tRNA-m1A modification promotes T cell expansion via efficient MYC protein synthesis.

Naive T cells undergo radical changes during the transition from dormant to hyperactive states upon activation, which necessitates de novo protein production via transcription and translation. However, the mechanism whereby T cells globally promote translation remains largely unknown. Here, we show that on exit from quiescence, T cells upregulate transfer RNA (tRNA) m1A58 'writer' proteins TRMT61A and TRMT6, which confer m1A58 RNA modification on a specific subset of early expressed tRNAs. These m1A-modified early tRNAs enhance translation efficiency, enabling rapid and necessary synthesis of MYC and of a specific group of key functional proteins. The MYC protein then guides the exit of naive T cells from a quiescent state into a proliferative state and promotes rapid T cell expansion after activation. Conditional deletion of the Trmt61a gene in mouse CD4+ T cells causes MYC protein deficiency and cell cycle arrest, disrupts T cell expansion upon cognate antigen stimulation and alleviates colitis in a mouse adoptive transfer colitis model. Our study elucidates for the first time, to our knowledge, the in vivo physiological roles of tRNA-m1A58 modification in T cell-mediated pathogenesis and reveals a new mechanism of tRNA-m1A58-controlled T cell homeostasis and signal-dependent translational control of specific key proteins.

Chromosome-level genome of putative autohexaploid Actinidia deliciosa provides insights into polyploidisation and evolution.

Actinidia ('Mihoutao' in Chinese) includes species with complex ploidy, among which diploid Actinidia chinensis and hexaploid Actinidia deliciosa are economically and nutritionally important fruit crops. Actinidia deliciosa has been proposed to be an autohexaploid (2n = 174) with diploid A. chinensis (2n = 58) as the putative parent. A CCS-based assembly anchored to a high-resolution linkage map provided a chromosome-resolved genome for hexaploid A. deliciosa yielded a 3.91-Gb assembly of 174 pseudochromosomes comprising 29 homologous groups with 6 members each, which contain 39 854 genes with an average of 4.57 alleles per gene. Here we provide evidence that much of the hexaploid genome matches diploid A. chinensis; 95.5% of homologous gene pairs exhibited >90% similarity. However, intragenome and intergenome comparisons of synteny indicate chromosomal changes. Our data, therefore, indicate that if A. deliciosa is an autoploid, chromosomal rearrangement occurred following autohexaploidy. A highly diversified pattern of gene expression and a history of rapid population expansion after polyploidisation likely facilitated the adaptation and niche differentiation of A. deliciosa in nature. The allele-defined hexaploid genome of A. deliciosa provides new genomic resources to accelerate crop improvement and to understand polyploid genome evolution.

The Genome of Vitis zhejiang-adstricta Strengthens the Protection and Utilization of the Endangered Ancient Grape Endemic to China.

Vitis zhejiang-adstricta (V. zhejiang-adstricta) is one of the most important and endangered wild grapes. It is a national key protected wild, rare and endangered ancient grape endemic to China and used as a candidate material for resistance breeding owing to its excellent significant disease resistance. Here, we present a high-quality chromosome-level assembly of V. zhejiang-adstricta (IB-VB-01), comprising 506.66 Mb assembled into 19 pseudo-chromosomes. The contig N50 length is 3.91 Mb with 31,196 annotated protein-coding genes. Comparative genome and evolutionary analyses illustrated that V. zhejiang-adstricta has a specific position in the evolution of East Asian Vitis and shared a common ancestor with Vitis vinifera during the divergence of the two species about 10.42 (between 9.34 and 11.12) Mya. The expanded gene families compared with those in plants were related to disease resistance, and constructed gene families were related to plant growth and primary metabolism. With the analysis of gene family expansion and contraction, the evolution of environmental adaptability and especially the NBS-LRR gene family of V. zhejiang-adstricta was elucidated based on the pathways of resistance genes (R genes), unique genes and structural variations. The near-complete and accurate diploid V. zhejiang-adstricta reference genome obtained herein serves as an important complement to wild grape genomes and will provide valuable genomic resources for investigating the genomic architecture of V. zhejiang-adstricta as well as for improving disease resistance breeding strategies in grape.

Discovery of a first-in-class CDK2 selective degrader for AML differentiation therapy.

The discovery of effective therapeutic treatments for cancer via cell differentiation instead of antiproliferation remains a great challenge. Cyclin-dependent kinase 2 (CDK2) inactivation, which overcomes the differentiation arrest of acute myeloid leukemia (AML) cells, may be a promising method for AML treatment. However, there is no available selective CDK2 inhibitor. More importantly, the inhibition of only the enzymatic function of CDK2 would be insufficient to promote notable AML differentiation. To further validate the role and druggability of CDK2 involved in AML differentiation, a suitable chemical tool is needed. Therefore, we developed first-in-class CDK2-targeted proteolysis-targeting chimeras (PROTACs), which promoted rapid and potent CDK2 degradation in different cell lines without comparable degradation of other targets, and induced remarkable differentiation of AML cell lines and primary patient cells. These data clearly demonstrated the practicality and importance of PROTACs as alternative tools for verifying CDK2 protein functions.

Safety, tolerability, pharmacokinetics and immunogenicity of an antibody-drug conjugate (SHR-A1201) in patients with HER2-positive advanced breast cancer: an open, phase I dose-escalation study.

SHR-A1201 is an antibody-drug conjugate (ADC) that combines trastuzumab with DM1 (a chemotherapeutic agent) using a chemical connector. This phase I study investigated the safety, tolerability and pharmacokinetics of SHR-A1201 in patients with human epidermal growth factor receptor 2-positive advanced breast cancer. This phase I study enrolled patients in a traditional 3 + 3 dose-escalation design to receive a single dose of SHR-A1201 (1.2 mg/kg, 2.4 mg/kg, 3.6 mg/kg or 4.8 mg/kg). The observation period of dose-limiting toxicity (DLT) was 21 days. A total of 12 patients were enrolled and received SHR-A1201. Most treatment-emergent adverse events (TEAEs) were grade 1 or 2 in severity, with elevated aspartate aminotransferase (75%), thrombocytopenia (75%), and nausea (66.7%) being reported most frequently. The common grade 3 TEAEs were thrombocytopenia and decreased lymphocyte count, and there were no grade 4 or above TEAEs. There were no serious adverse events or drug-related deaths. One DLT occurred in one patient treated with SHR-A1201 4.8 mg/kg (asymptomatic grade 3 increased γ-glutamyltransferase). The maximum tolerated dose of SHR-A1201 was not lower than that of T-DM1 (3.6 mg/kg). A total of 8.3% (1/12) of patients had ADA-positive reactions 504 h after administration, but no differences were observed in the type, incidence, or severity of TEAEs between patients with and without ADA. SHR-A1201 exhibited the pharmacokinetics characteristics of typical ADCs. An encouraging antitumor effect was observed in the 4.8 mg/kg dose group. SHR-A1201 was well tolerated and safe in patients with advanced HER2-positive breast cancer. The pharmacokinetics parameters showed a linear trend, and the immunogenicity results met the clinical expectations.

20(S)-protopanaxatriol inhibited D-galactose-induced brain aging in mice via promoting mitochondrial autophagy flow.

Previous reports have confirmed that saponins (ginsenosides) derived from Panax ginseng. C. A. Meyer exerted obvious memory-enhancing and antiaging effects, and the simpler the structure of ginsenosides, the better the biological activity. In this work, we aimed to explore the therapeutic effect and underlying molecular mechanism of 20(S)-protopanaxatriol (PPT), the aglycone of panaxatriol-type ginsenosides, by establishing D-galactose (D-gal)-induced subacute brain aging model in mice. The results showed that PPT treatment (10 and 20 mg/kg) for 4 weeks could significantly restore the D-gal (800 mg/kg for 8 weeks)-induced impaired memory function, choline dysfunction, and redox system imbalance in mice. Meanwhile, PPT also significantly reduced the histopathological changes caused by D-gal exposure. Moreover, PPT could increase TFEB/LAMP2 protein expression to promote mitochondrial autophagic flow. Importantly, the results from molecular docking showed that PPT had good binding ability with LAMP2 and TFEB, suggesting that TFEB/LAMP2 might play an important role in PPT to alleviate D-gal-caused brain aging.

Runoff, sediment, organic carbon, and nutrient loads from a Canadian prairie micro-watershed under climate variability and land management practices.

This study conducted a spatio-temporal analysis of runoff, total suspended sediment, suspended particulate carbon, nitrogen, and phosphorus loadings within the 2.06 km2 Steppler subwatershed in southern Manitoba of Canada based on 11 years of field monitoring data collected at nine stations. Results showed that the nutrient losses were very small because of the implementation of multiple BMPs in the study area. However, a high spatio-temporal variation of runoff and water quality parameters was found for the nine fields within the subwatershed. The average runoff coefficient was 0.19 at the subwatershed outlet with sediment, suspended particulate carbon, total nitrogen, and total phosphorus losses of 73.8, 6.10, 4.54, and 0.76 kg/ha respectively. Spring snowmelt runoff was about 74.5% of the annual runoff at the subwatershed outlet, while for sediment, suspended particulate carbon, total nitrogen, and total phosphorus, the proportions were 61.1%, 63.6%, 74.9%, and 81.2% respectively during the monitoring period, which suggests that BMPs designed for reducing nutrient loadings from snowmelt runoff would be more effective than BMPs designed for reducing pollutant loading from rainfall storms in the study area. Research findings from this study will benefit the enhancement of current BMPs and the development of new BMPs in the region to minimize soil and nutrient losses from agricultural fields and improve water quality in receiving water bodies.

Chromosome-level genome assembly and population genetic analysis of a critically endangered rhododendron provide insights into its conservation.

Rhododendrons are woody plants, famous throughout the world as having high horticultural value. However, many wild species are currently threatened with extinction. Here, we report for the first time a high-quality, chromosome-level genome of Rhododendron griersonianum, which has contributed to approximately 10% of all horticultural rhododendron varieties but which in its wild form has been evaluated as critically endangered. The final genome assembly, which has a contig N50 size of approximately 34 M and a total length of 677 M, is the highest-quality genome sequenced within the genus to date, in part due to its low heterozygosity (0.18%). Identified repeats constitute approximately 57% of the genome, and 38 280 protein-coding genes were predicted with high support. We further resequenced 31 individuals of R. griersonianum as well as 30 individuals of its widespread relative R. delavayi, and performed additional conservation genomic analysis. The results showed that R. griersonianum had lower genetic diversity (θ = 2.58e-3; π = 1.94e-3) when compared not only to R. delavayi (θ = 11.61e-3, π = 12.97e-3), but also to most other woody plants. Furthermore, three severe genetic bottlenecks were detected using both the Stairway plot and fastsimcoal2 analysis, which are thought to have occurred in the late Middle Pleistocene and the Last Glacial Maximum (LGM) period. After these bottlenecks, R. griersonianum recovered and maintained a constant effective population size (>25 000) until now. Intriguingly, R. griersonianum has accumulated significantly more deleterious mutations in the homozygous state than R. delavayi, and several deleterious mutations (e.g., in genes involved in the response to heat stress) are likely to have harmed the adaptation of this plant to its surroundings. This high-quality, chromosome-level genome and the population genomic analysis of the critically endangered R. griersonianum will provide an invaluable resource as well as insights for future study in this species to facilitate conservation and in the genus Rhododendron in general.

The genome of Shanputao (Vitis amurensis) provides a new insight into cold tolerance of grapevine.

Vitis amurensis (Shanputao) is the most cold tolerant Vitis species and so is of great interest to grape breeders and producers in areas with low winter temperatures. Here, we report its high-quality, chromosome-level genome assembly based on a combination of sequence data from Illumina and PacBio platforms, BioNano optical mapping and high-throughput chromosome conformation Capture (Hi-C) mapping. The 604.56-Mb genome contains 32 885 protein-coding genes. Shanputao was found to share a common ancestor with PN40024 (V. vinifera) approximately 2.17-2.91 million years ago, and gene expansion observed in Shanputao might contribute to the enhancement of cold tolerance. Transcriptome analysis revealed 17 genes involved in cold signal transduction, suggesting that there was a different response mechanism to chilling temperature and freezing conditions. Furthermore, a genome-wide association study uncovered a phosphoglycerate kinase gene that may contribute to the freezing resistance of buds in the winter. The Shanputao genome sequence not only represents a valuable resource for grape breeders, but also is important for clarifying the molecular mechanisms involved in cold tolerance.

First Demonstration of Novel Vertical Gate-All-Around Field-Effect-Transistors Featured by Self-Aligned and Replaced High-κ Metal Gates.

A novel n-type nanowire/nanosheet (NW/NS) vertical sandwich gate-all-around field-effect-transistor (nVSAFET) with self-aligned and replaced high-κ metal gates (HKMGs) is presented for the first time, aiming at a 3 nm technology node and beyond. The nVSAFETs were fabricated by an integration flow of Si/SiGe epitaxy, quasi-atomic layer etching (qALE) of SiGe selective to Si, formation of SiGe/Si core/shell NS/NW structure, building of nitride dummy gate, and replacement of the dummy gate. This fabrication method is complementary metal oxide semiconductor (CMOS)-compatible, simple, and reproducible, and NWs with a diameter of 17 nm and NSs with a thickness of 20 nm were obtained. Excellent control of short-channel-effects was presented. The device performance was also investigated and discussed. The proposed integration scheme has great potential for applications in chip manufacturing, especially with vertical channel devices.

Proteomic Analysis of Horseweed (Conyza canadensis) Subjected to Caprylic Acid Stress.

Caprylic acid (CAP) is anticipated to be a potential biocontrol herbicide in the control of weeds, however the molecular mechanism of how CAP affects weeds is poorly understood. Here, the physiological and biochemical (protein-level) changes in horseweed (Conyza canadensis L.) are studied under CAP treatment, with infrared gas analyzer and label-free quantitative proteomics methods. In total, 112 differentially-accumulated proteins (DAPs) (>1.5 fold change, p < 0.05) are present between treated horseweed and control samples, with 46 up-regulated and 66 down-regulated proteins. These DAPs are involved in 28 biochemical pathways, including photosynthesis pathways. In particular, six photosynthesis proteins show significant abundance changes in the CAP-treated horseweed. The qRT-PCR results confirm three of the six genes involved in photosynthesis. Moreover, by measuring photosynthesis characteristics, CAP was shown to decrease photosynthetic rate, stomatal conductance, intercellular CO2 concentration, and the transpiration rate of horseweed. These results suggest that photosystem I is one of the main biological processes involved in the response of horseweed to CAP.

Neighbouring crop diversity mediates the effect of Bt cotton on insect community and leaf damage in fields.

Effects of large-scale cultivation of transgenic crops on agricultural biodiversity remain unclear, particularly in the context of complex ecological interactions between transgenic crops and other organisms. Here we conducted a comprehensive survey to investigate the number of species, population abundance, community evenness and dominance of insects and weeds as well as leaf damage to weeds in Bt and non-Bt cotton fields at 27 sites across northern China. The role of neighbouring crop diversity around cotton fields in controlling insects and weeds in the cotton fields was also assessed. In addition, we conducted a 3-year field experiment to verify the results of the survey. Weed diversity in Bt and non-Bt cotton fields was similar, but the species number and diversity indices of insects are significantly decreased in Bt fields aligning with reduced leaf damage to broadleaf plant species including cotton as well as crops in neighbouring plots. The leaf damage to Bt and non-Bt cotton negatively associates with the diversity of neighbouring crops in cotton fields. Our study demonstrates the neighbouring crop diversity mediates the effects of Bt crops on agricultural diversity in complex interactions among transgenic crops, in-field weed and insect communities, and neighbouring crops.

One species to another: sympatric Bt transgene gene flow from Brassica napus alters the reproductive strategy of wild relative Brassica juncea under herbivore treatment.

Background and Aims: Since pollen flow or seed dispersal can contribute to transgene persistence in the environment, the sympatric presence of transgenic crops with their wild relatives is an ecological concern. In this study, we tested the hypothesis that proximate growth of a herbivore-resistant Bt crop and wild relatives coupled with the presence of herbivores can increase relative frequency of crop-to-wild transgene flow persistence outside of cultivation. Methods: We conducted a field experiment using insect enclosures with and without herbivores with cultivated Bt-transgenic Brassica napus (Bt OSR) and wild brown mustard (Brassica juncea) in pure and mixed stands. Low-density diamondback moth (Plutella xylostella) caterpillar infestation treatments were applied and transgene flow and reproductive organs were measured. Key Results: Bt-transgenic B. napus produced more ovules and pollen than wild mustard, but the pollen to ovule (P/O) ratio in the two species was not significantly different. Low-level herbivory had no effects on fitness parameters of Bt OSR or wild brown mustard or on the transgene flow frequency. All progeny from wild brown mustard containing the Bt transgene came from mixed stands, with a gene flow frequency of 0.66 %. In mixed stands, wild brown mustard produced less pollen and more ovules than in pure stands of brown mustard. This indicates a decreased P/O ratio in a mixed population scenario. Conclusions: Since a lower P/O ratio indicates a shift in sex allocation towards relatively greater female investment and a higher pollen transfer efficiency, the presence of transgenic plants in wild populations may further increase the potential transgene flow by altering reproductive allocation of wild species.

A novel sgRNA selection system for CRISPR-Cas9 in mammalian cells.

CRISPR-Cas9 mediated genome editing system has been developed as a powerful tool for elucidating the function of genes through genetic engineering in multiple cells and organisms. This system takes advantage of a single guide RNA (sgRNA) to direct the Cas9 endonuclease to a specific DNA site to generate mutant alleles. Since the targeting efficiency of sgRNAs to distinct DNA loci can vary widely, there remains a need for a rapid, simple and efficient sgRNA selection method to overcome this limitation of the CRISPR-Cas9 system. Here we report a novel system to select sgRNA with high efficacy for DNA sequence modification by a luciferase assay. Using this sgRNAs selection system, we further demonstrated successful examples of one sgRNA for generating one gene knockout cell lines where the targeted genes are shown to be functionally defective. This system provides a potential application to optimize the sgRNAs in different species and to generate a powerful CRISPR-Cas9 genome-wide screening system with minimum amounts of sgRNAs.

Anticancer agent pristimerin inhibits IL-2 induced activation of T lymphocytes.

Pristimerin (PM) is a quinonemethide triterpenoid with cytotoxic activity against a wide range of cancer cell lines. However, the effect of PM on IL-2 induced activation of T lymphocytes, which play a major role in antitumor immunity has not been studied. The objective of the present study was to evaluate the effect of PM on IL-2 induced proliferation of T cells, generation of lymphokine activated killer cells (LAK cells) and the signaling pathways involved in activation of T cells by IL-2. PM inhibited the IL-2 induced proliferation of mouse splenic T cells and the generation LAK cells at very low concentrations. The suppression of T cell proliferation by PM was associated with the inhibition of IL-2 induced Janus kinase/signal transducers and activators of transcription (Jak/STAT) and extracellular signal-regulated kinase 1 and 2 (Erk1/2) signaling pathways. PM also inhibited the proliferation and differentiation-related immediate early gene products such as p-c-fos, p-c-jun, c-myc and cyclin D1. In addition, antiapoptotic (prosurvival) NF-кB, p-Akt and p-mTOR were also inhibited by PM. These data demonstrated that PM inhibits IL-2 induced T cell activation and generation of LAK cells by disrupting multiple cell signaling pathways induced by IL-2.

Mycotoxin verrucarin A inhibits proliferation and induces apoptosis in prostate cancer cells by inhibiting prosurvival Akt/NF-kB/mTOR signaling.

Trichothecenes are powerful mycotoxins that inhibit protein synthesis and induce ribotoxic stress response in mammalian cells. Verrucarin A (VC-A) is a Type D macrocyclic mycotoxin which inhibits cell proliferation and induces apoptosis in cancer cells. However, the antitumor activity of VC-A for prostate cancer cells has not been investigated. The objective of the present study was to determine the anticancer activity and its mechanism of action in hormone-responsive (LNCaP) and hormone-refractory (PC-3) carcinoma of the prostate (CaP) cell lines. VC-A strongly inhibited the proliferation and induced cell cycle arrest in G2/M phase associated with the inhibition of cell cycle regulatory proteins cyclin D, cyclin E, cyclin-dependent kinases (cdks) cdk2, cdk4, cdk6 and cdk inhibitors WAF1/21 and KIP1/27. VC-A also induced apoptosis in CaP cells as characterized by the cleavage of poly (ADP-ribose) polymerase (PARP-1), procaspases-3, -8 and -9 and the inhibition of Bcl-2 family proteins that regulate apoptosis (Bcl-2, Bcl-xL, Bax, Bak and Bad). In addition, VC-A also down-regulated the expression of prosurvival phospho-AKT (p-AKT), nuclear factor kappa B (NF-kB) (p65) and phospho-mammalian target of rapamycin (p-mTOR) signaling proteins. Taken together, these results demonstrated strong antiproliferative and apoptosis-inducing activity of verrucarin A against CaP cells through cell cycle arrest and inhibition of the prosurvival (antiapoptotic) AKT/NF-kB/mTOR signaling pathway.

Gambogenic acid inhibits LPS-simulated inflammatory response by suppressing NF-κB and MAPK in macrophages.

Inflammation is a response of body tissues to injury and infection. Compounds that can inhibit inflammation have been shown to have potential therapeutic clinical application. Gambogenic acid (GEA) has potent antitumor and anti-inflammatory activities. Herein, the molecular mechanisms of GEA's anti-inflammatory effect were investigated in lipopolysaccharide (LPS)-stimulated macrophage cells. The results showed that pretreatment with GEA could markedly inhibit interleukin (IL)-1α, IL-1ß, tumor necrosis factor-α, IFN-ß, IL-12b, and IL-23a production in a dose-dependent manner in LPS-induced model. Furthermore, this drug significantly reduced the release of nitric oxide (NO), and impaired the protein level of inducible NO synthase and the cyclooxygenase 2. The finding also showed that the effect of GEA may be related to the suppression of the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathway. These results indicate that GEA could suppress LPS-simulated inflammatory response partially by attenuating NO synthesis and NF-κB and MAPK activation, suggesting that it may become a potent therapeutic agent for the treatment of inflammatory diseases.

The fate of fusion Cry1Ab/1Ac proteins from Bt-transgenic rice in soil and water.

Toxin proteins form transgenic crops entering into the environment are likely affect non-target organisms. To investigate the entry route and fate of fusion Cry1Ab/1Ac proteins from transgenic rice expressing insecticide toxins from Bacillus thuringiensis (Bt) in soil and water, we conducted greenhouse and field experiments in 2013 and 2014. Cry1Ab/1Ac proteins from Bt-transgenic rice in soil was found within a horizontal range of 25cm, where most of plant roots distributed. Concentration of Cry1Ab/1Ac proteins was lower in water than in soil in the greenhouse experiment, and no Cry1Ab/1Ac protein was detected in field water. Cry1Ab/1Ac concentration from rice straws was higher in ditch water than in distilled water due to the existence of aquatic organisms in ditch water. Bt proteins from transgenic crops enter into soil ecosystems mainly through root exudates and into aquatic ecosystems through plant residues, which determines Bt fate in the environment.

Examining water quality effects of riparian wetland loss and restoration scenarios in a southern ontario watershed.

Wetland conservation has two important tasks: The first is to halt wetland loss and the second is to conduct wetland restoration. In order to facilitate these tasks, it is important to understand the environmental degradation from wetland loss and the environmental benefits from wetland restoration. The purpose of the study is to develop SWAT based wetland modelling to examine water quality effects of riparian wetland loss and restoration scenarios in the 323-km(2) Black River watershed in southern Ontario, Canada. The SWAT based wetland modelling was set up, calibrated and validated to fit into watershed conditions. The modelling was then applied to evaluate various scenarios of wetland loss from existing 7590 ha of riparian wetlands (baseline scenario) to 100% loss, and wetland restoration up to the year 1800 condition with 11,237 ha of riparian wetlands (100% restoration). The modelling was further applied to examine 100% riparian wetland loss and restoration in three subareas of the watershed to understand spatial pattern of water quality effects. Modelling results show that in comparing to baseline condition, the sediment, total nitrogen (TN), and total phosphorus (TP) loadings increase by 251.0%, 260.5%, and 890.9% respectively for 100% riparian wetland loss, and decrease by 34.5%, 28.3%, and 37.0% respectively for 100% riparian wetland restoration. Modelling results also show that as riparian wetland loss increases, the corresponding environmental degradation worsens at accelerated rates. In contrast, as riparian wetland restoration increases, the environmental benefits improve but at decelerated rates. Particularly, the water quality effects of riparian wetland loss or restoration show considerable spatial variations. The watershed wetland modelling contributes to inform decisions on riparian wetland conservation or restoration at different rates. The results further demonstrate the importance of targeting priority areas for stopping riparian wetland loss and initiating riparian wetland restoration based on scientific understanding of watershed wetland effects.