Shading decreases lodging resistance of wheat under different planting densities by altering lignin monomer composition of stems.

To clarify the influences of shading stress and planting density on the lignin monomer composition of wheat stems and their relationship with lodging resistance, Lodging resistant variety Shannong 23 (SN23) and lodging sensitive variety Shannong 16 (SN16) were grown during 2018-2019 and 2019-2020 growing seasons. The planting densities were 150 × 104 plants ha-1 (D1), 225 × 104 plants ha-1 (D2) and 300 × 104 plants ha-1 (D3). At the jointing stage, an artificial shading shed was used to simulate shading stress. Then the effects of shading on stem morphological characteristics, lignin monomer composition and lodging resistance of wheat under different planting densities were studied. Results indicate that shading at the jointing stage increased the length of basal internodes and the plant height and moved the height of center of gravity (CG) upward. Moreover, the stem diameter and the wall thickness decreased by 0.10-0.53 mm and 0.18-0.40 mm, respectively. The stem filling degree was reduced accordingly. As indicated by the correlation analysis and the stepwise regression analysis, shading-induced lodging mainly resulted from changes in the stem morphological characteristics and lignin accumulation. The influential magnitude of these factors was ordered as follows: stem filling degree, wall thickness, lignin content, contents and proportions of monomers S and H, and length of the second internode. The expression abundance of TaPAL, TaCOMT, TaCCR, and TaCAD declined in response to shading stress and high planting density. As a result, the distribution ratios of photosynthetic carbon sources to lignin monomers S, G and H were changed. The lignin content of stems on the day 42 after the jointing stage decreased by 18.48%. The monomer S content decreased, while the content and proportion of monomer H increased, thus weakening the breaking strength of wheat stems.

Response of Lignin Metabolism to Light Quality in Wheat Population.

The low red/far-red (R/FR) light proportion at the base of the high-density wheat population leads to poor stem quality and increases lodging risk. We used Shannong 23 and Shannong 16 as the test materials. By setting three-light quality treatments: normal light (CK), red light (RL), and far-red light (FRL), we irradiated the base internodes of the stem with RL and FRL for 7h. Our results showed that RL irradiation enhanced stem quality, as revealed by increased breaking strength, stem diameter, wall thickness and, dry weight per unit length, and the total amount of lignin and related gene expression increased, at the same time. The composition of lignin subunits was related to the lodging resistance of wheat. The proportion of S+G subunits and H subunits played a key role in wheat lodging resistance. RL could increase the content of S subunits and G subunits and the proportion of S+G subunits, reduce the proportion of H subunits. We described here, to the best of our knowledge, the systematic study of the mechanism involved in the regulation of stem breaking strength by light quality, particularly the effect of light quality on lignin biosynthesis and its relationship with lodging resistance in wheat.

Identification of plant hormones and candidate hub genes regulating flag leaf senescence in wheat response to water deficit stress at the grain-filling stage.

In order to clarify the transcriptional regulatory network and physiological mechanisms governing leaf senescence response to drought stress in wheat, experiments were performed using two wheat varieties with contrasting drought tolerance: Fu287 (F287, a drought-sensitive genotype) and Shannong20 (SN20, a drought-resistant genotype). The latter has higher SPAD values, salicylic acid (SA), jasmonic acid (JA), zeatin (Z), zeatin riboside (ZR), and gibberellin (GA 3) content as well as higher expression levels of Cu/Zn-SOD, Mn-SOD, Fe-SOD,POD,CAT, and APX under various water deficit conditions. Conjoint analysis of physiological and biochemical indicators and transcriptome data by weighted gene co-expression network analysis (WGCNA) in the present study provides a useful genomic and molecular resource for studying drought adaptation in wheat. The flag leaf senescence process was changed by altering the concentration of phytohormones. SA, JA, abscisic acid (ABA), Z, ZR, and GA 3 coordinate with each other to control leaf senescence and plant adaptation under drought stress. Further, the leaf senescence process was divided into two phases: the persistence phase and the rapid loss phase. Shorter Chltotal (duration of the flag leaf being photosynthetically active), shorter Chlper (persistence phase), reduced M (inflection point cumulative temperature when senescence rate is the maximum), decreased r max (the maximum senescence rate), larger r 0 (the initial senescence rate), and increased r aver (the average senescence rate) were slightly associated with low grain mass. We speculated that extending the period of the persistence phase by cultivation or chemical control measures could further increase the drought survivability and productivity of wheat.

Deacetylation of ß-catenin by SIRT1 regulates self-renewal and oncogenesis of liver cancer stem cells.

Hepatocellular carcinoma (HCC) is a highly malignant liver tumor. The presence of cancer stem cells (CSCs) figures prominently in tumor invasion, therapeutic resistance and tumor recurrence resulting in poor outcome and limited therapeutic options. Wnt/ß-catenin signaling is essential for cancer stem cell regulation and tumorigenesis in HCC, but its molecular mechanisms are not fully understood. Here, we demonstrate that ß-catenin is overexpressed in liver CSCs, and its expression level is positively correlated with SIRT1 in HCC specimens. SIRT1 regulates the protein stability of ß-catenin, thereby affecting the transcriptional activity of Wnt/ß-catenin signaling in liver CSCs. Mechanistically, we show that nuclear accumulation of ß-catenin results from deacetylation mediated by SIRT1. Further, nuclear ß-catenin promotes the transcription of Nanog to help maintain self-renewal of liver CSCs. Taken together, our findings indicate that the deacetylation of ß-catenin by SIRT1 represents a critical mechanism for regulating liver CSCs self-renewal and tumorigenesis. It provides an improved understanding of molecular mechanisms underlying ß-catenin activation and tumorigenesis in HCC.

Aquaporin 1 is located on the intestinal basolateral membrane in Toxocara canis and might play a role in drug uptake.

BACKGROUND: Aquaporins (AQPs) are a family of integral membrane channel proteins that facilitate the transport of water and other small solutes across cell membranes. AQPs appear to play crucial roles in parasite survival and represent possible drug targets for novel intervention strategy. In this work, we investigated the tissue distribution and biological roles of an aquaporin TcAQP1 in the neglected parasitic nematode Toxocara canis. METHODS: Recombinant C-terminal hydrophilic domain of AQP1 of T. canis (rTcAQP1c) and polyclonal antibody against rTcAQP1c were produced to analyse the tissue expression of native TcAQP1 in adult (female and male) worms using an immunohistochemical approach. RNA interference (RNAi), quantitative real-time PCR (qRT-PCR) and nematocidal assays were performed to investigate the functional roles of TcAQP1 in the adult stage of T. canis. RESULTS: Immunofluorescence analysis showed that TcAQP1 was localised predominantly in the epithelial linings of the reproductive tract and basolateral membrane of the intestine in the adult stage (female and male) of T. canis, indicating important roles in reproduction, nutrient absorption and/or osmoregulation. Treatment with silencing RNA for 24 h resulted in a significant reduction of Tc-aqp-1 mRNA level in adult T. canis, though no phenotypical change was observed. The efficient gene knockdown compromised the nematocidal activity of albendazole in vitro, suggesting the role of TcAQP1 in drug uptake. CONCLUSIONS: The findings of this study provide important information about tissue expression and functional roles of TcAQP1 protein in adult T. canis. Understanding the biological functions of this protein in other developmental stages of T. canis and related parasitic nematodes would contribute to the discovery of novel diagnostic or anthelmintic targets.

Optimizing plant density and nitrogen application to manipulate tiller growth and increase grain yield and nitrogen-use efficiency in winter wheat.

The growth of wheat tillers and plant nitrogen-use efficiency (NUE) will gradually deteriorate in response to high plant density and over-application of N. Therefore, in this study, a 2-year field study was conducted with three levels of plant densities (75 ×104plants ha-1, D1; 300 ×104plants ha-1, D2; 525 ×104plants ha-1, D3) and three levels of N application rates (120 kg N ha-1, N1; 240 kg N ha-1, N2; 360 kg N ha-1, N3) to determine how to optimize plant density and N application to regulate tiller growth and to assess the contribution of such measures to enhancing grain yield (GY) and NUE. The results indicated that an increase in plant density significantly increased the number of superior tillers and the number of spikes per m2(SN), resulting in a higher GY and higher partial factor productivity of applied N (PFPN). However, there was no significant difference in GY and PFPN between plant densities D2 and D3. Increasing the N application rate significantly increased the vascular bundle number (NVB) and area (AVB), however, excess N application (N3) did not significantly improve these parameters. N application significantly increased GY, whereas there was a significant decrease in PFPN in response to an increase in N application rate. The two years results suggested that increasing the plant density (from 75 ×104plants ha-1to 336 ×104plants ha-1) in conjunction with the application of 290 kg N ha-1N will maximize GY, and also increase PFPN(39.7 kg kg-1), compared with the application of 360 kg N ha-1N. Therefore, an appropriate combination of increased planting density with reduced N application could regulate tiller number and favor the superior tiller group, to produce wheat populations with enhanced yield and NUE.

Unravelling mesosulfuron-methyl phytotoxicity and metabolism-based herbicide resistance in Alopecurus aequalis: Insight into regulatory mechanisms using proteomics.

Non-target-site based resistance (NTSR), a poorly understood multigenic trait, has evolved as the greatest threat to crop production worldwide, by endowing weed plants an unpredictable pattern of resistance to herbicides. Our recent work with multiple-herbicide-resistant shortawn foxtail (Alopecurus aequalis Sobol.) biotype has preliminary indicated that cytochrome P450s-involved enhanced rate of mesosulfuron-methyl metabolism may involve in the NTSR. Here by further determining the differences in glutathione S-transferase (GST) activity and uptake and metabolic rates of mesosulfuron between resistant (R) and susceptible (S) A. aequalis plants, and associating them with endogenous differently regulated proteins (DEPs) identified from combinational proteomics analyses, we provided direct evidences on the enhanced herbicide degradation in resistant plants. Subsequently, the physiological phenotypes of photosynthesis, chlorophyll fluorescence, and antioxidation were compared between R and S plants and linked with correlative DEPs, indicating a series of key pathways including solar energy capture, photosynthetic electron transport, redox homeostasis, carbon fixation, photorespiration, and reactive oxygen species scavenging in susceptible plants were broken or severely damaged by mesosulfuron stress. In comparison, resistant plants have evolved enhanced herbicide degradation to minimize the accumulation of mesosulfuron and protect the photosynthesis and ascorbate-glutathione cycle against the adverse effects of chemical injury, giving A. aequalis plants a NTSR phenotype. Additionally, three key proteins respectively annotated as esterase, GST, and glucosyltransferase were identified and enabled as potential transcriptional markers for quick diagnosing the metabolic mesosulfuron resistance in A. aequalis species.

Association Between Activation of the Programmed Cell Death-1 (PD-1)/Programmed Death-Ligand 1 (PD-L1) Pathway and Pain in Patients with Cancer.

BACKGROUND The aim of this study was to investigate the clinical correlation between sPD-1 (soluble programmed cell death-1) and PD-1 (programmed cell death-1) expression and cancer pain. MATERIAL AND METHODS sPD-1 content in peripheral blood was determined by enzyme-linked immunosorbent assay (ELISA). T cell surface-positive rate was determined by flow cytometry, and the correlation of clinical characteristics of patients with cancer pain was analyzed. RESULTS The positive expression rate of PD-1 in sPD-1 and T cells of patients with cancer pain was higher than that in normal patients. There was a significant correlation between sPD-1 and PD-1 positivity on T cell surface with tumor type, differentiation degree, and VAS scores of patients with cancer pain (P<0.05). Peripheral blood sPD-1 level and PD-1 positivity in patients with liver cancer and melanoma cancer were higher than those in patients with renal cell carcinoma and breast cancer. In addition, peripheral blood sPD-1 level and PD-1 positivity in patients with poorly-differentiated cancer pain were higher than those in patients with intermediately- to well-differentiated cancer. The sPD-1 content was lower and PD-1 positivity rate was higher in cancer pain patients with low VAS scores. CONCLUSIONS The positive expression rate of sPD-1 and PD-1 in patients with cancer pain is higher than that in normal people. The activation rate of the PD-1/PD-L1 pathway was mediated by sPD-1 and PD-1 positive expression, age, tumor type, and differentiation. There are correlations between clinical characteristics such as degree and pain level as shown by VAS score.

Preparation of pocket shaped microfiltration membranes with binary porous structures.

Highly permeable pocket-shaped microfiltration membranes with binary porous structures, which are composed of brominated poly(2,6-dimethyl-1,4-phenylene oxide) (BPPO), were prepared on needles by breath figure (BF) and colloidal crystal template (CCT) methods. In colloidal crystal templates, the membrane pore size in the bottom layer was adjusted by SiO2 microsphere diameter in the colloidal crystal template, while that in the top layer was adjusted by changing the BPPO concentration. The permeability of the binary porous membrane prepared by BF and CCT methods was higher than that of membranes only prepared by the BF method. Due to high permeability and antifouling properties, the pocket shaped binary porous membrane was connected to a syringe and used as a filter film in microfiltration and sample preparation fields.

Tissue expression pattern of ABCG transporter indicates functional roles in reproduction of Toxocara canis.

Toxocara canis is a zoonotic parasite with worldwide distribution. ATP-binding cassette (ABC) transporters are integral membrane proteins which involve in a range of biological processes in various organisms. In present study, the full-length coding sequence of abcg-5 gene of T. canis (Tc-abcg-5) was cloned and characterized. A 633 aa polypeptide containing two conserved Walker A and Walker B motifs was predicted from a continuous 1902 nt open reading frame. Quantitative real-time PCR was employed to determine the transcriptional levels of Tc-abcg-5 gene in adult male and female worms, which indicated high mRNA level of Tc-abcg-5 in the reproductive tract of adult female T. canis. Tc-abcg-5 was expressed to produce rabbit polyclonal antiserum against recombinant TcABCG5. Indirect-fluorescence immunohistochemical assays were carried out to detect the tissue distribution of TcABCG5, which showed predominant distribution of TcABCG5 in the uterus (especially in the germ cells) of adult female T. canis. Tissue transcription and expression pattern of Tc-abcg-5 indicated that Tc-abcg-5 might play essential roles in the reproduction of this parasitic nematode.

Development and validation of a sensitive UPLC-MS/MS instrumentation and alkaline nitrobenzene oxidation method for the determination of lignin monomers in wheat straw.

A method to determine the lignin monomers (p-hydroxybenzaldehyde, vanillin and syringaldehyde) in plant cell wall of wheat internode was developed and validated using a high-throughput nitrobenzene oxidation step and ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) for quantification. UPLC analyses were carried out using an reversed phase C18 column (ACQUITY UPLC BEH, 1.7µm, 2.1×100mm) and gradient elution with water and acetonitrile. This method was completely validated in terms of analyzing speed, linearity, sensitivity, limits of detection (LODs) and limits of quantification (LOQs).The three lignin monomers were successfully separated within 6min and only 2min were required to regain its equilibrium. The method linearity with regression coefficients values (R2) greater than 0.997. Additionally, LODs ranged from 0.21 to 0.89µgL-1 and LOQs ranged from 0.69 to 2.95µgL-1. The applicability of this analytical approach for determining the three lignin monomers was confirmed by the successful analysis of real samples of wheat stem internodes. The nitrobenzene oxidation method was used for the analysis of lignin monomers. We have optimized the treatment temperature (170°C, 1h) and realized the high-throughput using the microwave digestion instrument. Recovery of this extraction method ranged from 68.4% to 77.7%. The analysis result showed that the guaiacyl unit (G) was the major component of lignin and there was a higher content of the syringyl unit (S) than that of the hydroxybenzyl unit (H).

Phase I Escalating-Dose Trial of CAR-T Therapy Targeting CEA+ Metastatic Colorectal Cancers.

Chimeric antigen receptor T (CAR-T) cells have shown promising efficacy in treatment of hematological malignancies, but its applications in solid tumors need further exploration. In this study, we investigated CAR-T therapy targeting carcino-embryonic antigen (CEA)-positive colorectal cancer (CRC) patients with metastases to evaluate its safety and efficacy. Five escalating dose levels (DLs) (1 × 105 to 1 × 108/CAR+/kg cells) of CAR-T were applied in 10 CRC patients. Our data showed that severe adverse events related to CAR-T therapy were not observed. Of the 10 patients, 7 patients who experienced progressive disease (PD) in previous treatments had stable disease after CAR-T therapy. Two patients remained with stable disease for more than 30 weeks, and two patients showed tumor shrinkage by positron emission tomography (PET)/computed tomography (CT) and MRI analysis, respectively. Decline of serum CEA level was apparent in most patients even in long-term observation. Furthermore, we observed persistence of CAR-T cells in peripheral blood of patients receiving high doses of CAR-T therapy. Importantly, we observed CAR-T cell proliferation especially in patients after a second CAR-T therapy. Taken together, we demonstrated that CEA CAR-T cell therapy was well tolerated in CEA+ CRC patients even in high doses, and some efficacy was observed in most of the treated patients.

Involvement of ethylene and polyamines biosynthesis and abdominal phloem tissues characters of wheat caryopsis during grain filling under stress conditions.

Severe water deficit (SD) severely limited the photo-assimilate supply during the grain-filling stages. Although the ethylene and polyamines (PAs) have been identified as important signaling molecules involved in stress tolerance, it is yet unclear how 1-Aminocylopropane-1-carboxylic acid (ACC) and PA biosynthesis involving wheat abdominal phloem characters mitigate SD-induced filling inhibition. The results obtained indicated that the SD down-regulated the TaSUT1 expression and decreased the activities of sucrose synthase (SuSase, EC2.4.1.13), ADP glucose pyrophosphorylase (AGPase, EC2.7.7.27), soluble starch synthase (SSSase, EC2.4.1.21), then substantially limited grain filling. As a result, increased ACC and putrescine (Put) concentrations and their biosynthesis-related gene expression reduced spermidine (Spd) biosynthesis under SD condition. And, the ACC and PA biosynthesis in inferior grains was more sensitive to SD than that in superior grains. Intermediary cells (ICs) of caryopsis emerged prematurely under SD to compensate for the weakened photo-assimilate transport functions of sieve elements (SEs). Finally, plasmolysis and nuclear chromatin condensation of phloem parenchyma cells (PPC) and membrane degradation of SEs, as well as the decreased ATPase activity on plasma membranes of ICs and PPC at the later filling stage under SD were responsible for the considerably decreased weight of inferior grains.

Tissue distribution and functional analysis of vitellogenin-6 of Toxocara canis.

Toxocara canis is an common intestinal nematode of canids and the principal causative agent of human toxocariasis. Vitellogenin (Vg), a source of amino acids and lipids in the eggs, are considered to play an important role in embryo development of a wide range of organisms. In the present study, the transcriptional levels of Tc-vit-6 gene in male and female adult T. canis were determined by quantitative real-time PCR, which indicated high transcription of Tc-vit-6 in the intestine, reproductive tract and body wall of male and female adult T. canis. The fragment of Tc-vit-6 encoding a vWD domain, was cloned and expressed to produce a rabbit anti-TcvWD polyclonal antibody. Tissue distribution of TcVg6 was detected by immunohistochemical assays, which showed predominant distribution of TcVg6 in the tissues of intestine, as well as reproductive tract (including some of the germ cells) and musculature of male and female adult worms. Collectively, these results indicated multiple biological roles of TcVg6 apart from that in the reproduction of T. canis.

Manipulation of lignin metabolism by plant densities and its relationship with lodging resistance in wheat.

Increasing plant density is one of the most efficient ways of increasing wheat (Triticum aestivum L.) grain production. However, overly dense plant populations have an increased risk of lodging. We examined lignin deposition during wheat stem development and the regulatory effects of plant density using the wheat cultivars shannong23 and weimai8. Plants were cultivated at densities of 75, 225 and 375 plants per m2 during two growing seasons. Our results showed that decreasing plant density enhanced culm quality, as revealed by increased culm diameter, wall thickness and dry weight per unit length, and improved the structure of sclerenchyma and vascular bundles by increasing lignification. In addition, more lignins were deposited in the secondary cell walls, resulting in strong lodging resistance. The guaiacyl unit was the major component of lignin and there was a higher content of the syringyl unit than that of the hydroxybenzyl unit. Furthermore, we hypothesised that the syringyl unit may correlate with stem stiffness. We describe here, to the best of our knowledge, the systematic study of the mechanism involved in the regulation of stem breaking strength by plant density, particularly the effect of plant density on lignin biosynthesis and its relationship with lodging resistance in wheat.

Comparative transcriptomic analyses of male and female adult Toxocara canis.

Toxocariasis is an important, neglected zoonosis caused mainly by Toxocara canis. Although our knowledge of helminth molecular biology is improving through completed draft genome projects, there is limited detailed information on the molecular biology of Toxocara species. Here, transcriptomic sequencing of male and female adult T. canis and comparative analyses were conducted. For each sex, two-thirds (66-67%) of quality-filtered reads mapped to the gene set of T. canis, and at least five reads mapped to each of 16,196 (87.1%) of all 18,596 genes, and 321 genes were specifically transcribed in female and 1467 in male T. canis. Genes differentially transcribed between the two sexes were identified, enriched biological processes and pathways linked to these genes established, and molecules associated with reproduction and development predicted. In addition, small RNA pathways involved in reproduction were characterized, but there was no evidence for piwi RNA pathways in adult T. canis. The results of this transcriptomic study should provide a useful basis to support investigations of the reproductive biology of T. canis and related nematodes.2.

MEF2D Transduces Microenvironment Stimuli to ZEB1 to Promote Epithelial-Mesenchymal Transition and Metastasis in Colorectal Cancer.

Epithelial-mesenchymal transition (EMT) is an essential mechanism of metastasis, including in colorectal cancer. Although EMT processes are often triggered in cancer cells by their surrounding microenvironment, how EMT-relevant genes control these processes is not well understood. In multiple types of cancers, the transcription factor MEF2D has been implicated in cell proliferation, but its contributions to metastasis have not been addressed. Here, we show MEF2D is overexpressed in clinical colorectal cancer tissues where its high expression correlates with metastatic process. Functional investigations showed that MEF2D promoted cancer cell invasion and EMT and that it was essential for certain microenvironment signals to induce EMT and metastasis in vivo Mechanistically, MEF2D directly regulated transcription of the EMT driver gene ZEB1 and facilitated histone acetylation at the ZEB1 promoter. More importantly, MEF2D responded to various tumor microenvironment signals and acted as a central integrator transducing multiple signals to activate ZEB1 transcription. Overall, our results define a critical function for MEF2D in upregulating EMT and the metastatic capacity of colorectal cancer cells. Further, they offer new insights into how microenvironment signals activate EMT-relevant genes and deepen the pathophysiologic significance of MEF2D, with potential implications for the prevention and treatment of metastatic colorectal cancer. Cancer Res; 76(17); 5054-67. ©2016 AACR.

Exogenous Cytokinins Increase Grain Yield of Winter Wheat Cultivars by Improving Stay-Green Characteristics under Heat Stress.

Stay-green, a key trait of wheat, can not only increase the yield of wheat but also its resistance to heat stress during active photosynthesis. Cytokinins are the most potent general coordinator between the stay-green trait and senescence. The objectives of the present study were to identify and assess the effects of cytokinins on the photosynthetic organ and heat resistance in wheat. Two winter wheat cultivars, Wennong 6 (a stay-green cultivar) and Jimai 20 (a control cultivar), were subjected to heat stress treatment from 1 to 5 days after anthesis (DAA). The two cultivars were sprayed daily with 10 mg L-1 of 6-benzylaminopurine (6-BA) between 1 and 3 DAA under ambient and elevated temperature conditions. We found that the heat stress significantly decreased the number of kernels per spike and the grain yield (P < 0.05). Heat stress also decreased the zeatin riboside (ZR) content, but increased the gibberellin (GA3), indole-3-acetic acid (IAA), and abscisic acid (ABA) contents at 3 to 15 DAA. Application of 6-BA significantly (P < 0.05) increased the grain-filling rate, endosperm cell division rate, endosperm cell number, and 1,000-grain weight under heated condition. 6-BA application increased ZR and IAA contents at 3 to 28 DAA, but decreased GA3 and ABA contents. The contents of ZR, ABA, and IAA in kernels were positively and significantly correlated with the grain-filling rate (P < 0.05), whereas GA3 was counter-productive at 3 to 15 DAA. These results suggest that the decrease in grain yield under heat stress was due to a lower ZR content and a higher GA3 content compared to that at elevated temperature during the early development of the kernels, which resulted in less kernel number and lower grain-filling rate. The results also provide essential information for further utilization of the cytokinin substances in the cultivation of heat-resistant wheat.

Characterization and differential expression analysis of Toxocara canis aquaporin-1 gene.

Toxocara canis is an intestinal nematode of canids with a worldwide distribution, causing an important but neglected parasitic zoonosis in humans. Aquaporins (AQP) are a family of water channel proteins, which function as membrane channels to regulate water homeostasis. In this study, the coding sequence of aquaporin-1 gene of T. canis (Tc-aqp-1) was cloned and characterized. The obtained Tc-aqp-1 coding sequence was 933 bp in length, which predicted to encode 311 amino acids. Two conserved asparagine-proline-alanine (NPA) motifs were identified in the multiple sequence alignments. Phylogenetic analysis revealed the closest relationship between T. canis and Opisthorchis viverrini based on aquaporin-1 amino acid sequence. A structure was predicted with ligand binding sites predicted at H93, N95, N226, L94, I79, and I210 and with active sites predicted at I256 and G207. Gene Ontology (GO) annotations predicted its cellular component term of integral component of plasma membrane (GO: 0005887), molecular function term of channel activity (GO: 0015250), and biological process term of water transport (GO: 0006833). Tissue expression analysis revealed that the Tc-aqp-1 was highly expressed in the intestine of adult male. The findings of the present study provide the basis for further functional studies of T. canis aquaporin-1.

MicroRNAs of Toxocara canis and their predicted functional roles.

BACKGROUND: Toxocara canis is the causative agent of toxocariasis of humans and other animals. This parasitic nematode (roundworm) has a complex life cycle, in which substantial developmental changes and switches occur. As small non-coding RNAs (sRNAs) are key regulators of gene expression in a wide range of organisms, we explored these RNAs in T. canis to provide a basis for future studies of its developmental biology as well as host interactions and disease at the molecular level. METHODS: We conducted high-throughput RNA sequencing and bioinformatic analyses to define sRNAs in individual male and female adults of T. canis. RESULTS: Apart from snRNA and snoRNA, 560 and 619 microRNAs (miRNAs), including 5 and 2 novel miRNAs, were identified in male and female worms, respectively, without piRNAs being detected in either sex. An analysis of transcriptional profiles showed that, of 564 miRNAs predicted as being differentially transcribed between male and female individuals of T. canis, 218 miRNAs were transcribed exclusively in male and 277 in female worms. Functional enrichment analysis predicted that both male and female miRNAs were mainly involved in regulating embryonic morphogenesis, hemidesmosome assembly and genetic information processing. The miRNAs differentially transcribed between the sexes were predicted to be associated with sex determination, embryonic morphogenesis and nematode larval development. The roles of miRNAs were predicted based on gene ontology (GO) and KEGG pathway annotations. The miRNAs Tc-miR-2305 and Tc-miR-6090 are proposed to have roles in reproduction, embryo development and larval development, and Tc-let-7-5p, Tc-miR-34 and Tc-miR-100 appear to be involved in host-parasite interactions. Together with published information from previous studies, some miRNAs (such as Tc-miR-2861, Tc-miR-2881 and Tc-miR-5126) are predicted to represent drug targets and/or associated with drug resistance. CONCLUSIONS: This is the first exploration of miRNAs in T. canis, which could provide a basis for fundamental investigations of the developmental biology of the parasite, parasite-host interactions and toxocariasis as well as applied areas, such as the diagnosis of infection/disease, drug target discovery and drug resistance detection.