Taking spontaneous plants as a natural strategy for vegetation restoration in construction and demolition waste landfills: a case study in Suzhou, China.

Construction and demolition waste (CDW) landfills around the city have caused serious damage to the ecological environment and menaced the public health. Restoration of closed CDW landfills is critical to compensate for the degraded ecosystem and ensure safety in further development and utilization. Vegetation restoration is an essential part of the restoration of CDW landfills, in which the use of spontaneous plants is the foundation of the nature-based strategy. In this study, Fenghuangshan CDW landfill in Suzhou, China, was selected as the research site, and the species composition and diversity of the spontaneous plants were analyzed. Moreover, the types of habitats and growth indexes of 8 species with high frequency and 18 species with medium frequency in the CDW landfill were investigated, and a comprehensive evaluation of growth rate and expansion capacity of the 26 species was conducted. The results showed that, herbs were the main type of the spontaneous plants in the CDW landfill. The species and quantities of the spontaneous plants in the CDW landfill were obviously fewer than those in the surrounding areas of the CDW landfill, and the Shannon-Wiener index and Pielou index of the spontaneous plants were lower compared with the surrounding areas of the CDW landfill. Meanwhile, the differences of dominant families and the distribution of origins, life forms and growth types between these two fields were insignificant. The heliophilous and drought tolerance species were widely distributed in the CDW landfill while the shade-tolerant or hygrophilous species were few. The relatively large comprehensive evaluation indexes of Elymus dahuricus, Daucus carota, Sonchus asper, Geranium carolinianum, Rumex acetosa, Metaplexis japonica, Carex breviculmis, Erigeron canadensis, Trigonotis peduncularis, Lamium amplexicaule reflected their high growth rates and strong expansion capacity, demonstrating their great potentiality in the vegetation restoration of CDW landfills as indispensable components of the nature-based solution.

Identification of an Acinetobacter pittii acyltransferase involved in transformation of deoxynivalenol to 3-acetyl-deoxynivalenol by transcriptomic analysis.

Deoxynivalenol (DON), a mycotoxin primarily produced by Fusarium graminearum (F. graminearum), is widely present in food and feed, posing great hazards to human and livestock health. In this study, a strain of Acinetobacter pittii (A. pittii) S12 capable of degrading DON was isolated from soil samples and identified through morphological characterization, biochemistry analysis, and 16 S rRNA gene sequencing. The results of HPLC-MS indicated that the degradation products underwent a conversion from [M-H]- to [M+CH3CO], with concomitant transformation of the hydroxyl group into an acetyl moiety. Based on transcriptome sequencing analysis, the acyltransferase encoded by DLK06_RS13370 was predicted to be the pivotal gene responsible for DON biotransformation. The result of molecular docking analysis suggest a high affinity between the enzyme and DON. The recombinant protein encoded by DLK06_RS13370 was expressed in Escherichia coli (E. coli) and demonstrated the capacity to catalyze the conversion of DON into 3-Acetyl-deoxynivalenol (3-ADON), as confirmed by HPLC analysis. In conclusion, our findings confirm that the acyltransferase encoded by DLK06-RS13370 is responsible for the acetylation of DON. This sheds light on the co-occurrence of DON and its acetyl-derivatives in wheat-based products. DATA AVAILABILITY: Not applicable.

Dietary supplementation with Clostridium butyricum improves growth performance of broilers by regulating intestinal microbiota and mucosal epithelial cells.

Clostridium butyricum has been widely considered an antibiotic substitute in recent years. It can promote growth performance, improve the immune response and enhance the intestinal barrier function of the host. In the present study, 1-d-old Arbor Acres (AA) broilers were fed C. butyricum (1 × 109 cfu/kg) for 28 d. The transcriptomic characteristics of epithelial cells of the cecal mucosa were determined by RNA-sequence, and the cecal microbiota composition was explored by 16S ribosomal RNA gene sequencing. The changes in the intestinal mucosa of broilers were then analyzed by tissue staining. Gene Ontology (GO) annotations identified substance transport and processes and pathways that might participate in intestinal development and cell viability. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the differentially expressed genes are involved in numerous pathways related to amino acid and vitamin metabolism and antioxidant and defensive functions, among others. The relative expression of some genes associated with intestinal barrier function (claudins 2, 15, 19, and 23, tight junction proteins 1, 2, and 3 and mucin 1) was significantly increased in the treatment group (P < 0.05 or P < 0.01). Moreover, the proportion of Firmicutes was higher in the C. butyricum-treated group, whereas the proportion of Proteobacteria was higher in the control group. At the genus level, the relative abundances of Butyricicoccus and Lactobacillus, among other bacteria, were increased after C. butyricum supplementation. The tissue staining analysis showed that the cecal mucosa of broilers was significantly ameliorated after the addition of C. butyricum (P < 0.05 or P < 0.01). These results showed that dietary supplementation with C. butyricum can enhance the antioxidant capacity, mucosal barrier function, and stabilize the cecal microbiota, resulting in improving the growth performance.

Physiological and transcriptome changes induced by exogenous putrescine in anthurium under chilling stress.

BACKGROUND: Chilling stress is the major factor limiting plant productivity and quality in most regions of the world. In the present study, we aimed to evaluate the effects of putrescine (Put) and polyamine inhibitor D-arginine (D-arg) on the chilling tolerance of anthurium (Anthurium andraeanum). RESULTS: Anthurium seedlings were pretreated with five different concentrations of Put solution or D-arg solution. Subsequently, the seedlings were subjected to chilling stress at 6 °C for 3 days, followed by a recovery at 25 °C for 1 day. Relative permeability of the plasma membrane, as well as physiological and morphologic parameters was assessed during the experiments. Additionally, transcriptome sequencing and patterns of differential gene expression related to chilling response were analyzed by qRT-PCR in 1.0 mM Put-treated and untreated anthurium seedlings. Results indicated that the supplementation of exogenous Put decreased the extent of membrane lipid peroxidation and the accumulation of malondialdehyde (MDA), promoted the antioxidant activities and proline content and maintained the morphologic performances compared with the control group. This finding indicated that the application of exogenous Put could effectively decrease the injury and maintain the quality of anthurium under chilling conditions. In contrast, the treatment of D-arg exhibited the opposite effects, which confirmed the effects of Put. CONCLUSIONS: This research provided a possible approach to enhance the chilling tolerance of anthurium and reduce the energy consumption used in anthurium production.

Identification of a Bacillus amyloliquefaciens H6 Thioesterase Involved in Zearalenone Detoxification by Transcriptomic Analysis.

Zearalenone (ZEA), a nonsteroidal estrogenic mycotoxin produced by Fusarium graminearum, induces hyperestrogenic responses in mammals and can cause reproductive disorders in farm animals. In this study, a transcriptome analysis of Bacillus amyloliquefaciens H6, which was previously identified as a ZEA-degrading bacterium, was conducted with high-throughput sequencing technology, and the differentially expressed genes were subjected to gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses. Among the 16 upregulated genes, BAMF_RS30125 was predicted to be the key gene responsible for ZEA degradation. The protein encoded by BAMF_RS30125 was then expressed in Escherichia coli, and this recombinant protein (named ZTE138) significantly reduced the ZEA content, as determined by the enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC), and decreased the proliferating activity of ZEA in MCF-7 cells. What is more, the liquid chromatography-tandem mass spectrometry (LC-MS/MS) results showed that the relative molecular mass and the structure of ZEA also changed. Sequence alignment of the ZTE138 protein showed that it is a protease that belongs to the YBGC/FADM family of coenzyme A thioesterases, and thus, the protein can presumably cleave the ZEA lactone bond and break down its macrolide ring.

Gut microbiota profiles of commercial laying hens infected with tumorigenic viruses.

BACKGROUND: Studies have shown that some viral infections cause structural changes in the intestinal microflora, but little is known about the effects of tumorigenic viral infection on the intestinal microflora of chickens. RESULTS: A 29-week commercial layer flock positive for avian leukosis virus-J (ALV-J), Marek's disease virus (MDV) and avian reticuloendotheliosis virus (REV) was selected, and fresh fecal samples were collected and examined for the composition of the gut microflora by Illumina sequencing of the V3-V4 region of the 16S rRNA gene. The operational taxonomic units (OTUs) of the fecal microbiota differentiated the chickens infected with only ALV-J and those coinfected with ALV-J and MDV or REV from infection-negative chickens. The enrichment and diversity of cloacal microflora in chickens infected with ALV-J alone were slightly different from those in the infection-negative chickens. However, the diversity of cloacal microflora was significantly increased in chickens coinfected with both ALV-J and MDV or REV. CONCLUSIONS: The intestinal microbiota was more strongly disturbed in chickens after coinfection with ALV-J and MDV or REV than after infection with ALV-J alone, and there may be underlying mechanisms by which the capacity for the stabilization of the intestinal flora was impaired due to viral infection and tumorigenesis.

Characteristics of the fecal microbiota of high- and low-yield hens and effects of fecal microbiota transplantation on egg production performance.

The microbiota that resides in the digestive tract plays pivotal role in maintaining intestinal environmental stability by promoting nutrition digestion and intestinal mucosal immunity. However, whether the intestinal microbiota in laying hens affects egg laying- performance is not known. In this study, 16S rDNA gene sequencing and fecal microbiota transplantation were used to determine the structure of the intestinal microbiota and the effect of the intestinal microbiota on egg production. The results revealed that Firmicutes were dominant in both the H (high egg laying rates) and L (low egg laying rates) groups, while Bacteroides, Actinobacteria and Proteobacteria were significantly enriched in the L group compared to the H group. The laying rates were weakly affected in H hens transplanted with the fecal microbiota from L hens, except for temporary fluctuation, while the egg laying rates were significantly increased in L hens transplanted with the fecal microbiota from H hens. Therefore, we concluded that the population structure of the intestinal microbiota varied between the H and L groups, and the intestinal microbiota of high-yield laying hens had significant effects on low-yield laying hens performance.

Dataset on evolution analysis of splenic transcriptome in bighead carp and silver carp.

Bighead carp (Aristichthys nobilis) and silver carp (Hypophthalmichthys molitrix) are closely related species in the subfamily Xenocypridinae within Cyprinidae, and they are also two of the four most important pond-cultured fish species in China. The ability to resist some diseases often differs significantly in silver carp and bighead carp during fishery production. However, the evolutionary divergence of the immune defense functions in these two species is still not understood at the molecular level. The data presented in this article are related to the research article entitled "Comparative analysis of spleen transcriptome detects differences in evolutionary adaptation of immune defense functions in bighead carp and silver carp" (Li et al., 2018). Please refer to this data article for interpretation of the data. Data provided in this submission comprise the Ka/Ks ratios of orthologs as well as adaptive evolution genes, expression levels of orthologs, and TPM value of genes expressed only in spleen of bighead carp or silver carp. These data provide a better understanding of the differences in evolutionary adaptation of immune defense functions in bighead carp and silver carp.

MiR-330-3p suppresses phosphoglycerate mutase family member 5 -inducted mitophagy to alleviate hepatic ischemia-reperfusion injury.

Mitochondrial dysfunction plays a central role in hepatic ischemia-reperfusion injury (IRI). The significance of mitophagy in hepatic IRI remains poorly understood. The mechanisms that cause IRI are complex, and many factors are involved in the injury formation process. The miR-330-3p mediates cell proliferation, cell death, and metabolism in various organisms. In this study, the levels of miR-330-3p were significantly downregulated in hepatic IRI, and the number of autophagosomes was increased in response to IRI as obtained under both in vivo and in vitro conditions. These results demonstrate that a reduction in miR-330-3p expression represents an important factor involved with promoting hepatic IRI. Moreover, we found that miR-330-3p interacted with phosphoglycerate mutase family member 5 (PGAM5) to regulate mitophagy. In specific, an overexpression of miR-330-3p diminished PGAM5 levels, which promoted mitophagy in response to IRI. In contrast, a downregulation of miR-330-3p was associated with increased PGAM5 levels leading to increased mitophagy. In conclusion, miR-330-3p suppresses PGAM5-induced mitophagy to alleviate hepatic IRI. Such findings not only reveal some of the mechanistic basis for this microRNA in liver injury, but also provide a foundation for new therapeutic approaches in the treatment of this condition.

Comparative analysis of spleen transcriptome detects differences in evolutionary adaptation of immune defense functions in bighead carp and silver carp.

The evolutionary divergence of the immune defense functions in bighead carp (A. nobilis) and silver carp (H. molitrix) is still not understood at the molecular level. Here, we obtained 48,821,754 and 55,054,480 clean reads from spleen tissue libraries prepared for bighead carp and silver carp using Illumina paired-end sequencing technology, respectively, and identified 4976 orthologous genes from the transcriptome data sets by comparative analysis. Adaptive evolutionary analysis showed that 212 orthologous genes and 255 Gene Ontology (GO) terms were subjected to positive selection(Ka/Ks values > 1) only in bighead carp, and 195 orthologous genes and 309 GO terms only in silver carp. Among immune defense functions with significant evolutionary divergence, the positively selected biological processes in bighead carp mainly included B cell-mediated immunity, chemokine-mediated signaling pathway, and immunoglobulin mediated immune response, whereas those in silver carp mainly included the antigen processing and presentation, defense response to fungus, and detection of bacteria. Moreover, we found 2974 genes expressed only in spleen of bighead carp and 3494 genes expressed only in spleen of silver carp, where these genes were mostly enriched in the same biological processes or pathways. These results provide a better understanding of the differences in resistance to some diseases by bighead carp and silver carp, and also facilitate the identification of candidate genes related to disease resistance.

Formation of iron plaque in the roots of Spartina alterniflora and its effect on the immobilization of wastewater-borne pollutants.

Iron plaque (IP) plays an important role in the absorption of heavy metals (HMs) and nutrients in wetland plants. The present study aims to investigate the effect of IP in Spartina alterniflora on the immobilization of wastewater borne HMs and nutrients. The physiological responses and effect of IP formation on the uptake of HMs, nitrogen (N), and phosphorus (P) were studied in S. alterniflora subjected to different synthetic wastewater (SW) levels and waterlogging durations. Results showed that IP formed in roots of S. alterniflora increased significantly with increasing SW concentration but decreased under prolonged waterlogging. Increasing the waterlogging time enhanced the alcohol dehydrogenase activity and the ethylene content in the roots of S. alterniflora. HMs including Cu, Pb, and Cr, did not significantly accumulate in the IP, despite that the IP content increased with the increasing of SW levels. The SEM-EDX analysis revealed that IP formed on the surface of S. alterniflora did absorb HMs such as Cu, Zn, and Cr. At a fixed level of SW, the amount of HMs that accumulated in the DCB extract was substantially proportional to the IP concentration in the root. Increasing of the SW level enhanced the accumulation of P in the leaves and roots of S. alterniflora. In conclusion, IP formed on S. alterniflora helped immobilize SW pollutants, including HMs and P, and the formation of IP and its effect on pollutant immobilization were influenced by the waterlogging conditions.

Combined effect of water inundation and heavy metals on the photosynthesis and physiology of Spartina alterniflora.

The frequency and duration of tidal flooding significantly influence the bioavailability of heavy metals (HMs) in sediment and hence exert toxicological effects on coastal wetland plants. In this study, the combined effects of different water inundation times (3, 6, 9, and 12 h) and HMs (Cu, Zn, Pb, and Cr) on the photosynthesis and physiology of Spartina alterniflora were investigated under greenhouse conditions. Results showed that S. alterniflora was somehow tolerant to the combined HMs treatments, and only the highest level of HM treatment decreased leaf chlorophyll content. Furthermore, the plants did not show any signs of victimization. Different times of water inundation with HMs did not exert any significant effect on the malonaldehyde (MDA) and chlorophyll contents in the leaves of S. alterniflora at day 20. Prolonged water inundation time at day 60 significantly reduced leaf chlorophyll content with the decrease in leaf photosynthetic rate, which was accompanied by a significant increase in the intercellular concentration of CO2. At day 60, abscisic acid dose-dependently increased along the different water inundation times, indicating that this phytohormone is involved in plant responses to flooding stress. Peroxidase (POD), superoxide dismutase (SOD), and ascorbate peroxidase (APX), showed different responses to the combined treatment of water inundation and HMs at different times. At day 20, the long duration of water inundation and HMs treatments (9 h+HMs and/or 12 h+HMs) significantly increased enzyme activity in the leaves compared with the control group (6 h). At day 60, the POD and SOD activities in the leaves of S. alterniflora decreased with prolonged water inundation time, and root APX activity significantly decreased compared with the 6 h water inundation treatment.

Iron plaque formation and heavy metal uptake in Spartina alterniflora at different tidal levels and waterlogging conditions.

Tidal flat elevation in the estuarine wetland determines the tidal flooding time and flooding frequency, which will inevitably affect the formation of iron plaque and accumulations of heavy metals (HMs) in wetland plants. The present study investigated the formation of iron plaque and HM's (copper, zinc, lead, and chromium) accumulation in S. alterniflora, a typical estuarine wetland species, at different tidal flat elevations (low, middle and high) in filed and at different time (3, 6, 9, 12 h per day) of waterlogging treatment in greenhouse conditions. Results showed that the accumulation of copper, zinc, lead, and chromium in S. alterniflora was proportional to the exchangeable fraction of these metals in the sediments, which generally increased with the increase of waterlogging time, whereas the formations of iron plaque in roots decreased with the increase of waterlogging time. Under field conditions, the uptake of copper and zinc in the different parts of the plants generally increased with the tidal levels despite the decrease in the metals' exchangeable fraction with increasing tidal levels. The formation of iron plaque was found to be highest in the middle tidal positions and significantly lower in low and high tidal positions. Longer waterlogging time increased the metals' accumulation but decreased the formation of iron plaque in S. alterniflora. The binding of metal ions on iron plaque helped impede the uptake and accumulation of copper and chromium in S. alterniflora.

[Determination of guanidinoacetic acid in feed by ion chromatography coupled with ultraviolet detection].

A novel ion chromatography method for the determination of guanidinoacetic acid (GAA) in animal feed samples was developed. The optimal detection mode, chromatography column, gradient condition of mobile phases and sample pretreatment conditions were investigated. Using a linear gradient of methane sulfonic acid and ultraviolet detector at 200 nm, the interfering substances in feed samples and GAA could be separated successfully on a Dionex IonPacTM CS16 cation exchange analysis column. The calibration curves showed a range of linearity between 0.5 and 200 mg/L, and a high linear regression coefficient of 0.9999 was obtained for GAA. The limits of detection and limits of quantification of GAA in formula feed and concentrate feed were 4.5 and 15 mg/kg, respectively. The limit of detection and limit of quantification of GAA in compound-premix were 9.0 and 30 mg/kg, respectively. The recoveries of this method for GAA were investigated, and the recoveries were all more than 94% in formula feed, concentrate feed and compound-premix feed for pig and poultry. This method can meet the requirements of the determination of GAA in feed samples for the feed production enterprise and evaluation center.

Dataset on differential gene expression analysis for splenic transcriptome profiling and the transcripts related to six immune pathways in grass carp.

The data presented in this paper are related to the research article entitled "Transcriptome profiling of developing spleen tissue and discovery of immune-related genes in grass carp (Ctenopharyngodon idella)" (Li et al. 2016) [1]. Please refer to this article for interpretation of the data. Data provided in this submission are comprised of the expression levels of unigenes, significantly differentially expressed genes(DEGs), significant enrichment GO term and KEGG pathway of DEGs, and information of the transcripts assigned to six immune pathways.

Transcriptome profiling of developing spleen tissue and discovery of immune-related genes in grass carp (Ctenopharyngodon idella).

Grass carp Ctenopharyngodon idella is an important freshwater aquaculture species. However, studies regarding transcriptomic profiling of developing spleen tissue in the grass carp are lacking. Here, the transcriptome sequencing from the spleen tissue of one-year-old (cis1) and three-year-old (cis3) grass carp was performed using Illumina paired-end sequencing technology. The de novo assemblies yielded 48,970 unigenes with average lengths of 1264.51 bp from the two libraries. The assembled unigenes were evaluated and functionally annotated by comparing with sequences in major public databases including Nr, COG, Swiss-Prot, KEGG, Pfam and GO. Comparative analysis of expression levels revealed that a total of 38,254 unigenes were expressed in both the cis1 and cis3 libraries, while 4356 unigenes were expressed only in the cis1 library, and 3312 unigenes were expressed only in the cis3 library. Meanwhile, 1782 unigenes (including 903 down-regulated and 879 up-regulated unigenes) were differentially expressed between the two developmental stages of the grass carp spleen. Based on GO and KEGG enrichment analysis, these differentially expressed genes widely participated in the regulation of immunity and response in the grass carp. Moreover, the main components of six immune-related pathways were identified, including complement and coagulation cascades, Toll-like receptor signaling, B-cell receptor signaling, T-cell receptor signaling, antigen processing and presentation, and chemokine signaling. Finally, two identified transcripts including TLR 8 and complement component C8 were validated for reliability by RT-PCR. Collectively, the results obtained in this study will provide a basis for the study of molecular mechanisms in grass carp spleen development.

Dual-wavelength orthogonally polarized radiation generated by a tungsten thermal source.

Developing controllable radiation sources in the mid-infrared spectral region is significant in photonics technology because of rare available resources. Based on the thermal emission from a one-dimensional shallow tungsten grating, we propose a two-dimensional orthogonally-crossed shallow grating to produce an orthogonally-polarized dual-wavelength radiation with the emissivity as high as 78% and 91% from a single surface. The simulation shows that the field is intensively concentrated in vicinity of the air-tungsten interface when surface plasmon polaritons are excited. In addition, by optimizing the geometric parameters of the grating, the field is found to be more concentrated which leads to higher emissivity. The two wavelengths can be produced independently or simultaneously, depending on the polarization of the picking-up polarizer. Our investigations can help us developing controllable multi-wavelength thermal radiation sources from a single surface.

In-column "click" preparation of hydrophobic organic monolithic stationary phases for protein separation.

Two types of macroporous organic polymer monoliths based on glycidyl methacrylate (GMA), 4-vinylbenzyl chloride (VBC) and divinylbenzene (DVB) were prepared inside stainless-steel tubes. Azide functionalities were firstly introduced on the surfaces of poly(GMA-co-DVB) and poly(VBC-co-DVB) monoliths to provide reactive sites for click chemistry. With the application of copper(I)-catalyzed (3 + 2) azide-alkyne cycloaddition, an in-column click-modification approach for covalent attachment of long alkyl chains onto polymer monoliths was developed. The column morphology and surface chemistry of the fabricated monolithic columns were characterized by the scanning electron microscopy, mercury intrusion porosimeter, Fourier transform infrared spectroscopy, and elemental analyses, respectively. The chromatographic performances of the "clicked" stationary phases were demonstrated with the high separation efficiency for a variety of proteins within 4 min.

Room temperature ionic liquid-mediated molecularly imprinted polymer monolith for the selective recognition of quinolones in pork samples.

A novel molecularly imprinted polymer monolith was prepared by the room temperature ionic liquid-mediated in situ molecular imprinting technique, using norfloxacin (NOR) as the template, methacrylic acid as the functional monomer, ethylene glycol dimethacrylate as the cross-linker. The optimal synthesis conditions and recognition properties of NOR-imprinted monolithic column were investigated. The results indicated that the imprinted monoliths exhibited good ability of selective recognition against the template and its structural analog. Using the fabricated material as solid-phase extraction sorbent, a sample pre-treatment procedure of molecularly imprinted solid-phase extraction coupling with HPLC was developed for determination of trace quinolone residues in animal tissues samples. The recoveries ranging from 78.16 to 93.50% for eight quinolones antibiotics such as marbofloxacin, NOR, ciprofloxacin, danofloxacin, difloxacin, oxolinic acid, flumequine and enrofloxacin were obtained.

A facile and efficient strategy for one-step in situ preparation of hydrophobic organic monolithic stationary phases by click chemistry and its application on protein separation.

A simple one-step in situ "click" modification strategy was developed for the preparation of hydrophobic organic monolithic columns for the first time. The column morphology and surface chemistry of the fabricated monolithic columns were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, respectively. The chromatographic performances of the C8/C18 "click" monoliths were evaluated through the separation of a mixture of five proteins such as ribonuclease A, soybean trypsin inhibitor, cytochrome c, bovine haemoglobin and bovine serum albumin. Compared with the blank column, the higher hydrophobicity stationary phases obtained from the "clicked" modification have longer retention times and higher resolution for the five proteins. The separation of five proteins mixture on click C18 monolith with gradient elution at different flow rates was also investigated, the baseline separation of five proteins could be achieved at three different flow rates.