Unveiling unique microbial nitrogen cycling and nitrification driver in coastal Antarctica.

Largely removed from anthropogenic delivery of nitrogen (N), Antarctica has notably low levels of nitrogen. Though our understanding of biological sources of ammonia have been elucidated, the microbial drivers of nitrate (NO3-) cycling in coastal Antarctica remains poorly understood. Here, we explore microbial N cycling in coastal Antarctica, unraveling the biological origin of NO3- via oxygen isotopes in soil and lake sediment, and through the reconstruction of 1968 metagenome-assembled genomes from 29 microbial phyla. Our analysis reveals the metabolic potential for microbial N2 fixation, nitrification, and denitrification, but not for anaerobic ammonium oxidation, signifying a unique microbial N-cycling dynamic. We identify the predominance of complete ammonia oxidizing (comammox) Nitrospira, capable of performing the entire nitrification process. Their adaptive strategies to the Antarctic environment likely include synthesis of trehalose for cold stress, high substrate affinity for resource utilization, and alternate metabolic pathways for nutrient-scarce conditions. We confirm the significant role of comammox Nitrospira in the autotrophic, nitrification process via 13C-DNA-based stable isotope probing. This research highlights the crucial contribution of nitrification to the N budget in coastal Antarctica, identifying comammox Nitrospira clade B as a nitrification driver.

Community pattern of potential phenanthrene (PHE) degrading bacteria in PHE contaminated soil revealed by 13C-DNA stable isotope probing.

Quantification of polycyclic aromatic hydrocarbons (PAHs) in contaminated soil and identification of potential PAH degraders are essential for comprehending their environmental fate and conducting bioremediation. However, the microbial population responsible for the breakdown of phenanthrene (PHE) in polluted soil environments is frequently disregarded. In this study, via DNA-stable-isotope probing (DNA-SIP), we found that soil microbiota likely plays a crucial part in the PHE degradation. The PHE removal rates were 98% and 99%, in 13C-PHE and 12C-PHE microcosmic incubations, respectively. 13CO2 was produced along with the degradation of 13C-PHE. According to the analysis of 16S rRNA gene, there was a relatively higher presence of unidentified bacteria in the 'heavy' DNA fractions treated with 13C-PHE. Genus of Enterobacteriales, Acidobacteria, Alphaproteobacteria, Paenibacillaceae, Flavobacteriia, Chloroflexi, Cyanobacteria, Caldilineae, Latescibacteria, Armatimonadetes and Blastocatellia were succseesfully labeled during the degradation of 13C-PHE, indicating their capacity of utilizing PHE. Co-occurrence network of 13C-heavy fractions exhibited greater complexity compared with that of 12C-heavy fractions, revealling an enhancement of bacterial interspecies interactions. Collectivley, this study eluidated the soil microbes involed in the PHE degradation and offered fresh perspectives on the community pattern of potential PHE degrading bacteria.

Abundance, diversity and physiological preferences of comammox Nitrospira in urban groundwater.

Complete ammonia oxidizer (comammox Nitrospira), catalyze complete nitrification process in a single organism, are frequently detected in groundwater ecosystem. However, the ecological niches and environmental driving factors of comammox Nitrospira in urban groundwater are largely unknown. Here we investigated the communities of ammonia oxidizers in urban groundwater located in Shanghai city, China. Quantitative analysis demonstrated the dominance of comammox Nitrospira over classical ammonia oxidizers (ammonia-oxidizing archaea and bacteria, AOA and AOB). Phylogenetic analysis showed clades B and A2 comprise the majority of comammox Nitrospira groups. Temperature was one of the most vital factors affecting comammox Nitrospira community. Furthermore, clade A comammox Nitrospira can be enriched by urea substrate, which was in line with the ability of utilizing urea by the pure clade A comammox culture Nitrospira inopinata. In addition, we observed that relatively low temperature (<20 °C) and high copper levels (>0.04 mg L-1) can stimulate the growth of comammox Nitrospira. Overall, this study revealed the presence, diversity and physiological preferences of comammox Nitrospira in urban groundwater nitrification, shedding insights on the ecological roles of comammox Nitrospira in subsurface environment.

Combined Epimedii Folium and Ligustri Lucidi Fructus with dexamethasone alleviate the proliferation of airway smooth muscle cells by regulating apoptosis/autophagy.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine (TCM) theory believes kidney deficiency is the root cause of chronic refractory asthma with pathological changes of airway remodeling. Our previous experiments confirmed that the combination of Epimedii Folium and Ligustri Lucidi Fructus (ELL) with the effect of nourishing Yin and Yang of the kidney could improve the pathological changes of airway remodeling in asthmatic rats, but the specific mechanism remains unclear. AIM OF THE STUDY: This research was designed to reveal the synergy of ELL and dexamethasone (Dex) in the proliferation, apoptosis, and autophagy of airway smooth muscle cells (ASMCs). MATERIALS AND METHODS: Primary cultures of ASMCs from rats were prepared and induced with histamine (Hist), Z-DEVD-FMK (ZDF), rapamycin (Rap), or 3-Methyladenine (3-MA) at generation 3-7 for 24 or 48 h. Subsequently, the cells were treated with Dex, ELL, and ELL&Dex for 24 or 48 h. The effect of various concentrations of inducers and drugs on cell viability was detected by Methyl Thiazolyl Tetrazolium (MTT) assay, cell proliferation was tested using immunocytochemistry (ICC) by detecting Ki67 protein, cell apoptosis was measured by Annexin V-FITC/PI assay and Hoechst nuclear staining, cell ultrastructure was observed by transmission electron microscopy (TEM), and immunofluorescence (IF), Western blot (WB) combined with quantitative real-time PCR (qPCR) were used for measuring autophagy and apoptosis-related genes including protein 53 (P53), cysteinyl aspartate-specific proteinase (Caspase)-3, microtubule-associated protein 1 light chain 3 (LC3), Beclin-1, mammalian target of rapamycin (mTOR) and p-mTOR. RESULTS: In ASMCs, Hist and ZDF promoted cell proliferation, significantly decreased Caspase-3 protein expression, and up-regulated Beclin-1 levels; Dex alone and in combination with ELL promoted Beclin-1, Caspase-3, and P53 expression, enhancing autophagy activity and apoptosis in Hist and ZDF-induced AMSCs. In contrast, Rap inhibited cell viability, increased Caspase-3, P53, Beclin-1, and LC3-II/I and decreased the levels of mTOR and p-mTOR with promoting apoptosis and autophagy; ELL or ELL&Dex reduced P53, Beclin-1, and LC3-II/I to down-regulate apoptosis and the excessive autophagic state of ASMCs induced by Rap. In the 3-MA model, cell viability and autophagy were reduced; ELL&Dex significantly upgraded the expression of Beclin-1, P53, and Caspase-3 and promoted apoptosis and autophagy of ASMCs. CONCLUSIONS: These results suggest that ELL combined with Dex may regulate the proliferation of ASMCs by promoting apoptosis and autophagy and be a potential medicine for the treatment of asthma.

Pivotal Role of the Granularity Uniformity of the WO3 Film Electrode upon the Cyclic Stability during Cation Insertion/Extraction.

Delicate design and precise manipulation of electrode morphology has always been crucial in electrochemistry. Generally, porous morphology has been preferred due to the fast kinetic transport characteristics of cations. Nevertheless, more refined design details such as the granularity uniformity that usually goes along with the porosity regulation of film electrodes should be taken into consideration, especially in long-term cation insertion and extraction. Here, inorganic electrochromism as a special member of the electrochemical family and WO3 films as the most mature electrochromic electrode material were chosen as the research background. Two kinds of WO3 films were prepared by magnetron sputtering, one with a relatively loose morphology accompanied by nonuniform granularity and one with a compact morphology along with uniform particle size distribution, respectively. Electrochemical performances and cyclic stability of the two film electrodes were then traced and systematically compared. In the beginning, except for faster kinetic transport characters of the 50 W-deposited WO3 film, the two electrodes showed equivalent optical and electrochemical performances. However, after 5000 CV cycles, the 50 W-deposited WO3 film electrode cracked seriously. Strong stress distribution centered among boundaries of the nonuniform particle clusters together with the weak bonding among particles induced the mechanical damage. This discovery provides a more solid background for further delicate film electrode design.

Insight into mechanisms of biochar-fertilizer induced of microbial community and microbiology of nitrogen cycle in acidic soil.

Biochar has been shown to affect the nitrogen (N) cycle in soil, however, it is unknown how this occurs. Therefore, we used metabolomics, high-throughput sequencing, and quantitative PCR to explore biochar and nitrogen fertilizer effects on the mitigation mechanisms of adverse environments in acidic soil. In the current research, we used acidic soil and maize straw biochar (pyrolyzed at 400 °C with limited oxygen). Three maize straw biochar levels (B1; 0t ha-1, B2; 45 t ha-1, and B3; 90 t ha-1) along with three N fertilizer (urea) levels (N1; 0 kg ha-1, N2; 225 kg ha-1 mg kg-1, and N3; 450 kg ha-1 mg kg-1) were employed in a sixty-day pot experiment. We found that the formation of NH+ 4-N was faster at 0-10 days, while the formation of NO- 3-N occurred at 20-35 days. Furthermore, the combined application of biochar and N fertilizer most effectively boosted soil inorganic N contents compared to biochar and N fertilizer treatments alone. The B3 treatment increased the total N and total inorganic N by 0.2-24.2% and 55.2-91.7%, respectively. Soil microorganism, N fixation, and nitrification capabilities increased with biochar and N fertilizer addition in terms of N-cycling-functional genes. Biochar-N fertilizer had a greater impact on the soil bacterial community and their diversity and richness. Metabolomics revealed 756 distinct metabolites, including 8 substantially upregulated metabolites and 21 significantly downregulated metabolites. A significant amount of lipids and organic acids were formed by biochar-N fertilizer treatments. Thus, biochar and N fertilizer triggered soil metabolism by affecting bacterial community structure, and N-cycling of the soil micro-ecological environment.

Epimedii Folium and Ligustri Lucidi Fructus Promote Osteoblastogenesis and Inhibit Osteoclastogenesis against Osteoporosis via Acting on Osteoblast-Osteoclast Communication.

Osteoblast (OB) and osteoclast (OC) play important roles in bone formation and bone resorption, which can communicate with each other through cytokine paracrine. Previous studies have confirmed that Epimedii Folium (EF) and Ligustri Lucidi Fructus (LLF) used alone or in combination can treat osteoporosis (OP) through regulating bone remodeling, but the effects of EF and LLF on osteoblastogenesis, osteoclastogenesis, and OB-OC communication are unclear. In this study, we investigated the direct and indirect effects of EF and LLF on OBs and OCs via monoculture and coculture (transwell) models of OBs and OCs. We found that the combination of EF and LLF (EF&LLF) could promote osteoblastogenesis and inhibit osteoclastogenesis directly and indirectly. In order to study the mechanisms of EF&LLF on indirectly regulating osteoblastogenesis and osteoclastogenesis, we detected the expression of cytokines by which OBs and OCs could communicate with each other. We found that EF&LLF could downregulate the expression of RANKL and M-CSF and the protein ratio of RANKL/OPG of OBs and Atp6v0d2 expression of OCs and upregulate the expression of OPG and TGF-ß1 of OBs and the expression of TGF-ß1, BMP-2, and IGF-1 of OCs, indicating that EF&LLF could regulate cytokine expressions of OBs/OCs to affect OB-OC communication. In addition, EF&LLF had a better effect on regulating cytokines of OBs and OCs than EF or LLF in single use. This study suggested that EF&LLF exhibited the effects of promoting osteoblastogenesis and inhibiting osteoclastogenesis via acting on OB-OC communication and provided some scientific evidences for EF&LLF against OP.

Benzimidazole fungicide biotransformation by comammox Nitrospira bacteria: Transformation pathways and associated proteomic responses.

Benzimidazole fungicides are frequently detected in aquatic environments and pose a serious health risk. Here, we investigated the metabolic capacity of the recently discovered complete ammonia-oxidizing (comammox) Nitrospira inopinata and kreftii to transform a representative set of benzimidazole fungicides (i.e., benzimidazole, albendazole, carbendazim, fuberidazole, and thiabendazole). Ammonia-oxidizing bacteria and archaea, as well as the canonical nitrite-oxidizing Nitrospira exhibited no or minor biotransformation activity towards all the five benzimidazole fungicides. In contrast, the investigated comammox bacteria actively transformed all the five benzimidazole fungicides, except for thiabendazole. The identified transformation products indicated hydroxylation, S-oxidation, and glycosylation as the major biotransformation pathways of benzimidazole fungicides. We speculated that these reactions were catalyzed by comammox-specific ammonia monooxygenase, cytochrome P450 monooxygenases, and glycosylases, respectively. Interestingly, the exposure to albendazole enhanced the expression of the antibiotic resistance gene acrB of Nitrospira inopinata, suggesting that some benzimidazole fungicides could act as environmental stressors that trigger cellular defense mechanisms. Altogether, this study demonstrated the distinct substrate specificity of comammox bacteria towards benzimidazole fungicides and implies their significant roles in the biotransformation of these fungicides in nitrifying environments.

Stereotaxic Atlas of the Human Lumbar-Sacral Spinal Cord.

OBJECTIVE: A Stereotaxic Atlas of the Human Lumbar-Sacral Spinal Cord has been created to provide an anatomical basis for radiologic and ultrasonic imaging and electrophysiological examination, which are used to target the placement of lumbar-sacral epidural stimulating electrodes and cellular transplantation in order to restore movement in individuals with sustained spinal cord injury or a degenerative disorder of the spinal cord. Through the availability of an atlas that exhibits axial images of the cytoarchitecture of each cord segment with a stereotaxic millimeter grid of dorsal-ventral depth from the midline dorsal surface of the cord and right-left distances from the midline of the cord, neuromodulation, and cellular therapy would undoubtedly be made not only more precise but also safer for patients. METHODS: The atlas is based upon dimension measurements and subsequent serial sectioning, staining and high-resolution digital imaging of the lumbar-sacral enlargement of 20 adult human spinal cords. RESULTS: Nissl stained cross-sections from cord segments L1-S3 illustrate the cytoarchitecture and stereotactic coordinates. CONCLUSIONS: The atlas provides an anatomical basis for radiologic and physiologic confirmation of target localization in the lumbar-sacral spinal cord.

ERß overexpression may not be a direct prognostic factor in patients with NSCLC: A meta-analysis.

Overall survival of non-small cell lung cancer (NSCLC) patients remains disappointingly low. The estrogen receptor (ER) was considered a promising therapeutic target for NSCLC. Numerous studies have linked expression of ERß to lung cancer outcome. However, results are conflicting regarding the association of ERß with surviving lung cancer. The aim of this meta-analysis was to evaluate the prognostic aspect of ERß expression on survival among NSCLC patients. We performed a final analysis of prognostic value of overexpression ERß on 3500 patients from 18 evaluable studies (from January 1, 2000 to May 1, 2021). The reference category is specified as low ERß expression levels. Summarized hazard ratios were calculated. Our study showed that the pooled hazard ratios of ERß overexpression for overall survival in NSCLC was 0.81 (95% confidence interval (CI): 0.64-1.02, P = 0.07) by univariate analysis and 1.06 (95% CI: 0.83-1.36, P = 0.63) by multivariate analysis. Pooled hazard ratio by univariate analysis in Asian studies was 0.73 (95%CI: 0.59-0.89, P = 0.002). Pooled hazard ratio by univariate analysis was 0.75 (95% CI: 0.61-0.93, P = 0.009) from seven studies reported for nuclear ERß. No significant results were found in subgroups by multivariate analysis. No significant results were found in studies outside Asia or in studies reported for cytoplasmic ERß. Our results suggested that expression of ERß might not be a direct prognostic factor for NSCLC patients. More detailed prospective studies are needed to identify direct prognostic factors in these patients.

Dark carbon fixation in intertidal sediments: Controlling factors and driving microorganisms.

Dark carbon fixation (DCF) contributes approximately 0.77 Pg C y-1 to oceanic primary production and the global carbon budget. It is estimated that nearly half of the DCF in marine sediments occurs in estuarine and coastal regions, but the environmental factors controlling DCF and the microorganisms responsible for its production remain under exploration. In this study, we investigated DCF rates and the active chemoautotrophic microorganisms in intertidal sediments of the Yangtze Estuary, using 14C-labeling and DNA-stable isotope probing (DNA-SIP) techniques. The measured DCF rates ranged from 0.27 to 3.37 mmol C m-2 day-1 in intertidal surface sediments. The rates of DCF were closely related to sediment sulfide content, demonstrating that the availability of reductive substrates may be the dominant factor controlling DCF in the intertidal sediments. A significant positive correlation was also observed between the DCF rates and abundance of the cbbM gene. DNA-stable isotope probing (DNA-SIP) results further confirmed that cbbM-harboring bacteria, rather than cbbL-harboring bacteria, played a dominant role in DCF in intertidal sediments. Phylogenetic analysis showed that the predominant cbbM-harboring bacteria were affiliated with Burkholderia, including Sulfuricella denitrificans, Sulfuriferula, Acidihalobacter, Thiobacillus, and Sulfurivermis fontis. Moreover, metagenome analyses indicated that most of the potential dark-carbon-fixing bacteria detected in intertidal sediments also harbor genes for sulfur oxidation, denitrification, or dissimilatory nitrate reduction to ammonium (DNRA), indicating that these chemoautotrophic microorganisms may play important roles in coupled carbon, nitrogen, and sulfur cycles. These results shed light on the ecological importance and the underlying mechanisms of the DCF process driven by chemoautotrophic microorganisms in intertidal wetlands.

Corrigendum: Distribution and Diversity of Comammox Nitrospira in Coastal Wetlands of China.

[This corrects the article DOI: 10.3389/fmicb.2020.589268.].

Salinity gradients shape the nitrifier community composition in Nanliu River Estuary sediments and the ecophysiology of comammox Nitrospira inopinata.

The recent discovery of complete ammonia oxidizers (comammox), which convert ammonia to nitrate in a single organism, revolutionized the conventional understanding that two types of nitrifying microorganisms have to be involved in the nitrification process for more than 100 years. However, how different types of nitrifiers in response to salinity change remains largely unclear. This study not only investigated nitrifier community (including ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), comammox and nitrite-oxidizing Nitrospira) in the Nanliu estuary to find the ecological relationship between salinity and functional communities and also studied the physiology of a typical comammox Nitrospira inopinata in response to a salinity gradient. Based on sequences retrieved with four sets of functional gene primes, comammox Nitrospira was in general, mainly composed of clade A, with a clear separation of clade A1 subgroup in all samples and clade A2 subgroup in low salinity ones. As expected, group I.1b and group I.1a AOA dominated the AOA community in low- and high-salinity samples, respectively. Nitrosomonas-AOB were detected in all samples while Nitrosospira-AOB were mainly found in relatively high-salinity samples. Regarding general Nitrospira, lineages II and IV were the major groups in most of the samples, while lineage I Nitrospira was only detected in low-salinity samples. Furthermore, the comammox pure culture of N. inopinata showed an optimal salinity at 0.5‰ and ceased to grow at 12.8‰ for ammonia oxidation, but remained active for nitrite oxidation. These results show new evidence regarding niche specificity of different nitrifying microorganisms modulated mainly by salinity, and also a clear response by comammox N. inopinata to a wide range of simulated salinity levels.

Feedback mechanisms of periphytic biofilms to ZnO nanoparticles toxicity at different phosphorus levels.

The increasing use of nanoparticles (NPs) has raised concerns about their potential environmental risks. Many researches on NPs focused on the toxicity mechanism to microorganisms, but neglect the toxicity effects in relation to nutritional conditions. Here, we evaluated the interactive effects of zinc oxide (ZnO) NPs and phosphorus (P) levels on the bacterial community and functioning of periphytic biofilms. Results showed that long-term exposure to ZnO NPs significantly reduced alkaline phosphatase activity (APA) of periphytic biofilms just in P-limited conditions. Co-occurrence network analysis indicated that ZnO NPs exposure reduced network complexity between bacterial taxa in P-limited conditions, while the opposite trend was observed in P-replete conditions. Correlation analysis and random forest modeling suggested that excessive Zn2+ released and high reactive oxygen species (ROS) production might be mainly responsible for the inhibition of APA induced by ZnO NPs under P-limited conditions, while adjustment of bacterial diversity and improvement of keystone taxa cooperation were the main mechanisms in maintaining APA when subjected to weak toxicity of ZnO NPs in P-replete conditions. Taken together, our results provide insights into the biological feedback mechanism involved in ZnO NPs exposure on the ecological function of periphytic biofilms in different P nutritional conditions.

Biotransformation of lincomycin and fluoroquinolone antibiotics by the ammonia oxidizers AOA, AOB and comammox: A comparison of removal, pathways, and mechanisms.

In this study, we evaluated the biotransformation mechanisms of lincomycin (LIN) and three fluoroquinolone antibiotics (FQs), ciprofloxacin (CFX), norfloxacin (NFX), and ofloxacin (OFX), which regularly enter aquatic environments through human activities, by different ammonia-oxidizing microorganisms (AOM). The organisms included a pure culture of the complete ammonia oxidizer (comammox) Nitrospira inopinata, an ammonia oxidizing archaeon (AOA) Nitrososphaera gargensis, and an ammonia-oxidizing bacterium (AOB) Nitrosomonas nitrosa Nm90. The removal of these antibiotics by the pure microbial cultures and the protein-normalized biotransformation rate constants indicated that LIN was significantly co-metabolically biotransformed by AOA and comammox, but not by AOB. CFX and NFX were significantly co-metabolized by AOA and AOB, but not by comammox. None of the tested cultures transformed OFX effectively. Generally, AOA showed the best biotransformation capability for LIN and FQs, followed by comammox and AOB. The transformation products and their related biotransformation mechanisms were also elucidated. i) The AOA performed hydroxylation, S-oxidation, and demethylation of LIN, as well as nitrosation and cleavage of the piperazine moiety of CFX and NFX; ii) the AOB utilized nitrosation to biotransform CFX and NFX; and iii) the comammox carried out hydroxylation, demethylation, and demethylthioation of LIN. Hydroxylamine, an intermediate of ammonia oxidation, chemically reacted with LIN and the selected FQs, with removals exceeding 90%. Collectively, these findings provide important fundamental insights into the roles of different ammonia oxidizers and their intermediates on LIN and FQ biotransformation in nitrifying environments including wastewater treatment systems.

Quantification of Myelinated Nerve Fraction and Degeneration in Spinal Cord Neuropil by SHIFT MRI.

BACKGROUND: Neurodegeneration is a complex cellular process linked to prompt changes in myelin integrity and gradual neuron loss. Current imaging techniques offer estimations of myelin volumes in lesions/remyelinated areas but are limited to detect subtle injury. PURPOSE: To investigate whether measurements detected by a signal hierarchically isolated as a function of time-to-echo (SHIFT) MRI technique can determine changes in myelin integrity and fiber axolemma. STUDY TYPE: Prospective animal model. ANIMAL MODEL: Surgically demyelinated spinal cord (SC) injury model in rodents (n = 6). FIELD STRENGTH/SEQUENCE: Gradient-echo spin-echo at 3T. ASSESSMENT: Multicompartment T2 relaxations were computed by SHIFT MRI in 75-microns-resolution images of the SC injury penumbra region 2 weeks post-trauma. G-ratio and axolemma delamination were assessed by transmission electron microscopy (TEM) in intact and injured samples. SC myelinated nerve fraction was computed by SHIFT MRI prospectively and assessed histologically. STATISTICAL TESTS: Relations between SHIFT-isolated T2 -components and TEM measurements were studied using linear regression and t-tests. Pearson's correlation and significance were computed to determine the SHIFT's sensitivity to detect myelinated fibers ratio in gray matter. Regularized least-squares-based ranking analysis was employed to determine SHIFT MRI's ability to discern intact and injured myelinated nerves. RESULTS: Biexponential signals isolated by SHIFT MRI for intact vs. lesion penumbra exhibited changes in T2 , shifting from intermediate components (25 ± 2 msec) to long (43 ± 11 msec) in white matter, and similarly in gray matter regions-of-interest (31 ± 2 to 46 ± 16 msec). These changes correlated highly with TEM g-ratio and axon delamination measurements (P < 0.05). Changes in short T2 components were observed but not statistically significant (8.5 ± 0.5 to 7 ± 3 msec, P = 0.445, and 4.0 ± 0.9 to 7 ± 3 msec, P = 0.075, respectively). SHIFT MRI's ability to detect myelinated fibers within gray matter was confirmed (P < 0.001). DATA CONCLUSION: Changes detected by SHIFT MRI are associated with abnormal intermembrane spaces formed upon mild injury, directly correlated with early neuro integrity loss. Level of Evidence 1 Technical Efficacy Stage 2.

Comprehensive evaluation of the combined extracts of Epimedii Folium and Ligustri Lucidi Fructus for PMOP in ovariectomized rats based on MLP-ANN methods.

ETHNOPHARMACOLOGICAL RELEVANCE: Kidney deficiency is the main pathogenesis of osteoporosis based on the theory of "kidney governing bones" in traditional Chinese medicine (TCM). Osteoporosis is a systemic disease; kidney deficiency influences the growth, aging and reproduction of human body, reflecting in endocrine, nerve, immunity, metabolism and other functions. Multi-target drugs composed of natural non-toxic products from kidney-reinforcing herbs, are being investigated for the treatment of osteoporosis. Therefore, it is necessary and imperative to develop an objective and comprehensive method to evaluate and compare the effects of herbs with listed drugs. AIM OF THE STUDY: This study was designed to evaluate and compare the therapeutic effects and the underlying molecular mechanism of the combined extracts of Epimedii Folium and Ligustri Lucidi Fructus (EL) with Raloxifene hydrochloride (RH) in ovariectomy (OVX)-induced postmenopausal osteoporosis (PMOP) rats based on the multi-layer perception (MLP)-artificial neural network (ANN) model. MATERIALS AND METHODS: Female SD rats were subjected to either sham surgery (n = 8) or bilateral OVX (n = 48). One week after recovering from surgery, the OVX-induced rats were randomly divided into three groups: OVX model group (n = 32, every 8 rats were killed at the end of the 5th, 9th, 11th or 13th week after OVX), EL group (treated with EL 0.35 g/kg, n = 8), and RH group (treated with RH 6.25 mg/kg, n = 8). The rats in the treatment groups were administrated once a day for 12 weeks, then sacrificed. We observed bone mass and quality, bone remodeling, the function of estrogen and TGF-ß1/Smads pathway in all rats. RESULTS: Both EL and RH could increase bone mineral density, enhance bone strength, relieve bone micro-structure degeneration, re-balance bone remodeling, regulate estrogen dysfunction, and up-regulate TGF-ß1 expression. The evaluation of the MLP-ANN model showed that EL and RH had markedly anti-PMOP effects, and there was no significant difference in the comprehensive evaluation of anti-osteoporosis between the two drugs. However, RH had better effects on bone mass and quality and TGF-ß1/Smads pathway than EL; EL had better effects on estrogen function than RH. CONCLUSION: Combined extracts of Epimedii Folium and Ligustri Lucidi Fructus (EL) exhibited bone-protective effects on PMOP. The MLP-ANN method evaluated the efficacy of drugs more comprehensively, which provided a new direction for the evaluation and comparison of drugs.

Evaluation of Two Primer Sets for Amplification of Comammox Nitrospira amoA Genes in Wetland Soils.

After the discovery of complete ammonia-oxidizing (comammox) Nitrospira, detection and assessments of the contribution of comammox Nitrospira communities to nitrogen cycling are in great demand. PCR-based approach, a common method for the detection of comammox, depends strongly on accurate amplification of the amoA genes from the original DNA samples using appropriate primers. In this study, we reported an evaluation of the performance of two commonly used primer sets, Ntsp-amoA 162F/359R and comaA/B-244f/659r, for amplifying the comammox amoA genes from three representative wetland soils in China [Sangsang (SS), Sanjiang (SJ), and Xianghai (XH)]. Our results demonstrated the two primer sets could both successfully amplify the clades with high relative abundances (RA), and further revealed a broadly similar diversity and community composition of dominant comammox operational taxonomic units (OTUs) (RA ≥ 1%) in each of the three wetland soils. However, the clades with low RA, such as the clade A (1.26%) in SJ and the clade B (11.54%) in XH that were recovered by metagenomics analysis, failed to be amplified using comaA/B-244f/659r, but were successfully amplified and sequenced using Ntsp-amoA 162F/359R. It indicated that, compared to comaA/B-244f/659r, Ntsp-amoA 162F/359R was more sensitive to the clades with low RA. However, it is worth noting that Ntsp-amoA 162F/359R would overestimate the RA of some rare clades. For example, the RAs of clade B in XH were overestimated by 32-fold. Furthermore, high levels of non-target amplification were detected via gel electrophoresis using both primer sets, especially for comammox Clade B amoA genes, implying that we should treat qPCR results based on these primers with caution. Taken together, our study comprehensively compared the performance of the two primer sets on the sensitivity and specificity of amplifying comammox amoA genes in three wetland soils, pointing out the necessity of further development of new primers for the efficient and accurate detection of comammox in various environments.

Distribution and Diversity of Comammox Nitrospira in Coastal Wetlands of China.

Complete ammonia oxidizers (comammox), able to individually oxidize ammonia to nitrate, are considered to play a Complete ammonia oxidizers (comammox), able to individually oxidize ammonia to nitrate, are considered to play a significant role in the global nitrogen cycle. However, the distribution of comammox Nitrospira in estuarine tidal flat wetland and the environmental drivers affecting their abundance and diversity remain unknown. Here, we present a large-scale investigation on the geographical distribution of comammox Nitrospira along the estuarine tidal flat wetlands of China, where comammox Nitrospira were successfully detected in 9 of the 16 sampling sites. The abundance of comammox Nitrospira ranged from 4.15 × 105 to 6.67 × 106 copies/g, 2.21- to 5.44-folds lower than canonical ammonia oxidizers: ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA). Phylogenetic analysis based on the alpha subunit of the ammonia monooxygenase encoding gene (amoA) revealed that comammox Nitrospira Clade A, mainly originating from upstream river inputs, accounts for more than 80% of the detected comammox Nitrospira, whereas comammox Nitrospira clade B were rarely detected. Comammox Nitrospira abundance and dominant comammox Nitrospira OTUs varied within the estuarine samples, showing a geographical pattern. Salinity and pH were the most important environmental drivers affecting the distribution of comammox Nitrospira in estuarine tidal flat wetlands. The abundance of comammox Nitrospira was further negatively correlated with high ammonia and nitrite concentrations. Altogether, this study revealed the existence, abundance and distribution of comammox Nitrospira and the driving environmental factors in estuarine ecosystems, thus providing insights into the ecological niches of this recently discovered nitrifying consortium and their contributions to nitrification in global estuarine environments.

Systemic osteoprotective effects of Epimedii Folium and Ligustri Lucidi Fructus in senile osteoporosis rats by promoting the osteoblastogenesis and osteoclastogenesis based on MLP-ANN model.

BACKGROUND: Senile osteoporosis (SOP), which is caused by unbalanced bone remodeling, leads to significant economic and societal burdens globally. The combination of Epimedii Folium (EF) and Ligustri Lucidi Fructus (LLF) serves as a commonly-used prescription for SOP in Traditional Chinese Medicine (TCM). This study aimed to evaluate the osteoprotective effects of EF and LLF in combination on SOP rats based on the constructed multilayer perception (MLP)-artificial neural network (ANN) model. METHODS: 15 month old male Sprague-Dawley rats were administrated with EF, LLF or the combination of EF and LLF (EF&LLF) for 2 months, while 17 month old rats were used as the aging control group. All the rats were anesthetized with 25% ethyl carbamate, then their serum liver and bone tissues were taken. We detected bone mass, bone mineral density (BMD), biomechanics and the microstructure of bone trabecula by micro-CT and H&E staining to evaluate the degree of osteoporosis. Blood lipids and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and γ-glutamyl transferase (GGT) and liver pathology were use to assess the side effects of drugs. Levels of alkaline phosphatase (ALP) and Tartrate-resistant acid phosphatase (TRACP) and the ratio of ALP to TRACP both in serum and bone were measured for the evaluation of bone turnover rate. The bone mRNA and protein expression of osteoprotegerin (OPG), nuclear factor-kappa B ligand (RANKL), macrophage colony-stimulating factor (M-CSF), d2 isoform of vacuolar (H+) ATPase (ATP6V0d2), insulin-like growth factor (IGF-1), bone morphogenetic protein-2 (BMP2), M-CSF, Wnt5a, transforming growth factor-ß1 (TGF-ß1) were detected for evaluating bone metabolism. RESULTS: The results showed that EF&LLF improved bone mass and bone quality by preventing bone loss, increasing maximal load as well as protecting the micro-structural retrogressive change of trabecular bone in SOP rats; ameliorated the steatosis in the liver and decreased blood lipids and serum ALT, AST and GGT; enhanced bone remodeling by stimulating the expression of ALP and TRACP. At the molecular levels, EF&LLF stimulated the osteoclastogenesis by upregulating the protein and mRNA expression of OPG, RANKL, M-CSF and ATP6V0d2; meanwhile, EF&LLF stimulated osteoblastogenesis by enhancing the expression of TGF-ß1, BMP2, Wnt5a and IGF-1. According to our established MLP model, EF&LLF has a better effect on osteoclastogenesis or steoblastogenesis in SOP rats than EF or LLF. CONCLUSIONS: These findings demonstrate that the systemic bone protective effects of EF&LLF by promoting bone remodeling in aging rats might be a substitute medicine for the treatment of SOP.