Integrative multiomics enhancer activity profiling identifies therapeutic vulnerabilities in cholangiocarcinoma of different etiologies.

OBJECTIVES: Cholangiocarcinoma (CCA) is a heterogeneous malignancy with high mortality and dismal prognosis, and an urgent clinical need for new therapies. Knowledge of the CCA epigenome is largely limited to aberrant DNA methylation. Dysregulation of enhancer activities has been identified to affect carcinogenesis and leveraged for new therapies but is uninvestigated in CCA. Our aim is to identify potential therapeutic targets in different subtypes of CCA through enhancer profiling. DESIGN: Integrative multiomics enhancer activity profiling of diverse CCA was performed. A panel of diverse CCA cell lines, patient-derived and cell line-derived xenografts were used to study identified enriched pathways and vulnerabilities. NanoString, multiplex immunohistochemistry staining and single-cell spatial transcriptomics were used to explore the immunogenicity of diverse CCA. RESULTS: We identified three distinct groups, associated with different etiologies and unique pathways. Drug inhibitors of identified pathways reduced tumour growth in in vitro and in vivo models. The first group (ESTRO), with mostly fluke-positive CCAs, displayed activation in estrogen signalling and were sensitive to MTOR inhibitors. Another group (OXPHO), with mostly BAP1 and IDH-mutant CCAs, displayed activated oxidative phosphorylation pathways, and were sensitive to oxidative phosphorylation inhibitors. Immune-related pathways were activated in the final group (IMMUN), made up of an immunogenic CCA subtype and CCA with aristolochic acid (AA) mutational signatures. Intratumour differences in AA mutation load were correlated to intratumour variation of different immune cell populations. CONCLUSION: Our study elucidates the mechanisms underlying enhancer dysregulation and deepens understanding of different tumourigenesis processes in distinct CCA subtypes, with potential significant therapeutics and clinical benefits.

Berberine ameliorates iron levels and ferroptosis in the brain of 3 × Tg-AD mice.

BACKGROUND: Berberine (BBR) is a natural alkaloid extracted from the herb Coptis chinensis. This compound has the ability to penetrate the blood-brain barrier (BBB) and exhibit neuroprotective value in the treatment of Alzheimer's disease (AD). AD is a neurodegenerative disease characterized by ß-amyloid (Aß) deposition, hyperphosphorylated tau and other characters. Iron accumulation and ferroptosis were also detected in AD brain, which can result in neuronal damage. However, it is still unclear whether BBR can suppress ferroptosis in AD and alleviate its underlying pathology. PURPOSE: This study investigated whether BBR may affect ferroptosis and related signaling pathways in triple transgenic AD (3 × Tg-AD) mice. METHODS: Four-month-old 3 × Tg-AD mice received oral administration of BBR at a dose of 50 mg/kg for 7.5 months. Cognitive function and anxiety levels in mice were assessed using the morris water maze test, open field test, and novel object recognition test. Western blot, immunohistochemistry, and ICP-MS were employed to assess the pathology of AD, brain iron metabolism, and ferroptosis signaling pathways. Transmission electron microscopy was used to detect mitochondrial changes. The synergistic effects of BBR combined with Nrf2 were investigated using molecular docking programs and surface plasmon resonance technology. Co-inmunoprecipitation assay was used to examine the effect of BBR on the binding ability of Nrf2 and Keap1. RESULTS: The results indicated that chronic treatment of BBR mitigated cognitive disorders in 3 × Tg-AD model mice. Reductions in Aß plaque, hyperphosphorylated tau protein, neuronal loss, and ferroptosis in the brains of 3 × Tg-AD mice suggested that BBR could alleviate brain injury. In addition, BBR treatment attenuated ferroptosis, as evidenced by decreased levels of iron, MDA, and ROS, while enhancing SOD, GSH, GPX4, and SLC7A11. Consistent with the in vivo assay, BBR inhibited RSL3-induced ferroptosis in N2a-sw cells. BBR increased the expression levels of GPX4, FPN1 and SLC7A11 by regulating Nrf2 transcription levels, thereby inhibiting ferroptosis. Molecular docking programs and surface plasmon resonance technology demonstrated the direct combination of BBR with Nrf2. Co-inmunoprecipitation analysis showed that BBR inhibited the interaction between Keap1 and Nrf2. CONCLUSION: For the first time, these results showed that BBR could inhibit iron levels and ferroptosis in the brains of 3 × Tg-AD model mice and partially protect against RSL3-induced ferroptosis via the activation of Nrf2 signaling.

Cardiac-targeted delivery of nuclear receptor RORα via ultrasound targeted microbubble destruction optimizes the benefits of regular dose of melatonin on sepsis-induced cardiomyopathy.

BACKGROUND: Large-dose melatonin treatment in animal experiments was hardly translated into humans, which may explain the dilemma that the protective effects against myocardial injury in animal have been challenged by clinical trials. Ultrasound-targeted microbubble destruction (UTMD) has been considered a promising drug and gene delivery system to the target tissue. We aim to investigate whether cardiac gene delivery of melatonin receptor mediated by UTMD technology optimizes the efficacy of clinically equivalent dose of melatonin in sepsis-induced cardiomyopathy. METHODS: Melatonin and cardiac melatonin receptors in patients and rat models with lipopolysaccharide (LPS)- or cecal ligation and puncture (CLP)-induced sepsis were assessed. Rats received UTMD-mediated cardiac delivery of RORα/cationic microbubbles (CMBs) at 1, 3 and 5 days before CLP surgery. Echocardiography, histopathology and oxylipin metabolomics were assessed at 16-20 h after inducing fatal sepsis. RESULTS: We observed that patients with sepsis have lower serum melatonin than healthy controls, which was observed in the blood and hearts of Sprague-Dawley rat models with LPS- or CLP-induced sepsis. Notably, a mild dose (2.5 mg/kg) of intravenous melatonin did not substantially improve septic cardiomyopathy. We found decreased nuclear receptors RORα, not melatonin receptors MT1/2, under lethal sepsis that may weaken the potential benefits of a mild dose of melatonin treatment. In vivo, repeated UTMD-mediated cardiac delivery of RORα/CMBs exhibited favorable biosafety, efficiency and specificity, significantly strengthening the effects of a safe dose of melatonin on heart dysfunction and myocardial injury in septic rats. The cardiac delivery of RORα by UTMD technology and melatonin treatment improved mitochondrial dysfunction and oxylipin profiles, although there was no significant influence on systemic inflammation. CONCLUSIONS: These findings provide new insights to explain the suboptimal effect of melatonin use in clinic and potential solutions to overcome the challenges. UTMD technology may be a promisingly interdisciplinary pattern against sepsis-induced cardiomyopathy.

Response of microbial communities to nutrient removal in coastal sediment by using ecological concrete.

Ecological concrete (eco-concrete) is a kind of environment-friendly material with porous characteristics. In this study, the eco-concrete was used to remove the total nitrogen (TN), total phosphorus (TP), and total organic carbon (TOC) in marine coastal sediment. The bacterial communities in sediment and on eco-concrete surface were also investigated by using high-throughput sequencing and quantitative PCR of 16S rRNA gene. We found that the mean removal efficiencies of TN, TP, and TOC in treatment group were 8.3%, 8.4%, and 12.3% after 28 days. The bacterial community composition in the treatment group was significantly different from that in the control group on day 28. In addition, the bacterial community composition on eco-concrete surface was slightly different from that in sediment, and the copy numbers of 16S rRNA gene were higher on eco-concrete surface than in sediment. The types of eco-concrete aggregates (gravel, pebble, and zeolite) also had effects on the bacterial community composition and 16S rRNA gene copy numbers. Furthermore, we found the abundant genus Sulfurovum increased significantly on eco-concrete surface in the treatment group after 28 days. Bacteria belonging to this genus were found having denitrification ability and were commonly detected in bioreactors for nitrate removal. Overall, our study expands the application scopes of eco-concrete and suggests that the bacterial communities in eco-concrete can potentially enhance the removal efficiency of nutrients in coastal sediment.

Polyacetylene Isomers Isolated from Bidens pilosa L. Suppress the Metastasis of Gastric Cancer Cells by Inhibiting Wnt/ß-Catenin and Hippo/YAP Signaling Pathways.

(E)-7-Phenyl-2-hepten-4,6-diyn-1-ol (1) and (Z)-7-Phenyl-2-hepten-4,6-diyn-1-ol (2) are isomeric natural polyacetylenes isolated from the Chinese medicinal plant Bidens pilosa L. This study first revealed the excellent anti-metastasis potential of these two polyacetylenes on human gastric cancer HGC-27 cells and the distinctive molecular mechanisms underlying their activities. Polyacetylenes 1 and 2 significantly inhibited the migration, invasion, and adhesion of HGC-27 cells at their non-toxic concentrations in a dose-dependent manner. The results of a further mechanism investigation showed that polyacetylene 1 inhibited the expressions of Vimentin, Snail, ß-catenin, GSK3ß, MST1, YAP, YAP/TAZ, and their phosphorylation, and upregulated the expression of E-cadherin and p-LATS1. In addition, the expressions of various downstream metastasis-related proteins, such as MMP2/7/9/14, c-Myc, ICAM-1, VCAM-1, MAPK, p-MAPK, Sox2, Cox2, and Cyr61, were also suppressed in a dose-dependent manner. These findings suggested that polyacetylene 1 exhibited its anti-metastasis activities on HGC-27 cells through the reversal of the EMT process and the suppression of the Wnt/ß-catenin and Hippo/YAP signaling pathways.

Osteogenic Differentiation of Human Umbilical Cord Blood Mesenchymal Stem Cells Induced by Liu's Zhenggudan No. 2 Formula.

Aim: This study aimed to investigate the potential of Liu's Zhenggudan No. 2 Formula (LZF2) in inducing osteogenic differentiation of human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) and treating osteoporosis (OP), thereby providing new methods and ideas for the treatment of OP by traditional Chinese medicine. Methods: Forty sample rats were equally divided into five groups: high-concentration LZF2, low-concentration LZF2, the Eucommia ulmoides (EU) group, the classical osteogenesis induction (COI) group, and the blank control group. Eight rats in each group were routinely housed for 7 days. Subsequently, to induce hUCB-MSCs, drug-containing serum was extracted from the abdominal aorta of rats to prepare the osteogenic induction solution. In addition, alkaline phosphatase (ALP) activity and osteocalcin (OCN) content assays, and alizarin red staining were performed on days 3, 6, 9, and 12 after culture. Results: After induction of hUCB-MSCs, ALP activity and OCN content increased significantly in the high-concentration LZF2 group. Alizarin red staining also depicted numerous orange-red calcified nodules in rats in the high-concentration LZF2 group. Conclusion: High concentration of LZF2 can facilitate the differentiation of hUCB-MSCs to promote osteogenesis.

Application Value of Nutrition Support Team in Chemotherapy Period of Colon Cancer Based on Internet Multidisciplinary Treatment Mode.

Objective: To explore the application value of the nutrition support team in chemotherapy period of colon cancer based on the internet multidisciplinary treatment mode. Methods: For the method of retrospective study, 90 patients with colon cancer admitted to our hospital from August 2018 to August 2020 were selected as the study subjects. They were equally divided into the experimental group (n = 45) and the control group (n = 45) according to the order of initials and the method of parity group. The control group was given conventional nutrition support, and the experimental group was given the nutrition support under the internet multidisciplinary treatment mode. The serum tumor marker levels (CEA and CA19-9), immune function indexes, nutrition indicators, and the incidence of adverse reactions were compared between the two groups before and after intervention. Results: The serum tumor marker levels in the experimental group after intervention were significantly lower than those in the control group (P < 0.001). The immune function indexes in the experimental group after intervention were significantly better than those in the control group (P < 0.001). The nutrition indicators in the experimental group after intervention were significantly better than those in the control group (P < 0.001). The incidence of gastrointestinal adverse reactions above grade 2 in the experimental group was significantly lower than that in the control group (P < 0.05). There were 20 patients with myelosuppression, 2 patients with neurotoxicity, and 1 patient with hand and foot syndrome in the experimental group, while 22 patients with myelosuppression, 4 patients with neurotoxicity, and 2 patients with hand and foot syndrome in the control group, with no significant difference in the incidence of adverse reactions between the two groups (P > 0.05). Conclusion: The nutrition support team under the internet multidisciplinary treatment mode can improve the immune function of chemotherapy patients with colon cancer and enhance their nutritional level, thereby reducing the incidence of adverse reactions and improving the chemotherapy effects.

Efficacy and safety of long-term transcutaneous electroacupuncture versus sham transcutaneous electroacupuncture for delayed gastric emptying after distal gastrectomy: study protocol for a randomized, patient-assessor blinded, controlled trial.

BACKGROUND: Delayed gastric emptying (DGE) after distal gastrectomy impacts patients' nutritional status and quality of life. The current treatments of DGE seem unsatisfactory or need invasive interventions. It is unknown whether transcutaneous electroacupuncture (TEA) is effective in treating DGE. METHODS: A total of 90 eligible participants who underwent distal gastrectomy will be randomly allocated to either the TEA group (n = 60) or the sham transcutaneous electroacupuncture (sham-TEA) group (n = 30). Each participant will receive TEA on the bilateral acupoints of Zusanli (ST36) and Neiguan (PC6) for 4 weeks. The primary outcomes will be the residual rates of radioactivity in the stomach by gastric scintigraphy and total response rates. The secondary outcomes will be endoscopic features, autonomic function, nutritional and psychological status, serum examination, and quality of life (QoL). The adverse events will also be reported. The patients will be followed up 1 year after the treatment. DISCUSSION: The findings of this randomized trial will provide high-quality evidence regarding the efficacy and safety of long-term TEA for treating DGE after distal gastrectomy. TRIAL REGISTRATION: Chinese Clinical Trial Registry ChiCTR2000033965. Registered on 20 June 2020.

Nutrient Removal in Eutrophic Water Promotes Stability of Planktonic Bacterial and Protist Communities.

Nutrient (nitrogen and phosphorus) removal by using bioremediation technologies in eutrophic water alters bacterial and protist community structure and function, but how it changes the stability of community remains unclear. To fill this gap, in this study, bacterial and protist communities were investigated using 16S and 18S rRNA gene high-throughput sequencing during the nutrient removal by using ecological floating beds of Canna indica L. Our results showed that both bacterial and protist community compositions in the treatment group were similar to those in the control group at the beginning of the experiment (day 1 to day 11), but then bacterial and protist community compositions became more stable with the removal of nutrients in the treatment group than those in the control group (day 12 to day 18). We further explored the mechanisms for this increased stability and found that the contribution of the stochastic process to bacterial and protist community variations was higher in the control group than that in the treatment group. This suggests that the high nutrient concentration in the control group might increase the random colonization or extinction, and therefore resulted in the high temporal variability (i.e., unstable) of bacterial and protist communities. Our findings suggest that bioremediation for eutrophication can promote the stability of aquatic communities, and therefore potentially maintain aquatic ecosystem functions and services to humanity.

Herbal NF-κB Inhibitors Sensitize Rituximab-Resistant B Lymphoma Cells to Complement-Mediated Cytolysis.

BACKGROUND: Drug resistance remains a serious challenge to rituximab therapy in B-NHL (B cell non-Hodgkin's lymphoma). CDC (complement-dependent cytotoxicity) has been proposed as a major antitumor mechanism of rituximab, and direct abrogation of CD59 function partially restores rituximab sensitivity with high efficacy. However, universal blockade of CD59 may have deleterious effects on normal cells. Sp1 regulates constitutive CD59 expression, whereas NF-κB and CREB regulate inducible CD59 expression. METHODS: Immunohistochemistry (IHC) assay was used to detect the expression levels of CD59 and other related molecules. Quantitative Real-time PCR (RT-PCR) analysis was used to explore the levels of transcripts in the original and resistant cells. We chose LY8 cells to test the effects of NF-κB and CBP/p300 inhibition on CD59 expression using flow cytometry (FACS). Immunoblotting analysis was employed to detect the effects of curcumin and POH. The in vitro and in vivo experiments were used to evaluate the toxicity and combined inhibitory effect on tumor cells of curcumin and POH. RESULTS: We demonstrated that herbal (curcumin and perillyl alcohol) blockade of NF-κB specifically suppresses the expression of inducible CD59 but not CD20, thus sensitizing resistant cells to rituximab-mediated CDC. Moreover, activation of NF-κB and CREB is highly correlated with CD59 expression in B-NHL tissues. CONCLUSIONS: Our findings suggest the potential of CD59 expression as a predictor of therapeutic efficacy of NF-κB inhibitors in clinical application as well as the rationality of a NF-κB inhibitor-rituximab regimen in B-NHL therapy.

Protocol for fever-range whole-body hyperthermia (WBH) in mice to study febrile effect on T-cell adhesion and migration.

Fever is a complex physiological response enhancing immune surveillance during infection and inflammation. Fever-range whole-body hyperthermia (WBH) treatment can experimentally mimic the febrile condition in mice. Here, we describe a protocol for the treatment of mice with WBH and normothermia. We describe the isolation of T cells from mouse spleen followed by the evaluation of T-cell adhesion and transmigration. This animal model can be applied to studying the dysfunction of the immune system induced by fever. For complete details on the use and execution of this protocol, please refer to Lin et al. (2019).

Transformations from specialists to generalists cause bacterial communities are more stable than micro-eukaryotic communities under anthropogenic activity disturbance.

Different microbial components have different responses to environmental disturbances. Here, we found that the planktonic bacterial and micro-eukaryotic communities had different responses to anthropogenic activity disturbance in a subtropical river, because they had different survival strategies (generalist and specialist). We used nutrients (nitrogen and phosphorus) as indicators of anthropogenic activities. We found that river stretch 1 showed low nutrient concentrations from October 2018 to September 2019. However, a nutrient disturbance was observed in river stretch 2. The nutrient concentrations increased largely in December and January but recovered to low values in June. Bacterial communities had higher resilience under this disturbance than micro-eukaryotic communities in river stretch 2. The bacterial community composition were quite different between the two river stretches in December and January but were similar in June and July. However, the differences of micro-eukaryotic community composition between the two river stretches were always high during the study period. The bacterial communities in river stretch 2 contained more generalists and nutrient tolerant specialists. The bacterial nutrient tolerant specialists rapidly decreased in the low nutrient months and were replaced by the generalists. Bacteria which were involved in this shifts accounted for 29.3% of the total abundance. However, the micro-eukaryotic communities in river stretch 2 contained more moderate generalists. These moderate generalists were insensitive to the variation of nutrients and only 19.56% of the micro-eukaryotes had significant responses to the disturbance. The survival strategies caused bacterial communities had higher adaptability than eukaryotes to environmental fluctuation.

Use of ecological concrete for nutrient removal in coastal sediment and its effects on sediment microbial communities.

Ecological concrete (eco-concrete) can reduce excess nutrients (nitrogen and phosphorus) in water, but its effectiveness in removing nutrients in marine coastal sediments and the response of sediment microbial communities to its use are largely unknown. In this study, eco-concrete planted with Bruguiera gymnorrhiza was used to remove nutrients in marine coastal sediment. We found that the mean removal efficiencies of sediment total nitrogen and total phosphorus by using planted eco-concrete were 11.50% and 30.31% on day 60, and were higher than those obtained by only using B. gymnorrhiza (7.14% and 7.36%). the Diatoms and bacterial genera Fusibacter and Anoxynatronum (which belong to Firmicutes) increased and became the abundant microbes by day 60 when using planted eco-concrete, indicating their potential roles in nutrient removal. Moreover, the eco-concrete did not endanger the core microbes in sediment suggesting its environment-friendly character. Our results suggest a potential method to control marine coastal eutrophication.

Engineering of Long-Term Stable Transparent Nanoemulsion Using High-Gravity Rotating Packed Bed for Oral Drug Delivery.

BACKGROUND: Oil-in-water drug nanoemulsion forms drug delivery systems with high oral bioavailability. The conventional fabrication methods of nanoemulsion are low energy emulsification methods and high energy emulsification methods. However, both two methods are not ideal for industrial production. The problem of low energy emulsification methods is the high dosage of surfactant and co-surfactant which has potential biosecurity issues. What is more, high energy emulsification methods have some disadvantages, like the destruction of drug components, the price of equipment and the difficulties of industrial production. Hence, there have been a few commercial drug nanoemulsions so far. METHODS: In this work, we reported a novel method for the fabrication of stable and transparent drug nanoemulsion which contains hydrophilic drug rosuvastatin (ROS) calcium or hydrophobic drug silybinin (SYN) by using high-gravity rotating packed bed (RPB). The drug nanoemulsion was systematically characterized by droplet size, size distribution, stability and in vitro drug release as well as Caco-2 cells permeability. RESULTS: Compared with the self-emulsification method (SE), high-gravity technology could reduce 75% amount of mixed surfactants. The as-prepared nanoemulsion exhibited a very narrow droplet size distribution with a size of 13.53 ± 0.53 nm and a polydispersity index of 0.073 ± 0.018. Meanwhile, the drug nanoemulsion was physicochemically stable at 25°C and 4°C for one-year storage. Furthermore, both ROS and SYN nanoemulsion displayed higher cell permeability and in vitro dissolution than that of commercial formulations. CONCLUSION: These results demonstrate that RPB can be a potential device to facilitate the industrial production of drug nanoemulsion.

Therapeutic assessment of fractions of Gastrodiae Rhizoma on chronic atrophic gastritis by 1H NMR-based metabolomics.

ETHNOPHARMACOLOGICAL RELEVANCE: Gastrodiae Rhizoma (GR), a well-known and commonly-used TCM (Traditional Chinese Medicine) for treating headache, dizziness, tetanus, epilepsy, and etc., has been proven to relieve chronic atrophic gastritis (CAG). Due to its complex ingredients, the active fractions responsible for the treatment of CAG remain largely unknown. AIM OF THE STUDY: To explore the underlying material and interpret its underlying mechanism, the therapeutic effect of extract from different polar parts of Gastrodiae Rhizoma on autoimmune CAG was studied based on the 1H NMR metabolomics. MATERIALS AND METHODS: The rat model of CAG was established by autoimmune method. The modeled CAG rats were then treated with 4 polar parts (T1-4 in descending polarity, corresponding to water, n-butanol, ethyl acetate and petroleum ether extracts, respectively) of Gastrodiae Rhizoma for 21 consecutive days. The stomach and serum samples were collected and then subjected to histopathology observation, biochemical measurement (MDA, SOD, GSH, NO, XOD and pepsin), 1H NMR metabolic profiling and multivariate/univariate statistical analysis. RESULTS: The results showed that T1 had the best therapeutic effect, T2 the second, and T3 and T4 the poorest with no obvious therapeutic effect, demonstrating that the effective components of Gastrodiae Rhizoma should be compounds of high polarity. T1 achieved good therapeutic effects due to the anti-inflammatory and anti-oxidant activities, and by rectifying the disturbed energy and amino acid metabolism in CAG model. CONCLUSION: This integrated metabolomics approach proved the validity of the therapeutic effect of extract from different polar parts of Gastrodiae Rhizoma on autoimmune CAG, providing new insights into the underlying mechanisms, and demonstrating the feasibility of metabolomics to evaluate efficacy of herbal drug, which is often difficult by traditional means.

Effects of benthivorous fish disturbance and snail herbivory on water quality and two submersed macrophytes.

Benthivorous fish disturbance and snail herbivory are two important factors that determine the community structure of submersed macrophytes. We conducted an outdoor mesocosm experiment to examine the separate and combined effects of these two factors on water quality and the growth of two mixed-cultivation submersed macrophytes, Vallisneria natans and Hydrilla verticillata, with different growth forms. The experiment involved two levels of fish (Misgurnus anguillicaudatus) disturbance crossed with two levels of snail (Radix swinhoei) intensity. The results revealed that fish activity rather than snail activity significantly increased the overlying water concentrations of total suspended solids (TSS), total nitrogen (TN), ammonia nitrogen (N-NH4), total phosphorus (TP) and phosphate phosphorus (P-PO4). However, no differences among treatments were observed for chlorophyll a (chl a) concentrations. Fish disturbance or snail herbivory alone did not affect the relative growth rate (RGR) of H. verticillata, but their combined effects significantly decreased the RGR of H. verticillata. Although snail herbivory alone did not affect the RGR of V. natans, fish disturbance alone and the combined effects of these factors drastically reduced its RGR. Both species exhibited increased free amino acid (FAA) contents and decreased ramet numbers, soluble carbohydrate (SC) contents and starch contents in the presence of the fish. Moreover, compared to H. verticillata, V. natans showed exceedingly low ramet numbers and starch contents in the presence of the fish. H. verticillata had a higher RGR and summed dominance ratio (SDR2) than V. natans in all treatments; H. verticillata also displayed a larger competitive advantage in the presence of fish disturbance. The present study suggests that (1) fish disturbance rather than snail activity increases water nutrient concentrations, (2) low snail density may be harmful to submersed macrophyte growth when the plants are under other abiotic stress conditions and (3) the competitive advantage of H. verticillata over V. natans is more preponderant in a turbid environment.

Effects of benthivorous fish disturbance on chlorophyll a contents in water and the growth of two submersed macrophytes with different growth forms under two light regimes.

Low underwater light availability and benthivorous fish-mediated disturbance are two important factors that influence the growth of submersed macrophytes. However, the combined effects of these factors remain unclear. To determine the combined effects of low light and fish-mediated disturbance on the growth of two submersed macrophytes with contrasting growth forms, i.e., Vallisneria natans and Hydrilla verticillata, we conducted an outdoor mesocosm experiment with a two-by-two factorial design. The experiment involved two fish-mediated disturbance levels (0 and 1 Misgurnus anguillicaudatus) crossed with two levels of light intensity (ambient light and a low-light environment created by culturing the macrophytes under a shelter). The results showed that the chlorophyll a (chl a) concentration in the overlying water showed no difference among treatments for each macrophyte species. The fish-mediated disturbance significantly decreased the relative growth rate (RGR) of both species in the low-light environment but showed no effects in the ambient light environment. Low light availability and/or fish-mediated disturbance led to increased plant heights of both species compared with the heights under the ambient light regime. Low light availability combined with fish-mediated disturbance significantly reduced the ramet number and soluble carbohydrate (SC) content of both species; however, the free amino acid (FAA) content was not affected. Compared to V. natans, H. verticillata exhibited a high RGR and high ramet numbers in a low-light environment combined with fish-mediated disturbance. Our results indicated that the adaptability of H. verticillata is better than that of V. natans in turbid, shallow and hydrostatic water. Fish-mediated disturbance can negatively influence submersed macrophyte recovery in lakes when light is not abundant.

Characteristics of microbial eukaryotic community recovery in eutrophic water by using ecological floating beds.

Ecological floating beds can rapidly remove nutrients (nitrogen and phosphorus) from eutrophic water, but we still know little about whether this process can simultaneously recover microbial eukaryotic communities. To fill this gap, planktonic microbial eukaryotic communities were investigated using 18S rRNA high-throughput gene sequencing during nutrient removal by floating beds of Canna indica L. We found that nutrient concentrations were high in both the control and treatment groups during period 1 (days 0-5) but rapidly decreased in the treatment group during period 2 (days 6-9) and period 3 (days 10-18). However, the microbial eukaryotic species richness and community compositions were similar between the control and treatment groups during periods 1 and 2 but showed small differences during period 3. The microbial eukaryotic co-occurrence networks between the control and treatment groups also showed similar degree centrality and interconnected eukaryotic members. We found that some abundant fungi species significantly responded to nutrient variations, but a large number of abundant ciliates were insensitive to nutrient removal. Our findings suggest that ecological floating beds can rapidly remove nutrients in eutrophic waters but that it is difficult to quickly and simultaneously improve microbial eukaryotic communities. This result reveals the critical influence of nutrient pollution on aquatic ecosystems and therefore on long-term and comprehensive aquatic habitat restoration, as aquatic macrophyte recoveries should be conducted after nutrient controls have been implemented.

Hysteretic response of Microbial Eukaryotic Communities to Gradually Decreased Nutrient Concentrations in Eutrophic Water.

External environments to microbial eukaryotic communities often change gradually with time. However, whether the responses of microbial eukaryotic communities to these gradually changed environments are continuous or hysteretic and the mechanisms underlying these responses are largely unknown. Here, we used a microcosm to investigate the temporal variation of microbial eukaryotic communities with the gradually decreased nutrient concentrations (nitrogen and phosphorus). We found the differences of microbial eukaryotic community composition and species richness between the control and treatment groups were low during the days 0 to 12, although the nutrient concentrations decreased rapidly during this period in treatment group. However, these differences were clear during the days 14 to 18, although the nutrient concentrations decreased slowly during this period in treatment group. The mechanisms for these results are that the strong homogenous selection (perhaps due to the biotic factors) during the days 8 to 10 in treatment group might enhance the stability of microbial eukaryotic communities. However, the continuously decreased nutrient concentrations weakened the homogenous selection and promoted the strength of environmental filtering, and therefore resulted in the distinct change of microbial eukaryotic communities during the days 14 to 18 in treatment group. Fungi, Chlorophyta and Chrysophyta which associated with the nutrient removal played important roles in this hysteretic change of microbial eukaryotic communities. Overall, our findings suggest that disentangling the non-linear response of communities to gradual environmental changes is essential for understanding ecosystem restoration and degradation in future.

Potential hepatoxicity risk of the shell of Herpetospermum caudigerum Wall in rats based on 1H-NMR metabonomics.

The Herpetospermum caudigerum Wall (HCW) is a traditional Tibetan medicine and is widely used in clinical practice. However, the shell of the HCW (SHCW) has rarely been studied, and some researchers have suggested that the SHCW may be toxic. Therefore, in this study, SHCW was administered to rats at two doses (0.1 and 0.33 g/kg) once a day for 21 days. The hepatic stimuli induced by SHCW in rats were investigated for the first time by 1H-NMR-based metabolomics combined with histopathological observation and biochemical detection. Histopathological sections showed a certain degree of hepatocyte edema and hepatic sinus congestion in the liver tissue of the rats in the drug-administered group. Serum biochemical indicators revealed a significant increase in ALT, AST, and MDA, and a significant decrease in SOD. Metabolomic results showed that the metabolites in rats were changed after gavage administration of extracts from SHCW. By multivariate statistical analysis and univariate analysis, it was found that SHCW could cause the disorder of energy metabolism, oxidative stress and amino acid metabolism in rats, leading to liver damage. This comprehensive metabolomics approach demonstrates its ability to describe the global metabolic state of an organism and provides a powerful and viable tool for exploring drug-induced toxicity or side effects.