The Physiological Function of nNOS-Associated CAPON Proteins and the Roles of CAPON in Diseases.

In this review, the structure, isoform, and physiological role of the carboxy-terminal PDZ ligand of neuronal nitric oxide synthase (CAPON) are summarized. There are three isoforms of CAPON in humans, including long CAPON protein (CAPON-L), short CAPON protein (CAPON-S), and CAPON-S' protein. CAPON-L includes three functional regions: a C-terminal PDZ-binding motif, carboxypeptidase (CPE)-binding region, and N-terminal phosphotyrosine (PTB) structural domain. Both CAPON-S and CAPON-S' only contain the C-terminal PDZ-binding motif. The C-terminal PDZ-binding motif of CAPON can bind with neuronal nitric oxide synthase (nNOS) and participates in regulating NO production and neuronal development. An overview is given on the relationship between CAPON and heart diseases, diabetes, psychiatric disorders, and tumors. This review will clarify future research directions on the signal pathways related to CAPON, which will be helpful for studying the regulatory mechanism of CAPON. CAPON may be used as a drug target, which will provide new ideas and solutions for treating human diseases.

Neodymium affects the generation of reactive oxygen species via GSK-3ß/Nrf2 signaling in the gill of zebrafish.

Rare earth element neodymium (Nd) is widely used in industry and agriculture, which may result in the pollution of aquatic environment. In this study, we exposed zebrafish with 10, 50, and 100 µg/L Nd for four weeks. The results showed that Nd could be accumulated in fish gill and Nd accumulation affected the equilibrium of nutrient elements. Nd decreased the antioxidant enzymes' activity and gene expression level, but enhanced the generation of reactive oxygen species (ROS). Moreover, various concentration of Nd treatments inhibited Nrf2 signaling in gill. To examine the critical role of GSK-3ß/Nrf2 signaling on ROS generation under Nd stress, we further interfered gsk-3ß gene in zebrafish under 100 µg/L Nd exposure. The result showed that gsk-3ß gene interference induced Nrf2 signaling as well as the expression and activity of antioxidant enzymes in fish gill. In all, Nd could be accumulated in fish gill, and the signaling of GSK-3ß/Nrf2 was involved in regulating ROS generation under Nd treatments.

Effect of Dietary Manganese on the Growth Performance, Lipid Metabolism, and Antioxidant Capacity in the Post-Larval Coho Salmon (Oncorhynchus kisutch).

In this study, post-larval coho salmon Oncorhynchus kisutch (initial weight 0.37 ± 0.03 g) were fed with 6 experimental diets with increasing manganese (Mn) content (2.4, 8.5, 14.8, 19.8, 24.6, and 33.7 mg kg-1) for 12 weeks. Our results indicated that the feed conversion rate (FCR), specific growth rate (SGR), condition factor (CF), crude protein, moisture, crude lipid, ash, whole-body Mn, and vertebral Mn were affected by the elevation of Mn content in the diet. The activities of hepatic GSH-PX, Mn-SOD, and CAT were enhanced with increasing Mn content in the diet and reached the highest value at 19.8 mg kg-1 Mn. However, the level of hydrogen peroxide (H2O2), superoxide anion (O2·-), and malondialdehyde (MDA) was reduced with increasing Mn content in the diet. In addition, the activity of hepatic lipase (HL) and lipoprotein lipase (LPL) was increased with the elevation of dietary Mn content and reached a peak value at 14.8 mg kg-1 Mn. The activity of fatty acid synthetase (FAS) and the content of nonesterified fatty acid (NEFA) were increased following the elevation of Mn content from 2.4 to 19.8 mg kg-1 in the diet. The results indicated that the appropriate dietary Mn supplementation improved the feeding efficiency, lipid metabolism, and antioxidant capacity of coho salmon. The dietary Mn requirement for post-larval coho salmon was 17.35 mg kg-1 and 19.75 mg kg-1 based on the SGR and FCR, respectively. An optimal dietary level of Mn enhances hepatic lipid metabolism, and the signaling pathway of PI3K/AKT/mTOR may be involved in regulating the activity of enzymes related to lipid metabolism.

Characterization of the lipidomic profile of clam Meretrix petechialis in response to Vibrio parahaemolyticus infection.

Vibrio parahaemolyticus is a devastating pathogen of clam Meretrix petechialis, which brings about huge economic losses in aquaculture breeding industry. In our previous study, we have found that Vibrio infection is closely associated with lipid metabolism of clams. In this study, an untargeted lipidomics approach was used to explore the lipid profiling changes upon Vibrio infection. The results demonstrated that the hepatopancreas of clams was composed of five lipid categories including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids and sterol lipids. And the content of lipid classes altered during Vibrio infection, implying that Vibrio infection altered intracellular lipid homeostasis in clams. Meanwhile, a total of 200 lipid species including 82 up-regulated and 118 down-regulated significantly were identified in response to Vibrio infection, of which ceramide (Cer), phosphatidylcholine (PC) and triglyceride (TG) accounted for the largest proportion. Notably, all Cers showed a significantly decreased trend while nearly all TG species were increased significantly during Vibrio infection, which suggested that Cer and TG could be determined as effective biomarkers. Furthermore, these differentially expressed lipid species were enriched in 20 metabolic pathways and sphingolipid metabolism was one of the most enriched pathways. These results evidenced how the lipid metabolism altered in the process of Vibrio infection and opened a new perspective on the response of marine bivalves to pathogen infection.

Effect of cerium on the production of reactive oxygen species in the root of Arabidopsis thaliana: An in vitro study.

In the current study, the effect of trivalent cerium (Ce3+ ) on the production of reactive oxygen species (ROS) was investigated in the root of Arabidopsis thaliana by an in vitro study. The roots of A. thaliana were exposed with 0, 1, and 5 µmol/L Ce3+ for 12 h in vitro. It was found that the level of H2 O2 , O2 .- , and ·OH was enhanced by 5 µmol/L Ce3+ , but reduced by 1 µmol/L Ce3+ . The activities of peroxidase (POD), catalase (CAT), and superoxidase dismutase (SOD) were enhanced by 1 µmol/L Ce3+ , but reduced by 5 µmol/L Ce3+ . Moreover, we used a laser-scanning confocal microscopy to detect the changes of ROS in the root cells of A. thaliana by using a fluorochrome 2',7'-dichlorofluorescein diacetate (H2 DCF-DA). It showed that the level of ROS was declined in the root cells treated by 1 µmol/L Ce3+ , but the oscillation of ROS was found in the root cells treated with 5 µmol/L Ce3+ . In addition, REEs affect the uptake of mineral elements, which may be related to the oxidative stress in the cells of roots. In all, the data of our study indicated that the appropriate concentration of Ce3+ exhibited an anti-oxidation property and improved the defense system in the root cells of A. thaliana.

Dietary iron affects lipid deposition, nutritional element, and muscle quality in coho salmon (Oncorhynchus kisutch).

We investigated effect of dietary iron (Fe) on the lipid deposition, nutritional element, and muscle quality in coho salmon. Four level Fe diets at 23.7, 46.4, 77.3, and 127.7 mg/kg were fed to the post-larval coho salmon for 12 weeks. Our results showed that dietary Fe decreased the content of triglyceride and the activity of fatty acid synthetase, ATP-citrate lyase, and acetyl-CoA carboxylase. The content of Fe in muscle was increased with increasing dietary Fe levels, and dietary Fe affected the content of nutritional elements. In addition, dietary Fe levels affected the composition of fatty acids and the content of free amino acids, and increased muscle fiber size. The lower dietary Fe levels also affected the hardness, chewiness, resilience, springiness, cohesiveness, and gumminess of salmon muscle. In all, dietary Fe inhibited the lipid deposition and affected the content of nutritional element and muscle quality in coho salmon.

Betaine Regulates the Production of Reactive Oxygen Species Through Wnt10b Signaling in the Liver of Zebrafish.

When fish are under oxidative stress, levels of reactive oxygen species (ROS) are chronically elevated, which play a crucial role in fish innate immunity. In the present study, the mechanism by which betaine regulates ROS production via Wnt10b/ß-catenin signaling was investigated in zebrafish liver. Our results showed that betaine enrichment of diet at 0.1, 0.2 and 0.4 g/kg induced Wnt10b and ß-catenin gene expression, but suppressed GSK-3ß expression in zebrafish liver. In addition, the content of superoxide anion (O2 ·-), hydrogen peroxide (H2O2) and hydroxyl radical (·OH) was decreased by all of the experimental betaine treatments. However, betaine enrichment of diet at 0.1, 0.2 and 0.4 g/kg enhanced gene expression and activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) and catalase (CAT) in zebrafish liver. In addition, Wnt10b RNA was further interfered in zebrafish, and the results of Wnt10b RNAi indicated that Wnt10b plays a key role in regulating ROS production and antioxidant enzyme activity. In conclusion, betaine can inhibit ROS production in zebrafish liver through the Wnt10b/ß-catenin signaling pathway.

Roles of ubiquitination in the crosstalk between tumors and the tumor microenvironment (Review).

The interaction between a tumor and the tumor microenvironment (TME) plays a key role in tumorigenesis and tumor progression. Ubiquitination, a crucial post­translational modification for regulating protein degradation and turnover, plays a role in regulating the crosstalk between a tumor and the TME. Thus, identifying the roles of ubiquitination in the process may assist researchers to investigate the mechanisms underlying tumorigenesis and tumor progression. In the present review article, new insights into the substrates for ubiquitination that are involved in the regulation of hypoxic environments, angiogenesis, chronic inflammation­mediated tumor formation, and the function of cancer­associated fibroblasts and infiltrating immune cells (tumor­associated macrophages, T­cells, myeloid­derived suppressor cells, dendritic cells, and natural killer cells) are summarized. In addition, the potential targets of the ubiquitination proteasome system within the TME for cancer therapy and their therapeutic effects are reviewed and discussed.

The method of Yiqi Yangyin Tongluo can attenuate the pyroptosis of rheumatoid arthritis chondrocytes through the ASIC1a/NLRP3 signaling pathway.

Background: Based on the ASIC1a/NLRP3 signaling pathway, we explored the specific molecular mechanism of the pyroptosis of rheumatoid arthritis (RA) chondrocytes by the method of nourishing qi, nourishing yin, and dredging collaterals to provide new ideas for the treatment of this disease. Methods: A total of 50 rats were divided into a normal group, model group, methotrexate group, Yiqi Yangyin Tongluo group, and combined group. Except for the normal control group, the other groups used Freund's complete adjuvant (FCA) to make RA rat model. The arthritis index and ankle joint swelling of rats in each group were recorded. HE staining and ELISA were used to assess the pathology of the ankle joint of each group of rats and the content of IL-1ß and IL-18 in rat serum. Furthermore, immunofluorescence and qPCR methods were used to detect the protein and mRNA expression levels of NLRP3, caspase 1, ACS, and ASIC1a in the cartilage tissue of each group of rats. Results: Compared with the normal group, the right hind foot joint of the model group was significantly swollen, the levels of IL-18 and IL-1ß in the serum of rats increased significantly, and the mRNA and protein levels of NLRP3, caspase 1, ACS, and ASIC1a in the chondrocytes also increased significantly. Compared with the model group, the degree of ankle joint swelling and IL-18 and IL-1ß content in rat serum in each medication group was significantly reduced, and the combined group showed the greatest reduction compared with the other groups. After 8 weeks of treatment, compared with the model group, the mRNA and protein levels of NLRP3, caspase 1, ACS, and ASIC1a in the chondrocytes of each medication group were down-regulated. HE staining found that there were large numbers of infiltrating inflammatory cells and pannus in the joint tissue of the model group, while only a small amount of inflammatory cell infiltration and pannus was seen in the joint tissue of the rats in each treatment group. Conclusions: The method of Yiqi Yangyin Tongluo can attenuate the pyroptosis of RA chondrocytes through the ASIC1a/NLRP3 signaling pathway.

The Sensitive Genes in Response to Various Metal Ion Stresses in the Yeast Saccharomyces cerevisiae.

Yeast Saccharomyces cerevisiae is a good eukaryotic model for studying the molecular mechanism of toxic metal ion stress. Numerous studies have been performed on the signal transduction induced by toxic metal ion stress. The physiological process of eukaryotic cells has been studied, and various stress factors have been elucidated by constructing a gene deletion library. The sensitivity and tolerance mechanism of yeast under metal ion stress has been widely studied. The sensitive genes induced by metal ion stress will provide a key foundation for studying the gene function of eukaryotic organisms. In addition, the functions of genes in response to metal ion stress mainly participate in regulating ion homeostasis, high glycerin pathway, vacuole protein separation pathway, cell wall integrity pathway, and cell autophagy. However, the interaction of these signal pathways and the detailed response mechanism need to be further studied. In addition, the technique of genomics and proteomics will help study the detailed molecular mechanism induced by toxic metal ion stress. Thus, the sensitive genes related to various signal pathways under toxic metal ion stress will be reviewed in the yeast S. cerevisiae.

Protein Arginine Methyltransferase 1 and its Dynamic Regulation Associated with Cellular Processes and Diseases.

Post-translational modifications (PTMs) of proteins influence protein degradation, protein- protein interactions, expression of genes, and intracellular signal transduction, thereby regulating major life processes. Among the PTMs occurring within the cytoplasm and nucleus, the most commonly studied one is the arginine methylation of proteins catalyzed by PRMTs. PRMT1 is the most excellent and extensively studied member of the PRMT family. PRMT1 occurs in various isoforms, and the unique sequence splicing of each of these isoforms encodes differential proteins that exhibit different cellular localization, substrate specificity, and enzyme activity. In addition to methylating histones, PRMT1 also methylates a large number of non-histone substrates that regulate a broad range of cellular processes. In recent years, research has revealed an increasing number of pathological diseases caused by the misregulation and aberrant expression of PRMT1, demonstrating the potential of PRMT1 as an effective biomarker for drug targets. In this context, the present study discusses the structural characteristics and the biological functions of PRMT1. Practical Applications: Several diseases originate from aberrant post-translational modifications. The misregulation of the arginine methylation of proteins, which is regulated by PRMTs and influences a series of cellular activities, leads to developmental abnormalities and physiological diseases. PRMT1, which accounts for 85% of the activity of PRMTs, is involved in several cellular processes occurring in various diseases. Multiple inhibitors have been developed and studied for their potential as biomarkers and suitable drug targets in clinical application. The present report summarizes the findings of the most recent studies focusing on the structural characteristics, splicing, substrates, and biological functions of PRMT1, to contribute to future research for deciphering the molecular mechanisms of PRMT1 and drug improvement.

The toxicity of neodymium and genome-scale genetic screen of neodymium-sensitive gene deletion mutations in the yeast Saccharomyces cerevisiae.

The wide usage of neodymium (Nd) in industry, agriculture, and medicine has made it become an emerging pollutant in the environment. Increasing Nd pollution has potential hazards to plants, animals, and microorganisms. Thus, it is necessary to study the toxicity of Nd and the mechanism of Nd transportation and detoxification in microorganisms. Through genome-scale screening, we identified 70 yeast monogene deletion mutations sensitive to Nd ions. These genes are mainly involved in metabolism, transcription, protein synthesis, cell cycle, DNA processing, protein folding, modification, and cell transport processes. Furthermore, the regulatory networks of Nd toxicity were identified by using the protein interaction group analysis. These networks are associated with various signal pathways, including calcium ion transport, phosphate pathways, vesicular transport, and cell autophagy. In addition, the content of Nd ions in yeast was detected by an inductively coupled plasma mass spectrometry, and most of these Nd-sensitive mutants showed an increased intracellular Nd content. In all, our results provide the basis for understanding the molecular mechanisms of detoxifying Nd ions in yeast cells, which will be useful for future studies on Nd-related issues in the environment, agriculture, and human health.

Effect of rare earth element lanthanum on lipid deposition and Wnt10b signaling in the liver of male zebrafish.

This paper investigates the effect of lanthanum (La) on lipid deposition and Wnt10b signaling in the liver of male zebrafish with exposure of 0, 10, 20, and 30 µmol/L La. It suggests that La can be accumulated in liver, and its treatments decrease the activities and gene expression of enzymes related to fatty acid synthesis. The levels of total cholesterol (TC), triglyceride (TG), and nonesterified fatty acids (NEFA) as well as the size of lipid droplets are decreased by La treatments. Moreover, La treatments affect the composition of fatty acids and the content of nutrient elements. Meanwhile, they also induce the gene expression of wnt10b, ß-catenin, pparα, and pparγ, but inhibit gsk-3ß gene expression in liver. Further study on the result of wnt10b gene interference shows that Wnt10b/ß-catenin signaling plays a crucial role in the regulatory process of hepatic lipid deposition. Taken together, our observations suggest that La accumulation affects lipid deposition in the liver of male zebrafish, and Wnt10b signaling pathway may be involved in this process.

Vitamin C regulates the production of reactive oxygen species through Wnt10b signaling in the gill of zebrafish.

In this study, the mechanism that vitamin C (VC) regulates the production of reactive oxygen species (ROS) through Wnt10b signaling was investigated in the gill of zebrafish (Danio rerio). The results showed that 0.5 and 1.0 g/kg VC diets induced the gene expression of Wnt10b, ß-catenin, SOD, CAT, and GSH-PX in gill. In addition, VC decreased the levels of H2O2, O2·- and ·OH, whereas the activities of SOD, CAT, and GSH-PX were increased by VC in the gill of zebrafish. To evaluate the role of Wnt10b in regulating oxidative stress, Wnt10b RNA was further interfered and the gene expression and activities of antioxidant enzymes were detected in gill. The result of Wnt10b RNA interference showed that Wnt10b signaling played a key role in regulating the gene expression of SOD, CAT, and GSH-PX. In all, VC may regulate the production of ROS through Wnt10b signaling in the gill of zebrafish (Danio rerio).

The accumulation and effect of rare earth element neodymium on the root of rice seedlings.

Neodymium (Nd) potentially threatens ecological equilibrium for its wide usage in industries. In this study, the accumulation and effect of Nd on roots were investigated in the rice seedlings (Oryza sativa L.) exposed to different concentrations of Nd (0, 1, 10, 100, and 1000 µM). The toxic effect of Nd on rice growth was observed at the higher concentration, but the positive effects were found at the lower concentration. The accumulation of Nd was present in six different chemical forms, and the insoluble phosphate and oxalate Nd were the major forms of Nd in the roots. In addition, Nd was accumulated in the soluble fractions, organelles, and cell walls of rice seedlings, and the root cell wall was a major Nd sink site. The result of Fourier transform infrared spectrometer spectral analysis indicated that the functional groups of -OH and C-OH were the major binding sites of Nd in the cell wall of roots. Moreover, the level of reactive oxygen species (ROS) and the activity of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) were significantly increased with the increase of Nd concentration. The enhanced antioxidant capacity also played an important role in Nd detoxification of rice seedlings. In all, the results indicated that forming of inactive oxalate or phosphate and efficient sequestration into the root cell wall was a key process in Nd accumulation and detoxification of rice seedlings.

ß-Thymosin is an essential regulator of stem cell proliferation and neuron regeneration in planarian (Dugesia japonica).

ß-Thymosin is a multifunctional peptide ubiquitously expressed in vertebrates and invertebrates. Many studies have found ß-thymosin is critical for wound healing, angiogenesis, cardiac repair, hair regrowth, and anti-fibrosis in vertebrates, and plays an important role in antimicrobial immunity in invertebrates. However, whether ß-thymosin participates in the regeneration of organisms is still poorly understood. In this study, we identified a ß-thymosin gene in Dugesia japonica which played an important role in stem cell proliferation and neuron regeneration during the tissue repair process in D. japonica. Sequencing analysis showed that ß-thymosin contained two conserved ß-thymosin domains and two actin-binding motifs, and had a high similarity with other ß-thymosins of invertebrates. In situ or fluorescence in situ hybridization analysis revealed that Djß-thymosin was co-localized with DjPiWi in the neoblast cells of intact adult planarians and the blastema of regenerating planarians, suggesting Djß-thymosin has a potential function of regeneration. Disruption Djß-thymosin by RNA interference results in a slightly curled up head of planarian and stem cell proliferation defects. Additionally, we found that, upon amputation, Djß-thymosin RNAi-treated animals had impaired regeneration ability, including impaired blastema formation, delayed eyespot formation, decreased brain area, and disrupted central CNS formation, implying Djß-thymosin is an essential regulator of stem cell proliferation and neuron regeneration.

Evolution and Structure of API5 and Its Roles in Anti-Apoptosis.

Apoptosis, also named programmed cell death, is a highly conserved physiological mechanism. Apoptosis plays crucial roles in many life processes, such as tissue development, organ formation, homeostasis maintenance, resistance against external aggression, and immune responses. Apoptosis is regulated by many genes, among which Apoptosis Inhibitor-5 (API5) is an effective inhibitor, though the structure of API5 is completely different from the other known Inhibitors of Apoptosis Proteins (IAPs). Due to its high expression in many types of tumors, API5 has received extensive attention, and may be an effective target for cancer treatment. In order to comprehensively and systematically understand the biological roles of API5, we summarized the evolution and structure of API5 and its roles in anti-apoptosis in this review.

Planarian gamma-interferon-inducible lysosomal thiol reductase (GILT) is required for gram-negative bacterial clearance.

The powerful regenerative ability of planarians has long been a concern of scientists, but recently, their efficient immune system has attracted more and more attention from researchers. Gamma-interferon-inducible lysosomal thiol reductase (GILT) is related not only to antigen presentation but also to bacteria invasions. But the systematic studies are not yet to be conducted on the relationship between bacterial infection. Our study reveals for the first time that GILT of planarian (DjGILT) plays an essential role in the clearance of Gram-negative bacteria by conducting H2O2 concentration in planarians. In animals that DjGILT was silenced, it persisted for up to 9 days before all bacteria were cleared, compared with 6 days of the control group. When infected with E. coli and V. anguillarum, the level of H2O2 was significantly increased in DjGILT-silenced planarians, and concomitantly, mRNA level of C-type lectin DjCTL, which modulates agglutination and clearance efficiency of invading bacteria, was decreased. Further study showed that the decrease of H2O2 level led to a significant increase in DjCTL transcripts. Collectively, we proposed a mechanism model for the involvement of GILT gene in bacterial elimination. We have for the first time revealed the specific mechanism of GILT in innate immune response against bacterial infection.

Dietary vitamin E regulates the activity of antioxidant enzymes through Wnt10b signaling in the muscle of zebrafish.

Vitamin E (VE) plays a crucial role in regulating the physiological functions of animals. In the present study, the mechanism by which VE regulates the activities of antioxidant enzymes through Wnt10b signaling was investigated in the muscle of zebrafish. It was found that the gene expression of Wnt10b, ß-catenin, and PPARγ was induced, while the GSK-3ß expression was inhibited by 52.34 and 101.27 mg kg-1 VE treatments. The generation of a hydroxy radical (˙OH) and superoxide anion (O2˙-) and the content of hydrogen peroxide (H2O2) were decreased by VE treatments. However, the activities of superoxide dismutase (SOD), peroxidase (POD), and glutathione peroxidase (GSH-PX) were increased by 52.34 and 101.27 mg kg-1 VE treatments. In addition, the content of saturated fatty acids (SFA) was decreased, but that of polyunsaturated fatty acids (PUFA) was increased by VE treatment. To confirm the role of Wnt10b in regulating antioxidant functions, Wnt10b RNA was interfered in zebrafish fed with different concentrations of VE diets. The results showed that the GSK-3ß gene expression was induced but the ß-catenin expression was inhibited by Wnt10b RNA interference in the muscle of zebrafish. The levels of O2˙-, H2O2, and ˙OH were enhanced, but the activities of SOD, GSH-PX, and POD were decreased by the interference of Wnt10b RNA. In all, our results indicated that VE could induce the Wnt10b/ß-catenin signaling pathway, which may further regulate the activities of antioxidant enzymes in the muscle of zebrafish.

Matrix Metalloproteinases in Invertebrates.

Matrix Metalloproteinases (MMPs) belong to a family of metal-dependent endopeptidases which contain a series of conserved pro-peptide domains and catalytic domains. MMPs have been widely found in plants, animals, and microorganisms. MMPs are involved in regulating numerous physiological processes, pathological processes, and immune responses. In addition, MMPs play a key role in disease occurrence, including tumors, cardiovascular diseases, and other diseases. Compared with invertebrate MMPs, vertebrate MMPs have diverse subtypes and complex functions. Therefore, it is difficult to study the function of MMPs in vertebrates. However, it is relatively easy to study invertebrate MMPs because there are fewer subtypes of MMPs in invertebrates. In the present review, the structure and function of MMPs in invertebrates were summarized, which will provide a theoretical basis for investigating the regulatory mechanism of MMPs in invertebrates.