Geographical location influence 'Cabernet Franc' fruit quality in Shandong province.

Grape quality is a key factor in determining wine quality, and it depends not only on management skills, but also on the geographic location of the producing area. In China, Shandong is the province with the largest wine production, and 'Cabernet Franc' is widely planted. This study evaluated the 'Cabernet Franc' fruit quality in relation to geographical conditions in five 'Cabernet Franc' producing districts of Shandong province, China, including Dezhou Aodeman Winery (DZ), Tai'an Zhongqingsongshi Winery (TA), Penglai Longhu Winery (PL), Rushan Taiyihu Winery (RS), and Rizhao Taiyangcheng Winery (RZ). At the time of veraison and maturity, fruit was harvested from five areas, and compared for cosmetic and internal fruit quality. The soluble sugar content in the Rizhao area was rich, and the weight and volume of single fruit were relatively large. The titratable acid of the berries in Tai'an area was high. RNA-seq analysis showed that the number of genes in the véraison stage was 19,571-20,750, and the number of genes in the mature stage was 19,176-20,735. The analysis found that areas with multiple high-quality characteristics tended to have more DEGs (differential expressed genes). And the DEGs in different areas were mainly distributed on chromosome 7, and at least on chromosome 15. DEGs in 5 areas were enriched on 855 GO terms and 116 KEGG pathways during berries development. Among them, it was found that the up/down-regulation of DEGs was related to the formation of berry quality, which helps to explain the impact of environment on grape quality components. In summary, this study is helpful to understand the influence of cultivation location on the quality of 'Cabernet Franc' in different production areas in Shandong province, and further provide a reference for the production of high-quality wine grapes and winemaking.

The physiology of drought stress in two grapevine cultivars: Photosynthesis, antioxidant system, and osmotic regulation responses.

Drought stress impedes viticultural plant growth and development by modifying various metabolic pathways. However, the regulatory network response underlying drought stress is not yet clear. In this study, the leaves and roots of "Shine Muscat" ("SM," Vitis labruscana × Vitis vinifera) and "Thompson Seedless" ("TS," V. vinifera L. cv.) were subjected to drought stress to study the regulatory network used by drought stress. Morphophysiological results showed that the malondialdehyde content after 28 days of drought stress increased more significantly in "TS" than "SM." Furthermore, the multiomics analysis studies showed that a total of 3036-6714 differentially expressed genes and 379-385 differentially abundant metabolites were identified in "SM" and "TS" grapevine cultivars under drought stress. Furthermore, the retained intron was the major form of differential alternative splicing event under drought stress. The photosynthesis pathway, antioxidant system, plant hormone signal transduction, and osmotic adjustment were the primary response systems in the two grapevine cultivars under drought stress. We have identified GRIK1, RFS2, and LKR/SDH as the hub genes in the coexpression network of drought stress. In addition, the difference in the accumulation of pheophorbide-a reveals different drought resistance mechanisms in the two grapevine cultivars. Our study explained the difference in drought response between cultivars and tissues and identified drought stress-responsive genes, which provides reference data for further understanding the regulatory network of drought tolerance in grapevine.

The roles of brassinosteroids and methyl jasmonate on postharvest grape by regulating the interaction between VvDWF4 and VvTIFY 5 A.

Brassinosteroids (BRs) and methyl jasmonate (MeJA) are known for the regulation of plant development, and the crosstalk between them is important for plant growth. However, the interaction between them in the development of postharvest fruit is unresolved. We found that BR treatment enhanced the accumulation of sugar composition and aroma content, reduced the content of organic acids (such as tartaric acid) and promoted the coloring of grape callus. After the application of MeJA, the acidity increased and the sugar content decreased. The physiological data showed that exogenous BR also attenuated the JA inhibition of postharvest ripening in grape. DWF4 is a key enzyme in the BR biosynthetic pathway, and it can effectively regulate the content of endogenous BRs. TIFY 5 A, which belongs to the Jasmonate ZIM-domain (JAZ) family, can be baited by DWF4 through the Y2H experiment. TIFY 5 A represses the expression of dihydroflavonol-4-reductase (DFR) which plays a key role in the synthesis of anthocyanins, while this will be alleviated by VvDWF4. The interaction between TIFY 5 A and DWF4 contributes to the cross talk between JA and BR signalling pathways. This is also verified by the transgenic experimental results. The results in this paper provides a new insight into the relationship between BR and JA signalling pathways, which is important to the regulation of the postharvest ripening of grape.

Integrative analysis of grapevine (Vitis vinifera L) transcriptome reveals regulatory network for Chardonnay quality formation.

Anthocyanins, total phenols, soluble sugar and fruit shape plays a significant role in determining the distinct fruit quality and customer preference. However, for the majority of fruit species, little is known about the transcriptomics and underlying regulatory networks that control the generation of overall quality during fruit growth and ripening. This study incorporated the quality-related transcriptome data from 6 ecological zones across 3 fruit development and maturity phases of Chardonnay cultivars. With the help of this dataset, we were able to build a complex regulatory network that may be used to identify important structural genes and transcription factors that control the anthocyanins, total phenols, soluble sugars and fruit shape in grapes. Overall, our findings set the groundwork to improve grape quality in addition to offering novel views on quality control during grape development and ripening.

Assessment of 'Cabernet Sauvignon' Grape Quality Half-Véraison to Maturity for Grapevines Grown in Different Regions.

Grapes are widely cultivated around the world and their quality has distinct regional characteristics. In this study, the qualitative characteristics of the 'Cabernet Sauvignon' grape variety in seven regions, from half-véraison to maturity, were analyzed comprehensively at physiological and transcriptional levels. The results indicated that the quality traits of 'Cabernet Sauvignon' grapes in different regions were significantly different with obvious regionality. Total phenols, anthocyanins, and titratable acids were the main factors of the regionality of berry quality, which were very sensitive to changes in the environment. It should be noted that the changes in titrating acids and total anthocyanin of berries vary greatly from half-véraison to maturity between regions. Moreover, the transcriptional analysis showed that the co-expressed genes between regions characterized the core transcriptome of berry development, while the unique genes of each region reflected the regionality of berries. The differentially expressed genes (DEGs) between half-véraison and maturity can be used to demonstrate that the environment of the regions could promote or inhibit gene expression. The functional enrichment suggested that these DEGs help to understand the interpretation of the plasticity of the quality composition of grapes according to the environment. Taken together, the information generated by this study could contribute to the development of viticultural practices aimed at making better use of native varieties for the development of wines with regional characteristics.

Identification of GH17 gene family in Vitis vinifera and expression analysis of GH17 under various adversities.

Glycoside hydrolase (GH, EC 3.2.1) is a group of enzymes that hydrolyzes glycosidic bonds and play a role in the hydrolysis and synthesis of sugars in living organisms. Vitis vinifera is an important fruit crop and it harbors GH17 gene family however, their function in grapes has not been systematically investigated. In this study, a total of 870 GH17 genes were identified from 14 plant species and their structural domain, sequence alignment, phylogenetic tree, collinear analysis, with the expression profiles of VviGH17 gene family was performed. The promoter analysis of VviGH17 gene showed the presence of cis-acting elements, which are responsive to plant growth and development. In addition, elements for plant hormones were found that are triggered in response to abiotic/biological stress. Transcriptomic data led to the identification of several VviGH17 genes, which are associated with bud dormancy and in response to abiotic stress. Transcript analysis was carried out for some of the selected VviGH17 genes RT-qPCR. VviGH17-16 and VviGH17-30 genes were differentially expressed during bud dormancy, fruit development and different abiotic stresses. Moreover, VviGH17-37 and VviGH17-44 were differentially expressed at fruit development, in response to abiotic stress. In addition, subcellular localization predicts that the VviGH17-16, VviGH17-30, and VviGH17-37 genes were located in the cell membrane, while VviGH17-44 gene was located in the vacuole. In conclusion, our study led to the identification of several GH17s and their probable role in development and stress. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01014-1.

Transcriptional and metabolite analysis reveal a shift in fruit quality in response to calcium chloride treatment on "Kyoho" grapevine.

'Kyoho' grapevine (Vitis vinifera) treated by calcium ions solution has been proved as an effective treatment to extend grape quality during storage to reduce disease, but its molecular mechanism was not clear yet. In the current work, grape berries were treated with different concentration of Calcium chloride (CaCl2) solution, and their effects on antioxidant enzyme activity and transcriptome and metabolome in fruit were investigated. CaCl2 treatments reduced weight loss and inhibited the decrement of flesh firmness. 80 mM CaCl2 significantly increased the activity of antioxidant enzymes POD, SOD and CAT, which was the optimum experimental concentration. The study showed that the expression level of heat shock transcription factor and UBX which involved in endoplasmic reticulum stress and degradation pathway increased significantly. Moreover, the corresponding metabolites, such as heat shock protein and organic acid, also increased significantly. The misfolded proteins are transported to the cytosol for degradation, so that the preservation ability of grape is improved.

Retracted: Allopregnanolone restores the tyrosine hydroxylase-positive neurons and motor performance in a 6-OHDA-injected mouse model.

AIMS: It has been reported that allopregnanolone (APα) promotes the neurogenesis of the neural progenitor cells (NPCs) in the subventricular zone (SVZ) and prevents the decrease of dopaminergic neurons in 6-hydroxydopamine (6-OHDA)-treated mice by binding to γ-aminobutyric acid A receptor (GABAAR) and then opening voltage-gated L-type Ca2+ channel, but the underlying mechanisms remain elusive. The aim of this study was to explore the possible involvement of GABAAR and calcium/calmodulin-dependent protein kinase II delta 3 (CaMKIIδ3) in this process. METHODS: 6-OHDA-treated mice and primary cultured midbrain cells were administrated with APα and GABAAR antagonist bicuculline (Bic), and the proliferation and differentiation of NPCs, the tyrosine hydroxylase (TH)-positive neurons and their fibers, the expression levels of CaMKIIδ3 and brain-derived neurotrophic factor (BDNF), and motor functions were measured using ELISA, immunohistochemical staining, real-time RT-PCR, Western blot, and behavioral test. RESULTS: Allopregnanolone significantly promoted the phosphorylation of cytoplasmic CaMKIIδ3 and its nuclear translocation by binding to GABAAR, which, in turn, increased the expression levels of BDNF. This may account for the findings that the exogenous APα enhanced the proliferation and differentiation of NPCs, and ameliorated the nigrostriatal system and behavioral performance in 6-OHDA-treated mice. CONCLUSIONS: Allopregnanolone may directly activate GABAAR, which, in turn, enhance the proliferation and differentiation of NPCs via upregulating the expression levels of CaMKIIδ3, and finally contribute to the restoration of dopaminergic neurons in 6-OHDA-treated mice.

Tracing the evolution of fatty acid-binding proteins (FABPs) in organisms with a heterogeneous fat distribution.

The distribution of fat among both invertebrate and vertebrate groups is heterogeneous. Studies have shown that fatty acid-binding proteins (FABPs), which mainly bind and transport fatty acids, play important roles in the regulation of fat storage and distribution. However, the systematic and genome-wide investigation of FABP genes in organisms with a heterogeneous fat distribution remains in its infancy. The availability of the complete genomes of Caenorhabditis elegans, Callorhinchus milii, and other organisms with a heterogeneous fat distribution allowed us to systematically investigate the gene structure and phylogeny of FABP genes across a wide range of phyla. In this study, we analyzed the number, structure, chromosomal location, and phylogeny of FABP genes in 18 organisms from C. elegans to Homo sapiens. A total of 12 types of FABP genes were identified in the 18 species, and no single organism exhibited all 12 fatty acid-binding genes (FABPs). The absence of a specific FABP gene in tissue may be related to the absence of fat storage in the corresponding tissue. The genomic loci of the FABP genes were diverse, and their gene structures varied. The results of the phylogenetic analysis and the observation of conserved gene synthesis of FABP family genes/proteins suggest that all FABP genes may have evolved from a common ancestor through tandem duplication. This study not only lays a strong theoretical foundation for the study of fat deposition in different organisms, but also provides a new perspective regarding metabolic disease prevention and control and the improvement of agricultural product quality.

Qualification and Clinical Validation of an Immunodiagnostic Assay for Detecting 11 Additional Streptococcus pneumoniae Serotype-specific Polysaccharides in Human Urine.

BACKGROUND: Identifying Streptococcus pneumoniae serotypes by urinary antigen detection (UAD) assay is the most sensitive way to evaluate the epidemiology of nonbacteremic community-acquired pneumonia (CAP). We first described a UAD assay to detect the S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F, covered by the licensed 13-valent S. pneumoniae conjugate vaccine. To assess the substantial remaining pneumococcal disease burden after introduction of several pneumococcal vaccines, a UAD-2 assay was developed to detect 11 additional serotypes (2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F, and 33F) in individuals with radiographically confirmed CAP. METHODS: The specificity of the UAD-2 assay was achieved by capturing pneumococcal polysaccharides with serotype-specific monoclonal antibodies, using Luminex technology. Assay qualification was used to assess accuracy, precision, and sample linearity. Serotype positivity was based on cutoffs determined by nonparametric statistical evaluation of urine samples from individuals without pneumococcal disease. The sensitivity and specificity of the positivity cutoffs were assessed in a clinical validation, using urine samples obtained from a large study that measured the proportion of radiographically confirmed CAP caused by S. pneumoniae serotypes in hospitalized US adults. RESULTS: The UAD-2 assay was shown to be specific and reproducible. Clinical validation demonstrated assay sensitivity and specificity of 92.2% and 95.9% against a reference standard of bacteremic pneumonia. In addition, the UAD-2 assay identified a S. pneumoniae serotype in 3.72% of nonbacteremic CAP cases obtained from hospitalized US adults. When combined with bacteremic CAP cases, the proportion of pneumonias with a UAD-2 serotype was 4.33%. CONCLUSIONS: The qualified/clinically validated UAD-2 method has applicability in understanding the epidemiology of nonbacteremic S. pneumoniae CAP and for assessing the efficacy of future pneumococcal conjugate vaccines that are under development.

Natural variation and selection in GmSWEET39 affect soybean seed oil content.

Soybean (Glycine max) is a major contributor to the world oilseed production. Its seed oil content has been increased through soybean domestication and improvement. However, the genes underlying the selection are largely unknown. The present contribution analyzed the expression patterns of genes in the seed oil quantitative trait loci with strong selective sweep signals, then used association, functional study and population genetics to reveal a sucrose efflux transporter gene, GmSWEET39, controlling soybean seed oil content and under selection. GmSWEET39 is highly expressed in soybean seeds and encodes a plasma membrane-localized protein. Its expression level is positively correlated with soybean seed oil content. The variation in its promoter and coding sequence leads to different natural alleles of this gene. The GmSWEET39 allelic effects on total oil content were confirmed in the seeds of soybean recombinant inbred lines, transgenic Arabidopsis, and transgenic soybean hairy roots. The frequencies of its superior alleles increased from wild soybean to cultivated soybean, and are much higher in released soybean cultivars. The findings herein suggest that the sequence variation in GmSWEET39 affects its relative expression and oil content in soybean seeds, and GmSWEET39 has been selected to increase seed oil content during soybean domestication and improvement.

Assessing the Spatial Distribution of Soil PAHs and their Relationship with Anthropogenic Activities at a National Scale.

Soil polycyclic aromatic hydrocarbon (PAH) pollution is a major concern due to its negative impact on soil quality around the world. In China, accurate data on soil PAHs and information on the relationship with anthropogenic activities are limited. In this study, about 30,800 samples from 1833 soil sample sites were reviewed from 306 published reports to build a soil PAHs database. Based on the data obtained, the results demonstrated that 24.11% of surface soils in China are heavily contaminated. Meanwhile, the concentration of soil PAHs varied, in the order of independent mining and industrial areas (IMIA) > urban areas > suburban areas > rural areas, and the spatial distribution in China demonstrated a descending trend from north to south. Moreover, the characteristic ratio and PCA-MLR (principal component analysis-multiple linear regression) analysis demonstrated that coal combustion and vehicular exhaust emissions were the main sources of soil PAH pollution in China. On the other hand, provincial total Σ16PAHs in surface soil were significantly correlated with the per square kilometer GDP (gross domestic product) of industrial land, the per capita GDP, as well as the production and consumption of energy. These results indicate that anthropogenic factors have greatly affected the levels of soil PAHs in China. This study improves our understanding on the status and sources of soil PAH contamination in China, thereby facilitating the implementation of strategies of prevention, control, and remediation of soils.

Dopamine neuron loss by selective deletion of autophagy-related gene 5 is not exacerbated by MPTP toxicity in midbrain.

Parkinson's disease (PD) is a progressive neurological disease, one of the pathological characteristics is a gradual loss of midbrain dopaminergic (mDA) neurons in the substantia nigra pars compacta (SNpc). In animals, PD-like symptoms can be induced by genetic mutations or by neurotoxins such as 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP). It has been reported that deletion of autophagy-related gene 5 (Atg5) in the brain can disrupt neural function and is accompanied by the accumulation of cytoplasmic inclusions. However, the exact role of autophagy in PD etiology has not fully been asserted. In this study, we used tyrosine hydroxylase (TH)-Cre mice to generate conditional knockouts (CKO) with the specific deletion of Atg5 in mDA neurons, and found that adult Atg5 CKO mice contained ubiquitin- and p62-positive inclusions and fewer TH-positive mDA neurons compared with wild-type controls. Interestingly, MPTP-induced loss of mDA neurons was not observed in Atg5 CKO mice. Thus, Atg5-associated autophagy is required for the survival of mDA neurons, and may be involved in MPTP-induced neuronal degeneration.

SENP3 regulates the global protein turnover and the Sp1 level via antagonizing SUMO2/3-targeted ubiquitination and degradation.

SUMOylation is recently found to function as a targeting signal for the degradation of substrates through the ubiquitin-proteasome system. RNF4 is the most studied human SUMO-targeted ubiquitin E3 ligase. However, the relationship between SUMO proteases, SENPs, and RNF4 remains obscure. There are limited examples of the SENP regulation of SUMO2/3-targeted proteolysis mediated by RNF4. The present study investigated the role of SENP3 in the global protein turnover related to SUMO2/3-targeted ubiquitination and focused in particular on the SENP3 regulation of the stability of Sp1. Our data demonstrated that SENP3 impaired the global ubiquitination profile and promoted the accumulation of many proteins. Sp1, a cancer-associated transcription factor, was among these proteins. SENP3 increased the level of Sp1 protein via antagonizing the SUMO2/3-targeted ubiquitination and the consequent proteasome-dependent degradation that was mediated by RNF4. De-conjugation of SUMO2/3 by SENP3 attenuated the interaction of Sp1 with RNF4. In gastric cancer cell lines and specimens derived from patients and nude mice, the level of Sp1 was generally increased in parallel to the level of SENP3. These results provided a new explanation for the enrichment of the Sp1 protein in various cancers, and revealed a regulation of SUMO2/3 conjugated proteins whose levels may be tightly controlled by SENP3 and RNF4.

De-SUMOylation of FOXC2 by SENP3 promotes the epithelial-mesenchymal transition in gastric cancer cells.

The impact of cellular oxidative stress in promoting the epithelial-mesenchymal transition (EMT) has been noticed. Our previous study shows that SENP3, a redox-sensitive SUMO2/3-specific protease, accumulates in a variety of cancers, but whether SENP3 and SUMOylation involve in the regulation of EMT is unclear. The present study uncovers a novel role of SENP3 in promoting the EMT process in gastric cancer via regulating an EMT-inducing transcription factor, forkhead box C2 (FOXC2). We demonstrate that the expression of mesenchymal marker genes and cell migration ability are enhanced in SENP3-overexpressing gastric cancer cells and attenuated in SENP3-knockdown cells. A nude mouse model and a set of patient's specimens suggest the correlation between SENP3 and gastric cancer metastasis. Biochemical assays identify FOXC2 as a substrate of SENP3. Meanwhile N-cadherin is verified as a target gene of FOXC2, which is transcriptionally activated by a SUMO-less FOXC2. Additionally, reactive oxygen species-induced de-SUMOylation of FOXC2 can be blocked by silencing endogenous SENP3. In conclusion, SENP3, which is increased in gastric cancer cells, potentiates the transcriptional activity of FOXC2 through de-SUMOylation, in favor of the induction of specific mesenchymal gene expression in gastric cancer metastasis.

Parental THC exposure leads to compulsive heroin-seeking and altered striatal synaptic plasticity in the subsequent generation.

Recent attention has been focused on the long-term impact of cannabis exposure, for which experimental animal studies have validated causal relationships between neurobiological and behavioral alterations during the individual's lifetime. Here, we show that adolescent exposure to Δ(9)-tetrahydrocannabinol (THC), the main psychoactive component of cannabis, results in behavioral and neurobiological abnormalities in the subsequent generation of rats as a consequence of parental germline exposure to the drug. Adult F1 offspring that were themselves unexposed to THC displayed increased work effort to self-administer heroin, with enhanced stereotyped behaviors during the period of acute heroin withdrawal. On the molecular level, parental THC exposure was associated with changes in the mRNA expression of cannabinoid, dopamine, and glutamatergic receptor genes in the striatum, a key component of the neuronal circuitry mediating compulsive behaviors and reward sensitivity. Specifically, decreased mRNA and protein levels, as well as NMDA receptor binding were observed in the dorsal striatum of adult offspring as a consequence of germline THC exposure. Electrophysiologically, plasticity was altered at excitatory synapses of the striatal circuitry that is known to mediate compulsive and goal-directed behaviors. These findings demonstrate that parental history of germline THC exposure affects the molecular characteristics of the striatum, can impact offspring phenotype, and could possibly confer enhanced risk for psychiatric disorders in the subsequent generation.

Impaired periamygdaloid-cortex prodynorphin is characteristic of opiate addiction and depression.

Negative affect is critical for conferring vulnerability to opiate addiction as reflected by the high comorbidity of opiate abuse with major depressive disorder (MDD). Rodent models implicate amygdala prodynorphin (Pdyn) as a mediator of negative affect; however, evidence of PDYN involvement in human negative affect is limited. Here, we found reduced PDYN mRNA expression in the postmortem human amygdala nucleus of the periamygdaloid cortex (PAC) in both heroin abusers and MDD subjects. Similar to humans, rats that chronically self-administered heroin had reduced Pdyn mRNA expression in the PAC at a time point associated with a negative affective state. Using the in vivo functional imaging technology DREAMM (DREADD-assisted metabolic mapping, where DREADD indicates designer receptors exclusively activated by designer drugs), we found that selective inhibition of Pdyn-expressing neurons in the rat PAC increased metabolic activity in the extended amygdala, which is a key substrate of the extrahypothalamic brain stress system. In parallel, PAC-specific Pdyn inhibition provoked negative affect-related physiological and behavioral changes. Altogether, our translational study supports a functional role for impaired Pdyn in the PAC in opiate abuse through activation of the stress and negative affect neurocircuitry implicated in addiction vulnerability.

ELK1 transcription factor linked to dysregulated striatal mu opioid receptor signaling network and OPRM1 polymorphism in human heroin abusers.

BACKGROUND: Abuse of heroin and prescription opiate medications has grown to disturbing levels. Opioids mediate their effects through mu opioid receptors (MOR), but minimal information exists regarding MOR-related striatal signaling relevant to the human condition. The striatum is a structure central to reward and habitual behavior and neurobiological changes in this region are thought to underlie the pathophysiology of addiction disorders. METHODS: We examined molecular mechanisms related to MOR in postmortem human brain striatal specimens from a homogenous European Caucasian population of heroin abusers and control subjects and in an animal model of heroin self-administration. Expression of ets-like kinase 1 (ELK1) was examined in relation to polymorphism of the MOR gene OPRM1 and drug history. RESULTS: A characteristic feature of heroin abusers was decreased expression of MOR and extracellular regulated kinase signaling networks, concomitant with dysregulation of the downstream transcription factor ELK1. Striatal ELK1 in heroin abusers associated with the polymorphism rs2075572 in OPRM1 in a genotype dose-dependent manner and correlated with documented history of heroin use, an effect reproduced in an animal model that emphasizes a direct relationship between repeated heroin exposure and ELK1 dysregulation. A central role of ELK1 was evidenced by an unbiased whole transcriptome microarray that revealed ~20% of downregulated genes in human heroin abusers are ELK1 targets. Using chromatin immune precipitation, we confirmed decreased ELK1 promoter occupancy of the target gene Use1. CONCLUSIONS: ELK1 is a potential key transcriptional regulatory factor in striatal disturbances associated with heroin abuse and relevant to genetic mutation of OPRM1.