[Role and mechanism of cysteine and glycine-rich protein 2 in the malignant progression of neuroblastoma].

OBJECTIVE: To investigate the function and underlying mechanism of cysteine and glycine-rich protein 2 (CSRP2) in neuroblastoma (NB). METHODS: The correlation between the expression level of CSRP2 mRNA and the prognosis of NB children in NB clinical samples was analyzed in R2 Genomics Analysis and Visualization Platform. The small interfering RNA (siRNA) targeting CSRP2 or CSRP2 plasmid were transfected to NB cell lines SK-N-BE(2) and SH-SY5Y. Cell proliferation was observed by crystal violet staining and real-time cellular analysis. The ability of colony formation of NB cells was observed by colony-forming unit assay. Immunofluorescence assay was used to detect the expression of the proliferation marker Ki-67. Flow cytometry analysis for cell cycle proportion was used with cells stained by propidium iodide (PI). Annexin V/7AAD was used to stain cells and analyze the percentage of cell apoptosis. The ability of cell migration was determined by cell wound-healing assay. The level of protein and mRNA expression of CSRP2 in NB primary tumor and NB cell lines were detected by Western blot and quantitative real-time PCR (RT-qPCR). RESULTS: By analyzing the NB clinical sample databases, it was found that the expression levels of CSRP2 in high-risk NB with 3/4 stages in international neuroblastoma staging system (INSS) were significantly higher than that in low-risk NB with 1/2 INSS stages. The NB patients with high expression levels of CSRP2 were shown lower overall survival rate than those with low expression levels of CSRP2. We detected the protein levels of CSRP2 in the NB samples by Western blot, and found that the protein level of CSRP2 in 3/4 INSS stages was significantly higher than that in 1/2 INSS stages. Knockdown of CSRP2 inhibited cell viability and proliferation of NB cells. Overexpression of CSRP2 increased the proliferation of NB cells. Flow cytometry showed that the proportion of sub-G1, G0/G1 and S phase cells and Annexin V positive cells were increased after CSRP2 deficiency. In the cell wound-healing assay, the healing rate of NB cells was significantly attenuated after knockdown of CSRP2. Further mechanism studies showed that the proportion of the proliferation marker Ki-67 and the phosphorylation levels of extracellular signal-regulated kinases 1/2 (ERK1/2) were significantly decreased after CSRP2 knockdown. CONCLUSION: CSRP2 is highly expressed in high-risk NB with 3/4 INSS stages, and the expression levels of CSRP2 are negatively correlated with the overall survival of NB patients. CSRP2 significantly increased the proliferation and cell migration of NB cells and inhibited cell apoptosis via the activation of ERK1/2. All these results indicate that CSRP2 promotes the progression of NB by activating ERK1/2, and this study will provide a potential target for high-risk NB therapy.

Diurnal regulation of alternative splicing associated with thermotolerance in rice by two glycine-rich RNA-binding proteins.

Rice (Oryza sativa L.) production is threatened by global warming associated with extreme high temperatures, and rice heat sensitivity is differed when stress occurs between daytime and nighttime. However, the underlying molecular mechanism are largely unknown. We show here that two glycine-rich RNA binding proteins, OsGRP3 and OsGRP162, are required for thermotolerance in rice, especially at nighttime. The rhythmic expression of OsGRP3/OsGRP162 peaks at midnight, and at these coincident times, is increased by heat stress. This is largely dependent on the evening complex component OsELF3-2. We next found that the double mutant of OsGRP3/OsGRP162 is strikingly more sensitive to heat stress in terms of survival rate and seed setting rate when comparing to the wild-type plants. Interestingly, the defect in thermotolerance is more evident when heat stress occurred in nighttime than that in daytime. Upon heat stress, the double mutant of OsGRP3/OsGRP162 displays globally reduced expression of heat-stress responsive genes, and increases of mRNA alternative splicing dominated by exon-skipping. This study thus reveals the important role of OsGRP3/OsGRP162 in thermotolerance in rice, and unravels the mechanism on how OsGRP3/OsGRP162 regulate thermotolerance in a diurnal manner.

Genetic variation in the glycine-rich protein gene BnGRP1 contributes to low phosphorus tolerance in Brassica napus.

Lack of phosphorus (P) is a major environmental factor affecting rapeseed (Brassica napus. L) root growth and development. For breeding purposes, it is crucial to identify the molecular mechanisms underlying root system architecture traits that confer low-P tolerance in rapeseed. Natural variations in the glycine-rich protein gene BnGRP1 were analysed in the natural population of 400 rapeseed cultivars under low-P stress through genome-wide association study and transcriptome analysis. Based on 11 single nucleotide polymorphism mutations in the BnGRP1 sequence, 10 haplotypes (Hap) were formed. Compared with the other types, the cultivar BnGRP1Hap1 in the panel demonstrated the longest root length and heaviest root weight. BnGRP1Hap1 overexpression in rapeseed led to enhanced low-P tolerance. CRISPR/Cas9-derived BnGRP1Hap4 knockout mutations in rapeseed can lead to sensitivity to low-P stress. Furthermore, BnGRP1Hap1 influences the expression of the phosphate transporter 1 gene (PHT1) associated with P absorption. Overall, the findings of this study highlight new insights into the mechanisms of GRP1 enhancement of low-P tolerance in rapeseed.

A rice seed-specific glycine-rich protein OsDOR1 interacts with GID1 to repress GA signaling and regulates seed dormancy.

Seed dormancy is an important agronomic trait under the control of complex genetic and environmental interactions, which have not been yet comprehensively understood. From the field screening of rice mutant library generated by a Ds transposable element, we identified a pre-harvest sprouting (PHS) mutant dor1. This mutant has a single insertion of Ds element at the second exon of OsDOR1 (LOC_Os03g20770), which encodes a novel seed-specific glycine-rich protein. This gene successfully complemented the PHS phenotype of dor1 mutant and its ectopic expression enhanced seed dormancy. Here, we demonstrated that OsDOR1 protein binds to the GA receptor protein, OsGID1 in rice protoplasts, and interrupts with the formation OsGID1-OsSLR1 complex in yeast cells. Co-expression of OsDOR1 with OsGID1 in rice protoplasts attenuated the GA-dependent degradation of OsSLR1, the key repressor of GA signaling. We showed the endogenous OsSLR1 protein level in the dor1 mutant seeds is significantly lower than that of wild type. The dor1 mutant featured a hypersensitive GA-response of α-amylase gene expression during seed germination. Based on these findings, we suggest that OsDOR1 is a novel negative player of GA signaling operated in the maintenance of seed dormancy. Our findings provide a novel source of PHS resistance.

Molecular Analysis of MgO Nanoparticle-Induced Immunity against Fusarium Wilt in Tomato.

Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici (FOL), is a devastating soilborne disease in tomatoes. Magnesium oxide nanoparticles (MgO NPs) induce strong immunity against Fusarium wilt in tomatoes. However, the mechanisms underlying this immunity remain poorly understood. Comparative transcriptome analysis and microscopy of tomato roots were performed to determine the mechanism of MgO NP-induced immunity against FOL. Eight transcriptomes were prepared from tomato roots treated under eight different conditions. Differentially expressed genes were compared among the transcriptomes. The Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that in tomato roots pretreated with MgO NPs, Rcr3 encoding apoplastic protease and RbohD encoding NADPH oxidase were upregulated when challenge-inoculated with FOL. The gene encoding glycine-rich protein 4 (SlGRP4) was chosen for further analysis. SlGRP4 was rapidly transcribed in roots pretreated with MgO NPs and inoculated with FOL. Immunomicroscopy analysis showed that SlGRP4 accumulated in the cell walls of epidermal and vascular vessel cells of roots pretreated with MgO NPs, but upon FOL inoculation, SlGRP4 further accumulated in the cell walls of cortical tissues within 48 h. The results provide new insights into the probable mechanisms of MgO NP-induced tomato immunity against Fusarium wilt.

Exposure to environmental pollutant bisphenol A causes oxidative damage and lipid accumulation in Zebrafish larvae: Protective role of WL15 peptide derived from cysteine and glycine-rich protein 2.

Humans are exposed to obesity causing Bisphenol A in various ways, especially through diet and food containers. Bioactive peptides are already reported to have antioxidant, antidiabetic, and antiobesity properties, which can mimic the role of mediators involved in obesity prevention. The protective effect of a short molecule or peptide, WL15 from cysteine and glycine-rich protein 2 of a teleost of aquatic resource on Bisphenol A (BPA)-induced lipid accumulation in zebrafish larvae was investigated. BPA exposure disrupted the antioxidant enzymes, apoptosis, and nitric oxide and led to changes in biochemical markers including alkaline phosphatase, lactate dehydrogenase, lipid peroxidation, glutathione S-transferases, glutathione peroxidase, and reduced glutathione. However, WL15 inhibited the overproduction of oxidative stress, which correlates with its lipid-lowering potential. BPA-induced lipid accumulation in zebrafish showed an increase in triglyceride, cholesterol, and glucose level; simultaneously, WL15 treatment significantly reduced such accumulation in zebrafish. Evidenced by Oil red O staining and Nile red assay, WL15 inhibited lipid accumulation. At the same time, WL15 at 50 µM increases 2-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)-2-deoxy-d-glucose (2NBDG) glucose uptake in zebrafish. In addition, gene expression studies in zebrafish larvae demonstrated that the WL15 peptide could play a crucial role in preventing lipid accumulation by downregulating the expression of lipogenesis-specific genes. These results revealed an interesting and novel property of WL15, suggesting its potential application in preventing lipid accumulation through the hypolipidemic and antioxidant properties.

Cysteine and glycine-rich protein 3 (Crp3) as a critical regulator of elastolysis, inflammation, and smooth muscle cell apoptosis in abdominal aortic aneurysm development.

Abdominal aortic aneurysm (AAA) is a life-threatening vascular disease for which surgical or endovascular repair are the only currently available therapeutic strategies. The development of AAA involves the breakdown of elastic fibers (elastolysis), infiltration of inflammatory cells, and apoptosis of smooth muscle cells (SMCs). However, the specific regulators governing these responses remain unknown. We previously demonstrated that Cysteine and glycine-rich protein 3 (Crp3) sensitizes SMCs to apoptosis induced by stretching. Building upon this finding, we aimed to investigate the influence of Crp3 on elastolysis and apoptosis during AAA development. Using the elastase-CaCl2 rat model, we observed an increase in Crp3 expression, aortic diameter, and a reduction in wall thickness in wild type rats. In contrast, Crp3-/- rats exhibited a decreased incidence of AAA, with minimal or no changes in aortic diameter and thickness. Histopathological analysis revealed the absence of SMC apoptosis and degradation of elastic fibers in Crp3-/- rats, accompanied by reduced inflammation and diminished proteolytic capacity in Crp3-/- SMCs and bone marrow-derived macrophages. Collectively, our findings provide evidence that Crp3 plays a crucial role in AAA development by modulating elastolysis, inflammation, and SMC apoptosis. These results underscore the potential significance of Crp3 in the context of AAA progression and offer new insights into therapeutic targets for this disease.

BcGRP23: A novel gene involved in the chlorophyll metabolic pathway that is activated by BES1 in flowering Chinese cabbage.

Glycine-rich proteins (GRPs) are a large family of proteins that play vital roles in cell wall remodeling, metabolism and development, and abiotic stress response. Although the functions of GRPs in cell wall remodeling have been extensively characterized, only a few studies have explored their effects on chlorophyll metabolism and hormone response. Accordingly, we aimed to determine the molecular mechanism of BcGRP23 and its role in chlorophyll metabolism and the BRI1-EMS-SUPPRESSOR 1 (BES1) signaling pathway in flowering Chinese cabbage. The expression levels of BcGRP23 in the leaves and stems gradually decreased with increasing growth and development of flowering Chinese cabbage, while BcGRP23 was barely expressed after flowering. As plant growth continued, the GUS (ß-glucuronidase) stain gradually became lighter in hypocotyls and was largely free of growth points. The petioles and stems of BcGRP23-silenced plants lost their green color, and the contents of chlorophyll a (Chl a) and Chl b were significantly reduced. Further research revealed that the expression levels of chlorophyll degradation-related genes were significantly increased in silenced plants compared with the control; however, the opposite was noted for the BcGRP23-overexpressing lines. The BcGRP23 promoter sequence contains numerous hormone-responsive elements. In fact, the expression of BcGRP23 was upregulated in flowering Chinese cabbage following treatment with the hormones indole-3-acetic acid (IAA), gibberellin (GA), 6-benzylaminopurine (6-BA), methyl jasmonate (MeJA), and brassinosteroid (BR). Treatment with BR led to the most significant upregulation. BES1, in response to BRs, directly activated the BcGRP23 promoter. Overall, BcGRP23 regulated the expression of chlorophyll degradation-related genes, thereby affecting the chlorophyll content. Furthermore, the expression of BcGRP23 was significantly regulated by exogenous BR application and was directly activated by BES1. These findings preliminarily suggest the molecular mechanism and regulatory pathway of BcGRP23 in the growth and development of flowering Chinese cabbage plants and their response to environmental stress.

ß-cells regeneration by WL15 of cysteine and glycine-rich protein 2 which reduces alloxan induced ß-cell dysfunction and oxidative stress through phosphoenolpyruvate carboxykinase and insulin pathway in zebrafish in-vivo larval model.

BACKGROUND: Pancreatic ß-cells are susceptible to oxidative stress, leading to ß-cell death and dysfunction due to enhanced ROS levels and type 2 diabetes. To inhibit the ß-cells damages induced by the oxidative stress, the present study investigates the beneficial effect of various peptides (WL15, RF13, RW20, IW13 and MF18) of immune related proteins (cysteine and glycine-rich protein 2, histone acetyltransferase, vacuolar protein sorting associated protein 26B, serine threonine-protein kinase and CxxC zinc finger protein, respectively). Also, the molecular mechanism of WL15 from cysteine and glycine-rich protein 2 on ß-cell regeneration was identified through PEPCK and insulin pathway. MATERIALS AND METHODS: In this study, a total of five peptides including WL15, RF13, RW20, IW13, and MF18 were derived from immune-related proteins such as cysteine and glycine-rich protein 2, histone acetyltransferase, vacuolar protein sorting associated protein 26B, serine threonine-protein kinase and CxxC zinc finger protein, respectively. These protein sequences were obtained from an earlier constructed transcriptome database of a teleost Channa striatus. The identified peptides were evaluated for their antioxidant as well as antidiabetic activity. Based on the in silico analysis and in-vitro screening experiments, WL15 was predicted to have better antioxidant and antidiabetic activity among the five different peptides. Therefore, WL15 alone was further analyzed for apoptosis, antioxidant capacity, glucose metabolism, and gene expression performance, which was investigated on the alloxan (500 µM) induced zebrafish in vivo larval model. RESULTS: The results showed alloxan exposure to zebrafish larvae for a day, the ROS was generated in the ß-cells. Interestingly, WL15 treatment showed a protective effect by reducing the toxicity of alloxan exposed zebrafish larvae by increasing their survival and heart rate. Moreover, WL15 reduced the intracellular ROS level and apoptosis in alloxan-induced larvae. The superoxide anion and lipid peroxidation levels are also reduced by improving the glutathione content after the WL15 treatment. Besides, WL15 treatment increased the proliferation rate of ß-cells and decreased the glucose level. Further, the gene expression studies revealed that WL15 treatment normalized the PEPCK expression while upregulating the insulin expression in alloxan exposed larvae. CONCLUSION: Overall, the findings indicate that WL15 of cysteine and glycine-rich protein 2 can act as a potential antioxidant for type 2 diabetes patients in respect of improving ß-cell regeneration.

A cell wall-localized glycine-rich protein of dodder acts as pathogen-associated molecular pattern.

Cuscuta reflexa (giant dodder) is an obligate stem holoparasite withdrawing water, nutrients, and carbohydrates from its hosts. For a broad spectrum of host plants, C. reflexa usually stays unrecognized. The cultivated tomato Solanum lycopersicum, as one notable exception, possesses a leucine-rich repeat receptor protein (LRR-RP), Cuscuta receptor 1 (CuRe1), which enables tomato to recognize C. reflexa as a dangerous parasitic invader and to respond with plant immune responses. During the infection process, a glycine-rich protein (GRP) is freed from C. reflexa and gets detected by CuRe1. Here, we focus on the subcellular localization of the GRP within plant cell walls using a fluorescence based co-localization.

Intracellular ROS scavenging and antioxidant regulation of WL15 from cysteine and glycine-rich protein 2 demonstrated in zebrafish in vivo model.

Antioxidant peptides are naturally present in food, especially in fishes, and are considered to contain rich source of various bioactive compounds that are structurally heterogeneous. This study aims to identify and characterize the antioxidant property of the WL15 peptide, derived from Cysteine and glycine-rich protein 2 (CSRP2) identified from the transcriptome of a freshwater food fish, Channa striatus. C. striatus is already studied to contain high levels of amino acids and fatty acids, besides traditionally known for its pharmacological benefits in the Southeast Asian region. In our study, in vitro analysis of WL15 peptide exhibited strong free radical scavenging activity in 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), superoxide anion radical and hydrogen peroxide (H2O2) scavenging assay. Further, to evaluate the cytotoxicity and dose-response, the Human dermal fibroblast (HDF) cells were used. Results showed that the treatment of HDF cells with varying concentrations (10, 20, 30, 40 and 50 µM) of WL15 peptide was not cytotoxic. However, the treatment concentrations showed enhanced antioxidant properties by significantly inhibiting the levels of free radicals. For in vivo assessment, we have used zebrafish larvae for evaluating the developmental toxicity and for determining the antioxidant property of the WL15 peptide. Zebrafish embryos were treated with the WL15 peptide from 4 h of post-fertilization (hpf) to 96 hpf covering the embryo-larval developmental period. At the end of the exposure period, the larvae were exposed to H2O2 (1 mM) for inducing generic oxidative stress. The exposure of WL15 peptide during the embryo-larval period showed no developmental toxicity even in higher concentrations of the peptide. Besides, the WL15 peptide considerably decreased the intracellular reactive oxygen species (ROS) levels induced by H2O2 exposure. WL15 peptide also inhibited the H2O2-induced caspase 3-dependent apoptotic response in zebrafish larvae was observed using the whole-mount immunofluorescence staining. Overall results from our study showed that the pre-treatment of WL15 (50 µM) in the H2O2-exposed zebrafish larvae, attenuated the expression of activated caspase 3 expressions, reduced Malondialdehyde (MDA) levels, and enhanced antioxidant enzymes, including superoxide dismutase (SOD) and catalase (CAT). The gene expression of antioxidant enzymes such as glutathione S-transferase (GST), glutathione peroxide (GPx) and γ-glutamyl cysteine synthetase (GCS) was found to be upregulated. In conclusion, it can be conceived that pre-treatment with WL15 could mitigate H2O2-induced oxidative injury by elevating the activity and expression of antioxidant enzymes, thereby decreasing MDA levels and cellular apoptosis by enhancing the antioxidant response, demonstrated by the in vitro and in vivo experiments.

Inhibition of CSRP2 Promotes Leukemia Cell Proliferation and Correlates with Relapse in Adults with Acute Myeloid Leukemia.

BACKGROUND: Relapse is a major obstacle in the treatment of acute myeloid leukemia (AML). Refinement of risk stratification may aid the identification of patients who are likely to relapse. Abnormal cysteine and glycine-rich protein 2 (CSRP2) has been implicated in various cancers, but its function remains unclear. The purpose of this study was to explore the role of CSRP2 in predicting adult AML recurrence. METHODS: RT-PCR was used to detect the expression of CSRP2 in 193 newly diagnosed adult AML patients and 44 healthy controls. The competitive risk model was used to calculate the cumulative incidence of relapse rate (CIR), Kaplan-Meier to calculate the relapse-free survival rate (RFS), and the Cox regression model to perform multivariate analysis. Viral transfection was used to construct AML cell lines with stable knockdown of CSRP2, CCK8 to detect proliferation and drug resistance, flow cytometry to detect cell cycle and apoptosis, and Western blot to detect key molecules in signaling pathways. RESULTS: CSRP2 transcript levels were higher in 193 adult AML compared with 44 healthy controls. In 149 patients who achieved complete remission, those with high CSRP2 transcript levels displayed a lower 2-year CIR and higher 2-year RFS, especially when receiving only chemotherapy. In multivariate analysis, a high CSRP2 transcript level was independently associated with a better RFS. Knockdown of CSRP2 promoted proliferation and cell cycle progression, and reduced chemosensitivity. Western blot analysis showed upregulation of p-AKT and p-CREB in CSRP2-knockdown AML cell lines. Inhibition assays suggested these two signaling pathways participated in the CSRP2-mediated proliferation effects in AML cell lines. CONCLUSION: In summary, CSRP2 correlates with relapse in adult AML. Down-regulation of CSRP2 could promote the proliferation of AML cell lines by regulating the AKT and CREB signaling pathways. Therefore, CSRP2 may provide prognostic significance and potential therapeutic targets in the management of AML.

Discriminating aspects of global metabolism of neonatal cardiomyocytes from wild type and KO-CSRP3 rats using proton magnetic resonance spectroscopy of culture media samples.

Knockout of multifunction gene cysteine- and glycine-rich protein 3 (CSRP3) in cardiomyocytes (CMs) of mice leads to heart dilation, severely affecting its functions. In humans, CSRP3 mutations are associated with hypertrophic (HCM) and dilated cardiomyopathy (DCM). The absence of the CSRP3 expression produces unknown effects on in vitro neonatal CMs' metabolism. The metabolome changes in culture media conditioned by CSRP3 knockout (KO-CSRP3), and wild type (WT) neonatal cardiomyocytes were investigated under untreated or after metabolic challenging conditions produced by isoproterenol (ISO) stimulation, by in vitro high-resolution proton magnetic resonance spectroscopy (1H-MRS)-based metabolomics. Metabolic differences between neonatal KO-CSRP3 and WT rats' CMs were identified. After 72 h of culture, ISO administration was associated with increased CMs' energy requirements and increased levels of threonine, alanine, and 3-hydroxybutyrate in both neonatal KO-CSRP3 and WT CMs conditioned media. When compared with KO-CSRP3, culture media derived from WT cells presented higher lactate concentrations either under basal or ISO-stimulated conditions. The higher activity of ketogenic biochemical pathways met the elevated energy requirements of the contractile cells. Both cells are considered phenotypically indistinguishable in the neonatal period of animal lives, but the observed metabolic stress responses of KO-CSRP3 and WT CMs to ISO were different. KO-CSRP3 CMs produced less lactate than WT CMs in both basal and stimulated conditions. Mainly, ISO-stimulated conditions produced evidence for lactate overload within KO-CSRP3 CMs, while WT CMs succeeded to manage the metabolic stress. Thus, 1H-MRS-based metabolomics was suitable to identify early inefficient energetic metabolism in neonatal KO-CSRP3 CMs. These results may reflect an apparent lower lactate transport and consumption, in association with protein catabolism.

Cell wall Glycine-rich Protein2 is involved in tapetal differentiation and pollen maturation.

The tapetum plays important roles in anther development by providing materials for pollen-wall formation and nutrients for pollen development. Here, we report the characterization of a male-sterile mutant of glycine-rich protein 2 (OsGRP2), which exhibits irregular cell division and dysfunction of the tapetum. GRP is a cellwall structural protein present in the cell walls of diverse plant species, but its function is unclear in pollen development. We found that few GRP genes are expressed in rice and thus focused on one highly expressed gene, OsGRP2. The tapetal cell walls of an OsGRP2 mutant did not thicken at the pollen mothercell stage, as a result, pollen maturation and fertility rate decreased. High OsGRP2 expression was detected in male-floral organs, and OsGRP2 was distributed in the tapetum. OsGRP2 participated in establishment of the cellwall network during early tapetum development. In conclusion, our results indicate that OsGRP2 plays important roles in the differentiation and function of the tapetum.

Acute Cardiorenal Syndrome: Models and Heart-Kidney Connectors.

Cardiorenal syndrome type 1 (CRS-1) is an acute kidney injury (AKI) due to acute worsening of cardiac function. More than 20% of patients with acute heart failure develop AKI, and AKI predicts poor outcome. Although a number of potential pathways have been suggested as heart-kidney connectors which might drive the syndrome, there are significant barriers to investigation, such as a paucity of animal models, a lack of specific biomarkers, and an inconsistent temporal and causal relationship between changes in cardiac flow and development of renal dysfunction. Thus, mechanisms of heart-kidney interaction are still unclear, and there is no specific or effective therapy for CRS-1. This review, therefore, focuses on mitigating these challenges in the investigation of CRS-1. We review the available models and focus on mechanistic insights gained from those models. In particular, we focus on non-flow and endocrine mediators of CRS-1 such as heart-derived messengers which alter renal function and which may represent targetable pathways in this syndrome. As precise connectors of heart-kidney interaction remain unclear, the establishment of animal and relevant cell-culture models and further investigation are required.

LsGRP1, a class II glycine-rich protein of Lilium, confers plant resistance via mediating innate immune activation and inducing fungal programmed cell death.

Defence-related LsGRP1 is a leaf-specific plant class II glycine-rich protein (GRP) involved in salicylic acid-induced systemic resistance against grey mould caused by necrotrophic Botrytis elliptica in lily (Lilium) cultivar Stargazer. The C-terminal region of LsGRP1 (LsGRP1C ) can inhibit fungal growth in vitro via a mechanism of inducing fungal apoptosis programmed cell death (PCD). In this study, the role of LsGRP1 in induced defence mechanism was investigated using LsGRP1-silenced Stargazer lily and LsGRP1-transgenic Arabidopsis thaliana. LsGRP1 silencing in lily was found to slightly inhibit plant growth and greatly increase the susceptibility to B. elliptica by suppressing callose deposition and early reactive oxygen species (ROS) accumulation. In contrast, LsGRP1-transgenic Arabidopsis showed higher resistance to Botrytis cinerea and also to Pseudomonas syringae pv. tomato DC3000 as compared to the wild type, accompanied with the enhancement of callose deposition and ROS accumulation. Additionally, LsGRP1 silencing increased plant cell death caused by B. elliptica secretion and reduced pathogen-associated molecular pattern (PAMP)-triggered defence activation in Stargazer lily. Consistently, LsGRP1 expression boosted PAMP-triggered defence responses and effector recognition-induced hypersensitive response in Arabidopsis. Moreover, fungal apoptosis PCD triggered by LsGRP1 in an LsGRP1C -dependent manner was demonstrated by leaf infiltration with LsGRP1C -containing recombinant proteins in Stargazer lily. Based on these results, we presume that LsGRP1 plays roles in plant defence via functioning as a pathogen-inducible switch for plant innate immune activation and acting as a fungal apoptosis PCD inducer to combat pathogen attack.

A Plant SMALL RNA-BINDING PROTEIN 1 Family Mediates Cell-to-Cell Trafficking of RNAi Signals.

In plants, RNA interference (RNAi) plays a pivotal role in growth and development, and responses to environmental inputs, including pathogen attack. The intercellular and systemic trafficking of small interfering RNA (siRNA)/microRNA (miRNA) is a central component in this regulatory pathway. Currently, little is known with regards to the molecular agents involved in the movement of these si/miRNAs. To address this situation, we employed a biochemical approach to identify and characterize a conserved SMALL RNA-BINDING PROTEIN 1 (SRBP1) family that mediates non-cell-autonomous small RNA (sRNA) trafficking. In Arabidopsis, AtSRBP1 is a glycine-rich (GR) RNA-binding protein, also known as AtGRP7, which we show binds single-stranded siRNA. A viral vector, Zucchini yellow mosaic virus (ZYMV), was employed to functionally characterized the AtSRBP1-4 (AtGRP7/2/4/8) RNA recognition motif and GR domains. Cellular-based studies revealed the GR domain as being necessary and sufficient for SRBP1 cell-to-cell movement. Taken together, our findings provide a foundation for future research into the mechanism and function of mobile sRNA signaling agents in plants.

Craterostigma plantagineum cell wall composition is remodelled during desiccation and the glycine-rich protein CpGRP1 interacts with pectins through clustered arginines.

Craterostigma plantagineum belongs to the desiccation-tolerant angiosperm plants. Upon dehydration, leaves fold and the cells shrink which is reversed during rehydration. To understand this process changes in cell wall pectin composition, and the role of the apoplastic glycine-rich protein 1 (CpGRP1) were analysed. Cellular microstructural changes in hydrated, desiccated and rehydrated leaf sections were analysed using scanning electron microscopy. Pectin composition in different cell wall fractions was analysed with monoclonal antibodies against homogalacturonan, rhamnogalacturonan I, rhamnogalacturonan II and hemicellulose epitopes. Our data demonstrate changes in pectin composition during dehydration/rehydration which is suggested to affect cell wall properties. Homogalacturonan was less methylesterified upon desiccation and changes were also demonstrated in the detection of rhamnogalacturonan I, rhamnogalacturonan II and hemicelluloses. CpGRP1 seems to have a central role in cell adaptations to water deficit, as it interacts with pectin through a cluster of arginine residues and de-methylesterified pectin presents more binding sites for the protein-pectin interaction than to pectin from hydrated leaves. CpGRP1 can also bind phosphatidic acid (PA) and cardiolipin. The binding of CpGRP1 to pectin appears to be dependent on the pectin methylesterification status and it has a higher affinity to pectin than its binding partner CpWAK1. It is hypothesised that changes in pectin composition are sensed by the CpGRP1-CpWAK1 complex therefore leading to the activation of dehydration-related responses and leaf folding. PA might participate in the modulation of CpGRP1 activity.

The cardiac syndecan-4 interactome reveals a role for syndecan-4 in nuclear translocation of muscle LIM protein (MLP).

Costameres are signaling hubs at the sarcolemma and important contact points between the extracellular matrix and cell interior, sensing and transducing biomechanical signals into a cellular response. The transmembrane proteoglycan syndecan-4 localizes to these attachment points and has been shown to be important in the initial stages of cardiac remodeling, but its mechanistic function in the heart remains insufficiently understood. Here, we sought to map the cardiac interactome of syndecan-4 to better understand its function and downstream signaling mechanisms. By combining two different affinity purification methods with MS analysis, we found that the cardiac syndecan-4 interactome consists of 21 novel and 29 previously described interaction partners. Nine of the novel partners were further validated to bind syndecan-4 in HEK293 cells (i.e. CAVIN1/PTRF, CCT5, CDK9, EIF2S1, EIF4B, MPP7, PARVB, PFKM, and RASIP). We also found that 19 of the 50 interactome partners bind differently to syndecan-4 in the left ventricle lysate from aortic-banded heart failure (ABHF) rats compared with SHAM-operated animals. One of these partners was the well-known mechanotransducer muscle LIM protein (MLP), which showed direct and increased binding to syndecan-4 in ABHF. Nuclear translocation is important in MLP-mediated signaling, and we found less MLP in the nuclear-enriched fractions from syndecan-4-/- mouse left ventricles but increased nuclear MLP when syndecan-4 was overexpressed in a cardiomyocyte cell line. In the presence of a cell-permeable syndecan-4-MLP disruptor peptide, the nuclear MLP level was reduced. These findings suggest that syndecan-4 mediates nuclear translocation of MLP in the heart.

Characterization of a glycine-rich protein from Rhipicephalus microplus: tissue expression, gene silencing and immune recognition.

Salivary molecules, as glycine-rich proteins (GRPs), are essential to tick attachment and feeding on the host and are suggested to be involved in the host's immune system evasion, therefore representing natural candidates in the search for protective vaccine antigens. This work shows the molecular characterization of a GRP from Rhipicephalus microplus (RmGRP). The cDNA and putative amino acid sequences were analysed, as well as the transcription level in tick tissues/developmental stages, showing the highest levels of gene expression in 1-day-old larvae and salivary glands of fully engorged females. RmGRP gene silencing resulted in a lower hatching rate of larvae from treated females. In addition, recombinant RmGRP (rRmGRP) was recognized by sera from naturally and experimentally infested bovines, displaying considerable differences among the individuals tested. rRmGRP was recognized by anti-saliva and anti-salivary glands sera, while anti-rRmGRP serum recognized RmGRP in saliva and salivary glands, indicating its secretion into the host. The data collected indicate that RmGRP may present roles other than in the tick-host relationship, especially in embryo development. In addition, the high expression in adult females, antigenicity and presence of shared characteristics with other tick protective GRPs turns RmGRP a potential candidate to compose an anti-tick vaccine cocktail.