis and effective treatment are crucial to improving the survival rate of patients with pancreatic cancer. In a previous study, we employed the cell-SELEX strategy to obtain an ssDNA aptamer termed XQ-2d with high binding affinity for pancreatic cancer. Here, we first identify CD71 as the XQ-2d-binding target. We found that knockdown of CD71 abolished the binding of XQ-2d and that the binding affinity of XQ-2d is associated with membrane-bound CD71, rather than total CD71 levels. Competitive analysis revealed that XQ-2d shares the same binding site on CD71 with transferrin (Tf), but not anti-CD71 antibody. We then used a surface energy transfer (SET) nanoruler to measure the distance between the binding sites of XQ-2d and anti-CD71 antibody, and it was about 15 nm. Furthermore, we did molecular dynamics simulation to clarify that the spatial structure of XQ-2d and Tf competitively binding to CD71. We also engineered XQ-2d-mediated targeted therapy for pancreatic cancer, using an XQ-2d-based complex for loading doxorubicin (Dox). Because CD71 is overexpressed not only in pancreatic cancer but also in a variety of tumors, our work provides a systematic novel way of studying a potential biomarker and also promising tools for cancer diagnosis and therapy, opening new doors for effective cancer theranostics.

Identification of Vigilin as a Potential Ischemia Biomarker by Brain Slice-Based Systematic Evolution of Ligands by Exponential Enrichment.

Stroke is one of the leading causes of disability and death among adults worldwide and results in numerous biochemical alterations. However, few efficient biomarkers are clinically available to diagnose stroke because of the limitations of biomarkers and their probes. In this work, we utilized frozen brain slices of middle cerebral artery occlusion (MCAO) in a mouse model of ischemia to select a specific binding aptamer, termed LCW17, by tissue-based SELEX (systematic evolution of ligands by exponential enrichment). LCW17 was enhanced in binding in ischemic brain slices compared to sham control. We identified the binding target of LCW17 as vigilin. Vigilin is increased in ischemia brain slices and exhibits enhanced release from cultured hippocampal neurons after oxygen glucose deprivation in vitro. Taken together, ischemic brain slice-based aptamer selection will enable identification of more probes and potential target molecules for diagnosis and therapy of ischemic stroke. Aptamer LCW17 and vigilin may potentially be applied to define the molecular mechanism underlying ischemic stroke, as well as its diagnosis.

Enolase1 Alleviates Cerebral Ischemia-Induced Neuronal Injury via Its Enzymatic Product Phosphoenolpyruvate.

Stroke is a leading cause of disability and the second leading cause of death among adults worldwide, while the mechanisms underlying neuronal death and dysfunction remain poorly understood. Here, we investigated the differential proteomic profiles of mouse brain homogenate with 3 h of middle cerebral artery occlusion (MCAO) ischemia, or sham, using Coomassie Brilliant Blue staining, followed by mass spectrometry. We identified enolase1 (ENO1), a key glycolytic enzyme, as a potential mediator of neuronal injury in MCAO ischemic model. Reverse transcription polymerase chain reaction and western blotting data showed that ENO1 was ubiquitously expressed in various tissues, distinct regions of brain, and different postnatal age. Immunohistochemical analysis revealed that ENO1 is localized in neuronal cytoplasm and dendrites. Interestingly, the expression level of ENO1 was significantly increased in the early stage, but dramatically decreased in the late stage, of cerebral ischemia in vivo. This dynamic change was consistent with our finding in cultured hippocampal neurons treated with oxygen/glucose deprivation (OGD) in vitro. Importantly, ENO1 overexpression in cultured neurons alleviated dendritic and spinal loss caused by OGD treatment. Furthermore, the enzymatic product of ENO1, phosphoenolpyruvate (PEP), was also synchronously changed along with the dynamic ENO1 level. The neuronal injury caused by OGD treatment in vitro or ischemia in vivo was mitigated by the application of PEP. Taken together, our data revealed that ENO1 plays a novel and protective role in cerebral ischemia-induced neuronal injury, highlighting a potential of ENO1 as a therapeutic target of neuronal protection from cerebral ischemia.

Seroprevalence of severe fever with thrombocytopenia syndrome virus antibodies among inhabitants of Dachen Island, eastern China.

Severe fever with thrombocytopenia syndrome virus (SFTSV) has been emerging and has caused many human cases in China, Japan and Korea. Some studies speculated that SFTSV was transmitted with bird migration among these countries. Notably, SFTS cases have been identified in a Chinese island named Dachen which is situated southwest of Japan and Korea. In this study, we conducted a serum survey of SFTSV antibodies among inhabitants of the island. A total of 439 serum specimens were collected in June 2018. All serum samples were tested for total antibodies and IgM antibody with double-antigen sandwich ELISA method. The rates of seropositivity for SFTSV total antibodies and IgM antibody were 3.0% (95% CI 1.4-4.6) and 0.5% (2/439), respectively. The median age of all participants was 61 years and all seropositive samples were all from inhabitants aged >50 years. The differences of seroprevalence between different gender groups and different age groups were not significant. However, seroprevalence varied significantly among different villages (P = 0.033). Our results showed that some inhabitants of Dachen Island had been infected with SFTSV, and some ticks and host animals of the island carry SFTSV. Comprehensive measures should be conducted to prevent the occurrence of SFTS cases in the island.

Improved expression of recombinant fusion defensin gene plasmids packed with chitosan-derived nanoparticles and effect on antibacteria and mouse immunity.

In order to develop a secure and competent technique to express the human immune gene for fighting infections, we cloned and expressed the BD2/3 using VR1020 (a eukaryotic expression plasmid). BD2/3 contains human ß-defensin 2 (BD2) and human BD3. To explore safe and effective DNA delivery molecules in vitro and in vivo, the fusion genes of BD2/3 were used as an immune-labelled gene to verify transfection effectivness of modified chitosan (CS). Plasmid of VR1020-BD2/3 was packed with biomaterials: CS, average molecular weight: 25000D; polyethylene glycol-O-chitosan-polyethylenimine (PEG-O-CS-PEI); liposomes (LP); polyamine cationic liposomes (PCL); polyamine cationic liposomes of protamine (PCL-protamine) by ionotropic gelation. We observed that BD2/3 fusion gene showed high bioactivity in vitro and in vivo. The BD2/3 fusion protein inhibited the proliferation of bacteria (S. aureus, S. pneumoniae, P. aeruginosa and E. coli). The Kunming mice were immune to these nanoparticles and we analyzed their delivery efficiency and gene expression effect. BD2/3 results in multiple changes of innate and required immune system of mice. BD2/3 increases expression of IgG, IgG1, IgG2a, IL-2, IL-6, IFN-γ, as well as of lymphocytes and monocytes. Following challenge with virulent E. coli, CD4+ and CD8+ positive T-cell counts were highly elevated in the BD2/3 immunized mice, resulting in higher survival rates of mice. These results indicate that nanoparticles containing modified CS and BD2/3 are potentially safe and effective drugs in vivo to improve the immunity against bacterial infection and enhance innate immunity and adaptive immunity against infectious diseases.

Molecular Recognition and In-Vitro-Targeted Inhibition of Renal Cell Carcinoma Using a DNA Aptamer.

Renal cell carcinoma (RCC) is the most common malignant tumor of the urinary system, and it has a high frequency of local invasion and distant metastasis. Although multiple advances have been made in the diagnosis and therapy of RCC, the vast majority of patients with metastatic RCC remain incurable. In this study, an aptamer named SW-4 against RCC 786-O cells was identified from a known sequence pool. The identified aptamer exhibited high binding affinity for target cells with dissociation constants in the nanomolar range. Binding analysis revealed that SW-4 only bound to RCC 786-O cells, but not HEK293T cells or human proximal tubular HK-2 cells, indicating that SW-4 has excellent binding selectivity. By sequence optimization, the 26-nt truncated SW-4b demonstrated improved binding affinity, and it was internalized into target cells via caveolae-mediated endocytosis in a temperature-dependent manner. Furthermore, fluorescence imaging confirmed that SW-4b accumulated at tumor sites in 786-O xenograft nude mice models and specifically recognized clinical RCC tissues. Meanwhile, SW-4b inhibited proliferation of 786-O cells by arresting cell cycle progression at the S phase. Taken together, these results indicate that SW-4b is a potential candidate for development into a novel tool for diagnosis and targeted therapy of RCC.

Two winter PM2.5 pollution types and the causes in the city clusters of Sichuan Basin, Western China.

PM2.5 pollution types were analyzed and the causes were uncovered in Sichuan Basin using PM2.5 data from Chinese Ministry of Environmental Protection (MEP) and multiple meteorology data during January 2015 to February 2017. The events that PM2.5 increased gradually in the periods longer than 10 days and then decreased sharply were defined as "Type I", while the symmetrical variations of PM2.5 during increasing and decreasing periods were defined as "Type II" of PM2.5 pollution. Five cases of Type I and two cases of Type II were identified during the study period inside the basin. The increasing rates were almost comparable between the two PM2.5 pollution types with the range from 4 µg m-3 d-1 to 8 µg m-3 d-1, while the decreasing rates of Type I were between 25 µg m-3 d-1 and 40 µg m-3 d-1, which were 3-5 times higher than those of Type II (~8 µg m-3 d-1). The rapid reduction of PM2.5 for Type I was mainly related to improvement of vertical and horizontal diffusion conditions induced by invasion of cold air masses, while slowly decreased PM2.5 for Type II was due largely to elevated horizontal wind speeds and shifted wind directions in the city clusters of the basin.

Deubiquitylation and stabilization of p21 by USP11 is critical for cell-cycle progression and DNA damage responses.

p21WAF1/CIP1 is a broad-acting cyclin-dependent kinase inhibitor. Its stability is essential for proper cell-cycle progression and cell fate decision. Ubiquitylation by the multiple E3 ubiquitin ligase complexes is the major regulatory mechanism of p21, which induces p21 degradation. However, it is unclear whether ubiquitylated p21 can be recycled. In this study, we report USP11 as a deubiquitylase of p21. In the nucleus, USP11 binds to p21, catalyzes the removal of polyubiquitin chains conjugated onto p21, and stabilizes p21 protein. As a result, USP11 reverses p21 polyubiquitylation and degradation mediated by SCFSKP2, CRL4CDT2, and APC/CCDC20 in a cell-cycle-independent manner. Loss of USP11 causes the destabilization of p21 and induces the G1/S transition in unperturbed cells. Furthermore, p21 accumulation mediated by DNA damage is completely abolished in cells depleted of USP11, which results in abrogation of the G2 checkpoint and induction of apoptosis. Functionally, USP11-mediated stabilization of p21 inhibits cell proliferation and tumorigenesis in vivo. These findings reveal an important mechanism by which p21 can be stabilized by direct deubiquitylation, and they pinpoint a crucial role of the USP11-p21 axis in regulating cell-cycle progression and DNA damage responses.

The Receptor-Like Cytoplasmic Kinase STRK1 Phosphorylates and Activates CatC, Thereby Regulating H2O2 Homeostasis and Improving Salt Tolerance in Rice.

Salt stress can significantly affect plant growth and agricultural productivity. Receptor-like kinases (RLKs) are believed to play essential roles in plant growth, development, and responses to abiotic stresses. Here, we identify a receptor-like cytoplasmic kinase, salt tolerance receptor-like cytoplasmic kinase 1 (STRK1), from rice (Oryza sativa) that positively regulates salt and oxidative stress tolerance. Our results show that STRK1 anchors and interacts with CatC at the plasma membrane via palmitoylation. CatC is phosphorylated mainly at Tyr-210 and is activated by STRK1. The phosphorylation mimic form CatCY210D exhibits higher catalase activity both in vitro and in planta, and salt stress enhances STRK1-mediated tyrosine phosphorylation on CatC. Compared with wild-type plants, STRK1-overexpressing plants exhibited higher catalase activity and lower accumulation of H2O2 as well as higher tolerance to salt and oxidative stress. Our findings demonstrate that STRK1 improves salt and oxidative tolerance by phosphorylating and activating CatC and thereby regulating H2O2 homeostasis. Moreover, overexpression of STRK1 in rice not only improved growth at the seedling stage but also markedly limited the grain yield loss under salt stress conditions. Together, these results offer an opportunity to improve rice grain yield under salt stress.

How many proteins can be identified in a 2DE gel spot within an analysis of a complex human cancer tissue proteome?

Two-dimensional gel electrophoresis (2DE) in proteomics is traditionally assumed to contain only one or two proteins in each 2DE spot. However, 2DE resolution is being complemented by the rapid development of high sensitivity mass spectrometers. Here we compared MALDI-MS, LC-Q-TOF MS and LC-Orbitrap Velos MS for the identification of proteins within one spot. With LC-Orbitrap Velos MS each Coomassie Blue-stained 2DE spot contained an average of at least 42 and 63 proteins/spot in an analysis of a human glioblastoma proteome and a human pituitary adenoma proteome, respectively, if a single gel spot was analyzed. If a pool of three matched gel spots was analyzed this number further increased up to an average of 230 and 118 proteins/spot for glioblastoma and pituitary adenoma proteome, respectively. Multiple proteins per spot confirm the necessity of isotopic labeling in large-scale quantification of different protein species in a proteome. Furthermore, a protein abundance analysis revealed that most of the identified proteins in each analyzed 2DE spot were low-abundance proteins. Many proteins were present in several of the analyzed spots showing the ability of 2DE-MS to separate at the protein species level. Therefore, 2DE coupled with high-sensitivity LC-MS has a clearly higher sensitivity as expected until now to detect, identify and quantify low abundance proteins in a complex human proteome with an estimated resolution of about 500 000 protein species. This clearly exceeds the resolution power of bottom-up LC-MS investigations.

Co-expression of mFat-1 and pig IGF-1 genes by recombinant plasmids in modified chitosan nanoparticles and its synergistic effect on mouse immunity.

To develop a cost-effective molecular regulator to improve growth metabolism and immunity of animals, a recombinant plasmid co-expressing fatty acid desaturase (mFat-1) and pig insulin growth like factor 1 (IGF-1) genes was constructed by the 2 A self-cleavage technique. After entrapment within modified chitosan nanoparticles (chitosan modified with polyethyleneglycol-polyethylenimine, CPP), the recombinant plasmid was injected intramuscularly into mice. Compared with controls, co-expression of mFat-1 and IGF-1 significantly raised the level of serum IGF-1, and increased the liver and muscle docosa hexaenoic acid (DHA) content. Th and Tc cell levels were also elevated, as were expression levels of serum IL-4 and IL-6 genes. These results demonstrate that the immunity and metabolism of an animal can be effectively improved by co-expression of mFat-1 and IGF-1 genes in vivo, which may contribute to further development of novel immunomodulators with beneficial effects on growth metabolism and immunity.

Selection and characterization of DNA aptamer against glucagon receptor by cell-SELEX.

Excessive secretion of glucagon, a functional insulin antagonist, significantly contributes to hyperglycemia. Glucagon exerts its physiological functions through activation of the glucagon receptor (GCGR). Inhibition of GCGR activity represents a potential therapeutic approach for reducing excess glucose production in diabetes mellitus. Aptamers are short DNA or RNA oligonucleotides evolved from systematic evolution of ligands by exponential enrichment (SELEX). Here, we have successfully selected a DNA aptamer against GCGR by cell-SELEX, which can specifically bind membrane protein of CHO-GCGR cells with a K d of 52.7 ± 5.1 nM. Aptamer-mediated pull-down and gcgr knockdown assay verified that GCGR was the target of aptamer GR-3. Binding analysis revealed that GR-3 could recognize other cells with different affinity according to the level of GCGR protein expressed in these cells. Hepatic tissue imaging suggested that GR-3 could bind the cell membrane of hepatic tissues. With the advantages of small size, high binding affinity, good stability, lack of immunogenicity, and easy synthesis, aptamer GR-3 against GCGR can be a promising tool with the potential to attenuate hyperglycemia in diabetes mellitus.

Long-Term Durability of Basalt Fiber-Reinforced Polymer (BFRP) Sheets and the Epoxy Resin Matrix under a Wet⁻Dry Cyclic Condition in a Chloride-Containing Environment.

Basalt fiber-reinforced polymer (BFRP) composites are receiving increasing attention as they represent a low-cost green source of raw materials. FRP composites have to face harsh environments, such as chloride ions in coastal marine environments or cold regions with salt deicing. The resistance of FRPs subjected to the above environments is critical for the safe design and application of BFRP composites. In the present paper, the long-term durability of BFRP sheets and the epoxy resin matrix in a wet⁻dry cyclic environment containing chloride ions was studied. The specimens of the BFRP sheet and epoxy resin matrix were exposed to alternative conditions of 8-h immersion in 3.5% NaCl solution at 40 °C and 16-h drying at 25 °C and 60% relative humidity (RH). The specimens were removed from the exposure chamber at the end of the 180th, 270th and 360th cycles of exposure and were analyzed for degradation with tensile tests, scanning electron microscopy (SEM) and void volume fractions. It was found that the tensile modulus of the BFRP sheet increased by 3.4%, and the tensile strength and ultimate strain decreased by 45% and 65%, respectively, after the 360th cycle of exposure. For the epoxy resin matrix, the tensile strength, tensile modulus and ultimate strain decreased by 27.8%, 3.2% and 64.8% after the 360th cycle of exposure, respectively. The results indicated that the degradation of the BFRP sheet was dominated by the damage of the interface between the basalt fiber and epoxy resin matrix. In addition, salt precipitate accelerated the fiber⁻matrix interfacial debonding, and hydrolysis of the epoxy resin matrix resulted in many voids, which accelerated the degradation of the BFRP sheet.

Determination of trace thiophanate-methyl and its metabolite carbendazim with teratogenic risk in red bell pepper (Capsicumannuum L.) by surface-enhanced Raman imaging technique.

Surface-enhanced Raman scattering (SERS) imaging coupling with multivariate analysis in spectral region of 200 to 1800cm-1 was developed to quantify and visualize thiophanate-methyl (TM) and its metabolite carbendazim residues in red bell pepper (Capsicum annuum L.). Least squares support vector machines (LS-SVM) and support vector machines (SVM) models based on seven optimized characteristic peaks that showed SERS effects of TM and its metabolite carbendazim residues were employed to establish prediction models. SERS spectra with first derivative (1st) and second derivative (2nd) method were subsequently compared and the optimized model of 1st-LS-SVM acquired showed the best performance (RPD=6.08, R2P=0.986 and RMSEP=0.473). The results demonstrated that SERS imaging with multivariate analysis had the potential for rapid determination and visualization of the trace TM and its metabolite carbendazim residues in complex food matrices.

Unexpected role for p19INK4d in posttranscriptional regulation of GATA1 and modulation of human terminal erythropoiesis.

Terminal erythroid differentiation is tightly coordinated with cell-cycle exit, which is regulated by cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors (CDKI), yet their roles in erythropoiesis remain to be fully defined. We show here that p19INK4d, a member of CDKI family, is abundantly expressed in erythroblasts and that p19INK4d knockdown delayed erythroid differentiation, inhibited cell growth, and led to increased apoptosis and generation of abnormally nucleated late-stage erythroblasts. Unexpectedly, p19INK4d knockdown did not affect cell cycle. Rather, it led to decreased expression of GATA1 protein. Importantly, the differentiation and nuclear defects were rescued by ectopic expression of GATA1. Because the GATA1 protein is protected by nuclear heat shock protein family (HSP) member HSP70, we examined the effects of p19INK4d knockdown on HSP70 and found that p19INK4d knockdown led to decreased expression of HSP70 and its nuclear localization. The reduced levels of HSP70 are the result of reduced extracellular signal-regulated kinase (ERK) activation. Further biochemical analysis revealed that p19INK4d directly binds to Raf kinase inhibitor PEBP1 and that p19INK4d knockdown increased the expression of PEBP1, which in turn led to reduced ERK activation. Thus we have identified an unexpected role for p19INK4d via a novel PEBP1-p-ERK-HSP70-GATA1 pathway. These findings are likely to have implications for improved understanding of disordered erythropoiesis.

Receptor kinase complex transmits RALF peptide signal to inhibit root growth in Arabidopsis.

A number of hormones work together to control plant cell growth. Rapid Alkalinization Factor 1 (RALF1), a plant-derived small regulatory peptide, inhibits cell elongation through suppression of rhizosphere acidification in plants. Although a receptor-like kinase, FERONIA (FER), has been shown to act as a receptor for RALF1, the signaling mechanism remains unknown. In this study, we identified a receptor-like cytoplasmic kinase (RPM1-induced protein kinase, RIPK), a plasma membrane-associated member of the RLCK-VII subfamily, that is recruited to the receptor complex through interacting with FER in response to RALF1. RALF1 triggers the phosphorylation of both FER and RIPK in a mutually dependent manner. Genetic analysis of the fer-4 and ripk mutants reveals RIPK, as well as FER, to be required for RALF1 response in roots. The RALF1-FER-RIPK interactions may thus represent a mechanism for peptide signaling in plants.