Development and validation of a clinical prediction model for the risk of distal metastasis in intrahepatic cholangiocarcinoma: a real-world study.

BACKGROUND: Cholangiocarcinoma (CCA) is a highly malignant and easily metastatic bile duct tumor with poor prognosis. We aimed at studying the associated risk factors affecting distal metastasis of CCA and using nomogram to guide clinicians in predicting distal metastasis of CCA. METHODS: Based on inclusion and exclusion criteria, 345 patients with CCA were selected from the Fifth Medical Center of Chinese PLA General Hospital and were divided into distal metastases (N = 21) and non-distal metastases (N = 324). LASSO regression models were used to screen for relevant parameters and to compare basic clinical information between the two groups of patients. Risk factors for distal metastasis were identified based on the results of univariate and multivariate logistic regression analyses. The nomogram was established based on the results of multivariate logistic regression, and we drawn the corresponding correlation heat map. The predictive accuracy of the nomogram was evaluated by receiver operating characteristic (ROC) curves and calibration plots. The utility of the model in clinical applications was illustrated by applying decision curve analysis (DCA), and overall survival(OS) analysis was performed using the method of Kaplan-meier. RESULTS: This study identified 4 independent risk factors for distal metastasis of CCA, including CA199, cholesterol, hypertension and margin invasion, and developed the nomogram based on this. The result of validation showed that the model had significant accuracy for diagnosis with the area under ROC (AUC) of 0.882 (95% CI: 0.843-0.914). Calibration plots and DCA showed that the model had high clinical utility. CONCLUSIONS: This study established and validated a model of nomogram for predicting distal metastasis in patients with CCA. Based on this, it could guide clinicians to make better decisions and provide more accurate prognosis and treatment for patients with CCA.

An ethylene-responsive transcription factor and a flowering locus KH domain homologue jointly modulate photoperiodic flowering in chrysanthemum.

The timely transition from vegetative to reproductive development is coordinated through the quantitative regulation of floral pathway genes in response to physiological and environmental cues. The function of ethylene-responsive element-binding protein (ERF) transcription factors in the regulation of flowering in chrysanthemum (Chrysanthemum morifolium Ramat.) is not well understood. Here, chrysanthemum overexpressing CmERF110 flowered earlier than the wild-type plants, while those in which CmERF110 was suppressed flowered later. RNA-seq results revealed that several genes involved in the circadian rhythm were transcribed differently in CmERF110 transgenic plants from that of the wild-type plants. The rhythm peak of the circadian clock genes in transgenic plants was delayed. Yeast two-hybrid screening of CmERF110 interactors identified a chrysanthemum FLOWERING LOCUS KH DOMAIN (FLK) homologue CmFLK, which was further confirmed with both in vitro and in vivo assays. KEGG pathway enrichment also revealed that CmFLK is involved in the regulation of circadian rhythm-related genes. CmFLK transgenic plants showed a change in flowering time and delayed rhythm peak of the circadian rhythm genes. Taken together, the present data not only suggest that CmERF110 interacts with CmFLK to promote floral transition by tuning the circadian clock, but also provides evidence for the evolutionary conservation of the components in the autonomous pathway in chrysanthemum.

Physiological and Transcripts Analyses Reveal the Mechanism by Which Melatonin Alleviates Heat Stress in Chrysanthemum Seedlings.

Heat stress limits the growth and development of chrysanthemum seedlings. Although melatonin (MT) has been linked to the heat stress response in plants, research on the underlying molecular mechanisms is scarce. In this study, the regulatory networks of MT on heat stress in chrysanthemum seedlings were explored. Physiological measurements suggested that MT not only reduced malondialdehyde accumulation, hydrogen peroxide content, and superoxide anion free radical generation rate, but also significantly promoted osmotic regulation substance synthesis (proline and soluble protein), antioxidant accumulation (GSH and AsA), and the antioxidant enzyme activities (SOD, POD, CAT, and APX) in chrysanthemum leaves under heat stress. Furthermore, MT increased the fresh weight, dry weight, chlorophyll content, photosynthesis rate, and gas exchange indexes. Further, RNA-seq results revealed 33,497 and 36,740 differentially expressed genes in the S/Con and SMT/ConMT comparisons, respectively. The differences in the comparisons revealed that MT regulated heat shock transcription factors (HSFs) and heat shock proteins (HSPs), and the genes involved in Ca2+ signal transduction (CNGCs and CAM/CMLs), starch and sucrose metabolism (EDGL, BGLU, SuS, and SPS), hormone (PP2Cs, AUX/IAAs, EBFs, and MYC2), chlorophyll metabolism (HEMA and PORA), flavonoid biosynthesis (CHS, DFR, and FNS), and carotenoid biosynthesis (DXPS, GGDP, and PSY). MT effectively improved chrysanthemum seedling heat-resistance. Our study, thus, provides novel evidence of a gene network regulated by MT under heat stress.

Integrated Signals of Jasmonates, Sugars, Cytokinins and Auxin Influence the Initial Growth of the Second Buds of Chrysanthemum after Decapitation.

Decapitation is common in horticulture for altering plant architecture. The decapitation of chrysanthemum plants breaks apical dominance and leads to more flowers on lateral branches, resulting in landscape flowers with good coverage. We performed both third- and second-generation transcriptome sequencing of the second buds of chrysanthemum. This third-generation transcriptome is the first sequenced third-generation transcriptome of chrysanthemum, revealing alternative splicing events, lncRNAs, and transcription factors. Aside from the classic hormones, the expression of jasmonate-related genes changed because of this process. Sugars also played an important role in this process, with upregulated expression of sucrose transport-related and TPS genes. We constructed a model of the initial growth of the second buds after decapitation. Auxin export and sugar influx activated the growth of these buds, while the JA-Ile caused by wounding inhibited the expression of CycD genes from 0 h to 6 h. After wound recovery, cytokinins accumulated in the second buds and might have induced ARR12 expression to upregulate CycD gene expression from 6 h to 48 h, together with sugars. Therefore, jasmonates, cytokinins, sugars, and auxin work together to determine the fate of the buds of plants with short internodes, such as chrysanthemum.

A single residue change in the product of the chrysanthemum gene TPL1-2 leads to a failure in its repression of flowering.

The TPL/TPR co-repressor is involved in many plant signaling pathways, including those regulating the switch from vegetative to reproductive growth. Here, a TPL homolog (TPL 1-2) was isolated from chrysanthemum. Its product was found to be deposited in the nucleus. The abundance of TPL1-2 transcript varied across the plant, with its highest level being recorded in the stem apex, and its lowest in the root and stem. In the leaf, the abundance of TPL1-2 transcript was highest at dusk in plants exposed to long days, and at dawn in those exposed to short days. Site-directed mutagenesis was used to induce an N176H mutation in TPL1-2. The constitutive expression in Arabidopsis thaliana of the wild type and the mutated alleles of TPL1-2 had a contrasting effect on flowering time, with the mutant transgene expressors flowering later than the wild type transgene expressors. The flowering-related genes FT, TSF, FUL and AP1 were all more strongly transcribed in the mutant transgene expressors than in the wild type transgene expressors.

Strigolactone represses the synthesis of melatonin, thereby inducing floral transition in Arabidopsis thaliana in an FLC-dependent manner.

The transition from vegetative to reproductive growth is a key developmental event in a plant's life cycle. The process is mediated by a combination of phytohormones, including melatonin (MT) and strigolactone (SL). Here, the Arabidopsis mutants, d14-1 and max4-1, which are compromised with respect to either SL synthesis or signaling, were shown to flower earlier than wild types. The tissue MT content in both mutants was higher than in wild types, as a result of the up-regulation of various genes encoding enzymes involved in MT synthesis. The abundance in the mutants of transcripts derived from each of the genes SPLs, AP1, and SOC1 was reduced with exogenously supplied MT, while FLC was induced. Plants exposed to a high concentration of MT did not flower earlier than wild types. The tissue MT content of a mutant unable to synthesize caffeic acid O-methyltransferase was less than that of wild type and flowered earlier than did wild types. The suggestion is that the flowering time of Arabidopsis is altered if the tissue content of MT is either higher than ~ 8 ng/g F.W, or lower than ~ 0.9 ng/g. Within this range, SL acts to determine flowering time by its regulation of SPL genes. The application of exogenous SL reduces tissue MT content. The flowering time of the flc-3 mutant was unaffected by exogenously supplying either MT or/and SL. It is proposed that MT acts downstream of SL to activate FLC, inducing a delay to flowering if its concentration lies outside a certain range.

The Constitutive Expression of a Chrysanthemum ERF Transcription Factor Influences Flowering Time in Arabidopsis thaliana.

AP2/ERF transcription factors (TFs) represent valuable targets for the genetic manipulation of crop plants, as they participate in the control of metabolism, growth and development, as well as in the plants' response to environmental stimuli. Here, an ERF TF encoded by the chrysanthemum (Chrysanthemum morifolium) genome, designated CmERF110, was cloned and functionally characterized. The predicted CmERF110 polypeptide included a conserved DNA-binding AP2/ERF domain. A transient expression experiment revealed that the protein was deposited in the nucleus, and a transactivation experiment in yeast suggested that it had no transcriptional activity. The gene was transcribed in the chrysanthemum root, stem and leaf, with its transcript level following a circadian rhythm under both long and short days. The effect of constitutively expressing the gene in Arabidopsis thaliana was to accelerate flowering. Transcriptional profiling implied that its effect on floral initiation operated through the photoperiod pathway.

Transgenic Arabidopsis Plants Expressing Grape Glutathione S-Transferase Gene (VvGSTF13) Show Enhanced Tolerance to Abiotic Stress.

Although glutathione S-transferase (GST, EC 2.5.1.18) is thought to play important roles in abiotic stress, limited information is available regarding the function of its gene in grapes. In this study, a GST gene from grape, VvGSTF13, was cloned and functionally characterized. Transgenic Arabidopsis plants containing this gene were normal in terms of growth and maturity compared with control plants but had enhanced resistance to salt, drought, and methyl viologen stress. The increased tolerance of the transgenic plants correlated with changes in activities of antioxidative enzymes. Our results indicate that the gene from grape plays a positive role in improving tolerance to salinity, drought, and methyl viologen stresses in Arabidopsis.

Differentiation of glycated residue numbers on heat-induced structural changes of bovine serum albumin.

BACKGROUND: Glycation is an approach in dealing with heat-induced protein aggregation. The relationship between degree of glycation and heat-induced structural changes is still unclear. The present work investigates the effect of different numbers and sites of glycated residues on heat-induced structural changes of bovine serum albumin (BSA). Glycation of BSA was carried out with xylose (Xyl) and galactose (Gal) by Maillard reaction. Glycated residues in BSA were identified by liquid chromatography-tandem mass spectrometry, and heat-induced protein structural changes were characterized by fluorescence emission and synchronous fluorescence spectra, 8-anilino-1-naphthalenesulfonic acid fluorescence, Fourier transform infrared (FTIR) and circular dichroism (CD) spectra. RESULTS: The numbers of glycated residues were 2 and 13 when BSA was glycated by Gal (BSA - Gal) and Xyl (BSA - Xyl), respectively. There were shifts of maximum wavelengths and decreases in fluorescence intensities for both intrinsic and extrinsic fluorescences; shifts of FTIR amide I, III, and A bands; and decrease or increase of CD band intensities, α-helix and ß-sheet percentages when BSA was heated. Glycation with Gal or Xyl restrained in similar degrees those changes, including fluorescence wavelengths, amide I band, CD band intensities, and α-helix and ß-sheet percentages. CONCLUSION: Xyl glycated more residues than Gal, while their effects were similar in restraining heat-induced BSA structural changes. © 2017 Society of Chemical Industry.

Over-expression of AtGSTU19 provides tolerance to salt, drought and methyl viologen stresses in Arabidopsis.

The plant-specific tau class of glutathione S-transferases (GSTs) is often highly stress-inducible and expressed in a tissue-specific manner, thereby suggesting its important protective roles. Although activities associated with the binding and transport of reactive metabolites have been proposed, little is known about the regulatory functions of GSTs. Expression of AtGSTU19 is induced by several stimuli, but the function of this GST remains unknown. In this study, we demonstrated that transgenic over-expressing (OE) plants showed enhanced tolerance to different abiotic stresses and increased percentage of seed germination and cotyledon emergence. Transgenic plants exhibited an increased level of proline and activities of antioxidant enzymes, along with decreased malonyldialdehyde level under stress conditions. Real-time polymerase chain reaction (PCR) analyses revealed that the expression levels of several stress-regulated genes were altered in AtGSTU19 OE plants. These results indicate that AtGSTU19 plays an important role in tolerance to salt/drought/methyl viologen stress in Arabidopsis.

Identification of a phosphinothricin-resistant mutant of rice glutamine synthetase using DNA shuffling.

To date, only bar/pat gene derived from Streptomyces has been used to generate the commercial PPT-resistant crops currently available in the market. The limited source of bar/pat gene is probably what has caused the decrease in PPT-tolerance, which has become the main concern of those involved in field management programs. Although glutamine synthetase (GS) is the target enzyme of PPT, little study has been reported about engineering PPT-resistant plants with GS gene. Then, the plant-optimized GS gene from Oryza sativa (OsGS1S) was chemically synthesized in the present study by PTDS to identify a GS gene for developing PPT-tolerant plants. However, OsGS1S cannot be directly used for developing PPT-tolerant plants because of its poor PPT-resistance. Thus, we performed DNA shuffling on OsGS1S, and one highly PPT-resistant mutant with mutations in four amino acids (A63E, V193A, T293A and R295K) was isolated after three rounds of DNA shuffling and screening. Among the four amino acids substitutions, only R295K was identified as essential in altering PPT resistance. The R295K mutation has also never been previously reported as an important residue for PPT resistance. Furthermore, the mutant gene has been transformed into Saccharomyces cerevisiae and Arabidopsis to confirm its potential in developing PPT-resistant crops.

Transgenic Arabidopsis Plants Expressing Tomato Glutathione S-Transferase Showed Enhanced Resistance to Salt and Drought Stress.

Although glutathione S-transferases (GST, EC 2.5.1.18) are involved in response to abiotic stress, limited information is available regarding gene function in tomato. In this study, a GST gene from tomato, designated LeGSTU2, was cloned and functionally characterized. Expression profile analysis results showed that it was expressed in roots and flowers, and the transcription was induced by salt, osmotic, and heat stress. The gene was then introduced to Arabidopsis by Agrobacterium tumefaciens-mediated transformation. Transgenic Arabidopsis plants were normal in terms of growth and maturity compared with wild-type plants. Transgenic plants also showed an enhanced resistance to salt and osmotic stress induced by NaCl and mannitol. The increased tolerance of transgenic plants was correlated with the changes in proline, malondialdehyde and antioxidative emzymes activities. Our results indicated that the gene from tomato plays a positive role in improving tolerance to salinity and drought stresses in Arabidopsis.

Outcomes of Coaxial Micro-incision Phacoemulsification in Nanophthalmic Eyes: Report of Retrospective Case Series.

PURPOSE: The surgical risk and complication rate after cataract surgery are extremely high in patients with nanophthalmos. This study is designed to compare the visual and refractive outcomes before and after coaxial micro-incision phacoemulsification and evaluate postoperative complications. METHODS: Fifty nine patients (89 eyes) with axial length (AL) < 21 mm diagnosed with nanophthalmos were enrolled in this retrospective study. All patients underwent coaxial micro-incision phacoemulsification and IOL implantation. The main outcome measures included anterior chamber depth (ACD), anterior chamber volume (ACV), anterior chamber angle (A CA), intraocular pressure (IOP) and best corrected visual acuity (BCVA). Wilcoxon signed rank test or Mann-Whitney test, and Chi-square test and logistic regression analysis were performed for statistical tests as appropriate. RESULTS: The median AL was 19.63 mm. Sixty-six eyes (74.16%) had a history of surgical intervention. Postoperative ACD, ACV and ACA were increased significantly (all P < 0.001), whereas postoperative IOP was decreased significantly (P < 0.001) after surgery. Previous surgical intervention was related to a reduction in the postoperative ACD and ACA (P < 0.01), and both preoperative and postoperative IOP (P < 0.001). Postoperative BCVA was improved in 94.38% of the cases. Intraoperative complications mainly included iridoschisis (6 eyes, 6.74%). Early postoperative complications included temporary corneal edema (TCE) (23 eyes, 25.84%), anterior inflammatory response (AIR) (19 eyes, 21.35%), cystoid macular edema (CME) (14 eyes, 15.73%), and uveal effusion (4 eyes, 4.49%). Late postoperative complications included CME (8 eyes, 8.99%), uveal effusion (8 eyes, 8.99%), malignant glaucoma (2 eyes, 2.25%) and posterior capsular opacification (PCO) (10 eyes, 11.24%). The majority of complications (80%) were successfully resolved by pharmacotherapy or operation. The risk of surgical complications was greater in patients with lower AL, ACD, ACV or ACA and higher nuclear hardness or mean keratometry (Km). CONCLUSION: With reasonable preoperative management, prudent selection of the lens, rigorous surgical technique and unerring cognition of potential complications, coaxial microincision phacoemulsification lens surgery can be performed in patients with nanophthalmos and yield favorable outcomes and a low incidence of complications.

Functional characterization of 5-enopyruvylshikimate-3-phosphate synthase from Alkaliphilus metalliredigens in transgenic Arabidopsis.

Although a large number of AroA enzymes (EPSPS: 5-enopyruvylshikimate-3-phosphate synthase) have been identified, cloned, and tested for glyphosate resistance, only two AroA variants, derived from Agrobacterium tumefaciens strain CP4 and Zea mays, have been utilized to produce the commercial glyphosate-resistant crops. Here, we have used a PCR-based twostep DNA synthesis method to synthesize an aroA gene (aroAA. metalliredigens) from Alkaliphilus metalliredigens, encoding a new EPSPS. Furthermore, transgenic Arabidopsis with the new aroAA. metalliredigens gene was obtained to confirm the potential of the novel aroA gene in developing glyphosate-resistant crops.

Inhibition of ROS elevation and damage to mitochondrial function prevents lead-induced neurotoxic effects on structures and functions of AFD neurons in Caenorhabditis elegans.

Here we investigated the possible roles of oxidative stress in the formation of decreased thermotaxis to cultivation temperature in lead (Pb)-exposed nematodes Caenorhabditis elagans. Exposure to Pb at the examined concentrations decreased thermotaxis behaviors, and induced severe deficits in the structural properties of AFD sensory neurons. Meanwhile, Pb exposure caused the induction of severe oxidative damage, reactive oxygen species (ROS) production, and mitochondrial dysfunction in young adults. Moreover, pre-treatment with the antioxidants dimethyl sulfoxide (DMSO), ascorbate and N-acetyl-L-cysteine (NAC), used to inhibit both the ROS elevation and the mitochondrial dysfunction caused by Pb exposure, at the L2-larval stage prevented the induction of oxidative damage and the formation of severe deficits in thermotaxis and structural properties of AFD sensory neurons in Pb-exposed young adults. Therefore, the formation of oxidative stress caused by Pb exposure may be due to both the induction of ROS elevation and damage to mitochondrial function, and oxidative stress may play a key role in inducing the neurotoxic effects on the structures and function of AFD sensory neurons in Pb-exposed nematodes.

MicroRNA-145 targets vascular endothelial growth factor and inhibits invasion and metastasis of osteosarcoma cells.

MicroRNAs are important gene regulators that play a profound role in tumorigenesis. MicroRNA-145 (miR-145), an important member in the family of microRNAs, is under-expressed in several types of tumors and acts as a tumor suppressor. The role and probable pathways of miR-145 in osteosarcoma carcinogenesis are still unknown. In this study, we found that miR-145 was significantly under-expressed in osteosarcoma tissues, and the over-expression of miR-145 could inhibit invasion and angiopoiesis of osteosarcoma cells. Furthermore, the results showed that vascular endothelial growth factor (VEGF) expression was down-regulated in osteosarcoma cells after miR-145 transfection. On the basis of these results, we performed the luciferase assay and verified that miR-145 could down-regulate VEGF at the translational level by partially binding to VEGF 3' untranslated region (3'UTR). Therefore, it can be concluded that miR-145 can inhibit invasion and metastasis of osteosarcoma cells. One of the mechanisms is the down-regulation of VEGF expression by miR-145 by binding to the 3'UTR of VEGF mRNA specifically. These novel findings may have extensive implications for an effective gene therapy of osteosarcoma.

Targeted silencing of heparanase gene by small interfering RNA inhibits invasiveness and metastasis of osteosarcoma cells.

The effects of targeted silencing of heparanase gene by small interfering RNA (siRNA) on invasiveness and metastasis of osteosarcoma cells (MG63 cells) were investigated in the present study. Two complementary oligonucleotide strands were synthesized and inserted into pGenesil-1 vector based on the mRNA sequence of heparanase gene. The expression vector containing short hairpin RNA (pGenesil-shRNA) was constructed successfully. MG63 cells were randomly allocated into 3 groups: blank group, empty vector (pGenesil) transfected group and expression vector (pGenesil-shRNA) transfected group. Under the induction of Lipofectamine 2000, the recombinants were transfected into MG63 cells. Heparanase gene expression level was detected by RT-PCR and Western blotting. Cell proliferation was measured by MTT assay. Cell invasiveness and metastasis were examined by cell adhesion and Transwell-ECM assays. HUVECs migration assay was applied for the detection of angiogenesis. As compared with negative controls, the mRNA and protein expression levels of heparanase were down-regulated by 76.1% (P<0.01) and 75.3% (P<0.01) respectively in the pGenesil-shRNA transfected group. Meanwhile, the proliferation, adhesiveness, invasiveness and angiogenesis properties of MG63 cells were all significantly inhibited. It was suggested that targeted silencing of heparanase gene by siRNA could dramatically inhibit the invasiveness and metastasis of osteosarcoma cells.

Pre-treatment with mild UV irradiation suppresses reproductive toxicity induced by subsequent cadmium exposure in nematodes.

In nematodes, 10 J/m(2)/min of UV irradiation induced a mild reproductive toxicity. Pre-treatment with UV irradiation at 10 J/m(2)/min suppressed the formation of reproductive defects, and activated a noticeable reduction of percentage of population with hsp-16.2::gfp expression, an obvious elevation of superoxide dismutase activities, and decrease of oxidative damage in 50 and 100 microM Cd exposed nematodes; however, pre-treatment with UV irradiation at 20 J/m(2)/min caused a significant decrease of brood sizes or increase of generation times in Cd-exposed nematodes. Pre-treatment with mild UV irradiation did not suppress the formation of reproductive defects in 150 microM Cd-exposed nematodes. Furthermore, the adaptive response to reproductive toxicity from Cd exposure was not observed in a reactive oxygen species sensitive mev-1(kn1) mutant. Therefore, pre-treatment with mild UV irradiation triggers the resistance to reproductive toxicity from Cd exposure by at least partially inducing adaptation to oxidative stress and through a mev-1-dependent pathway.

Pre-treatment with mild UV irradiation increases the resistance of nematode Caenorhabditis elegans to toxicity on locomotion behaviors from metal exposure.

UV irradiation at 10J/m(2)/min induced a mild toxicity on locomotion behaviors and stress response in Caenorhabditis elegans. Pre-treatment with UV irradiation at 10J/m(2)/min at L2-larva stage prevented the formation of locomotion behavioral defects, and activated a noticeable reduction of stress response and oxidative damage in 50 and 100µM metal (Hg, Pb, and Cr) exposed nematodes. Pre-treatment with UV irradiation at 20J/m(2)/min caused a significant decrease of locomotion behaviors in metal exposed nematodes, and pre-treatment with mild UV irradiation could not prevent the formation of locomotion behavioral defects in 200µM metal exposed nematodes. Moreover, the adaptive response to toxicity on locomotion behaviors induced by metal exposure was not formed in mev-1 mutants. Therefore, pre-treatment to mild UV irradiation activates the cross-adaptation response to toxicity on locomotion behaviors induced by metal exposure, and this kind of adaptive response may be under the control of MEV-1 function.

Using the larvae nematode Caenorhabditis elegans to evaluate neurobehavioral toxicity to metallic salts.

In this study, we investigated the locomotion behavior changes at different developmental stages in Caenorhabditis elegans exposed to metals for 4h. No obvious differences could be observed in young adults exposed to examined metals, and only exposure to 100 microM of examined metals could significantly decrease the locomotion behaviors of L4 larvae. In contrast, exposure to 50 and 100 microM of examined metals induced noticeable repression of locomotion behaviors at L1-L3 larval stages, and a significant decrease of locomotion behaviors could be observed in L1 larvae exposed to Pb and Hg, and in L2 larvae exposed to Hg at the concentration of 2.5 microM. Moreover, the L1-, L2-, and L3-larvae exposed to metals for 4h exhibited similar neurobehavioral toxicity manner to L4-larvae exposed to metals for 24h. Therefore, younger larvae showed more severe deficits in neurobehavioral phenotypes than L4 larvae and young adults in metal-exposed nematodes.