USP25 promotes hepatocellular carcinoma progression by interacting with TRIM21 via the Wnt/ß-catenin signaling pathway.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. The ubiquitin-specific peptidase 25 (USP25) protein has been reported to participate in the development of several cancers. However, few studies have reported its association with HCC. In this study, we aimed to investigate the function and mechanism of USP25 in the progression of HCC. METHODS: We analyzed USP25 protein expression in HCC based on The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) database cohorts. Then, we constructed USP25-overexpressing and USP25-knockdown HepG2, MHCC97H, and L-O2 cells. We detected the biological function of USP25 by performing a series of assays, such as Cell Counting Kit-8 (CCK-8), colony formation, transwell, and wound healing assays. Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) analyses were performed to detect the interaction between USP25 and the Wnt/ß-catenin signaling pathway. The relationship between USP25 and tripartite motif-containing 21 (TRIM21) was assessed through mass spectrometry and co-immunoprecipitation (Co-IP) analysis. Finally, we constructed a mouse liver cancer model with the USP25 gene deletion to verify in vivo role of USP25. RESULTS: USP25 was highly expressed in HCC tissue and HCC cell lines. Importantly, high expression of USP25 in tissues was closely related to a poor prognosis. USP25 knockdown markedly reduced the proliferation, migration, and invasion of HepG2 and MHCC97H cells, whereas USP25 overexpression led to the opposite effects. In addition, we demonstrated that USP25 interacts with TRIM21 to regulate the expression of proteins related to epithelial-mesenchymal transition (EMT; E-cadherin, N-cadherin, and Snail) and the Wnt/ß-catenin pathway (ß-catenin, Adenomatous polyposis coli, Axin2 and Glycogen synthase kinase 3 beta) and those of their downstream proteins (C-myc and Cyclin D1). Finally, we verified that knocking out USP25 inhibited tumor growth and distant metastasis in vivo . CONCLUSIONS: In summary, our data showed that USP25 was overexpressed in HCC. USP25 promoted the proliferation, migration, invasion, and EMT of HCC cells by interacting with TRIM21 to activate the ß-catenin signaling pathway.

KIFC3 Regulates the progression and metastasis of gastric cancer via Notch1 pathway.

INTRODUCTION: KIFC3 is a member of the kinesin family which has shown great promise in cancer therapy recently. In this study, we sought to elucidate the role of KIFC3 in the development of GC and its possible mechanisms. METHODS: Two databases and a tissue microarray were used to explore the expression of KIFC3 and its correlation with patients' clinicopathological characteristics. Cell proliferation was examined by cell counting kit-8 assay and colony formation assay. Wound healing assay and transwell assay were performed to examine cell metastasis ability. EMT and Notch signaling related proteins were detected by western blot. Additionally, a xenograft tumor model was established to investigate the function of KIFC3 in vivo. RESULTS: The expression of KIFC3 was upregulated in GC, and was associated with higher T stage and poor prognosis in GC patients. The proliferation and metastasis ability of GC cells were promoted by KIFC3 overexpression while inhibited by KIFC3 knockdown in vitro and in vivo. Furthermore, KIFC3 might activate the Notch1 pathway to facilitate the progression of GC, and DAPT, an inhibitor of Notch signaling, could reverse this effect. CONCLUSION: Together, our data revealed that KIFC3 could enhance the progression and metastasis of GC by activating the Notch1 pathway.

KIFC3 regulates progression of hepatocellular carcinoma via EMT and the AKT/mTOR pathway.

INTRODUCTION: Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related deaths worldwide. Despite an overall downward trend in cancer mortality, HCC-related mortality continues to increase. KIFC3 is involved in cell division and cancers. However, the role of KIFC3 in HCC has yet to be elucidated. METHODS: A total of 36 cases of HCC tissues, 4 HCC cell lines, and TCGA databases were searched to explore the expression of KIFC3 in HCC. Subsequently, Western blot analysis, immunofluorescence, bioinformatic analysis, molecular docking, and Co-IP were performed to investigate the molecular mechanisms of KIFC3 in HCC. RESULT: We found that the expression of KIFC3 was upregulated in HCC, and high KIFC3 expression was related to poor overall survival. In addition, the knockdown of KIFC3 inhibited the proliferation, migration, and invasion of HCC cells in vitro, and impeded the growth of HCC in vivo, while overexpression of KIFC3 in HCC cells revealed the opposite effect. Mechanistically, KIFC3 promotes the progression of HCC through the PI3K/AKT/mTOR signalling. And KIFC3 had slight effect on the protein expression of p-PI3K, p-AKT and p-mTOR in TRIP13-ablated or LY294002-treated HCC cells. The KIFC3 knockdown could further enhance the inhibitory effect of LY294002. CONCLUSION: Our data revealed that KIFC3 is upregulated in HCC and may serve as a novel biomarker for predicting survival in HCC patients. Targeting KIFC3 may serve as a novel therapeutic strategy for HCC patients.

KIFC3 Promotes Proliferation, Migration, and Invasion in Colorectal Cancer via PI3K/AKT/mTOR Signaling Pathway.

Background: KIFC3, belongs to kinesin superfamily proteins (KIFs), is well known for its role in intracellular cargo movement. KIFC3 has been identified as a docetaxel resistance gene in breast cancer cells, however, the role of KIFC3 and its potential mechanism in colorectal cancer (CRC) remains elusive. Objectives: We aims to investigate the effects of KIFC3 in proliferation, migration, and invasion in CRC as well as the potential mechanism inside. Methods: We investigated the expression of KIFC3 in the Oncomine, Gene Expression Profiling Interactive Analysis databases. The KIFC3 protein expression and mRNA level in CRC cells were evaluated by western blot and qRT-PCR. Cell proliferation ability was detected by CCK-8, EdU, colony formation assay and xenograft tumor in nude mice. Flow cytometry was used to detect the cell cycle. The effect of KIFC3 on the epithelial-to-mesenchymal transition (EMT) was investigated by transwell and wound healing assay. The association of KIFC3 with EMT and PI3K/AKT/mTOR signaling pathway were measured by western blot and immunofluorescence staining. Results: The expression of KIFC3 was higher in CRC tissues than normal colorectal tissue, and was negatively correlated with the overall survival of patients with CRC. KIFC3 silencing inhibited the proliferation, migration and invasion of CRC cells. Meanwhile, it could decrease the number of cells in S phase. KIFC3 silencing inhibited the expression of proliferating cell nuclear antigen, Cyclin A2, Cyclin E1, and CDK2 and increased the expression of p21 and p53. KIFC3 overexpression promoted the G1/S phase transition. KIFC3 silencing inhibited the EMT process, which decreased the level of N-cadherin, Vimentin, SNAIL 1, TWIST, MMP-2, MMP-9 and increased E-cadherin, while KIFC3 overexpression show the opposite results. Furthermore, the knockdown of KIFC3 suppressed the EMT process by modulating the PI3K/AKT/mTOR signaling pathway. KIFC3 silencing decreased the expression of phosphorylated PI3K, AKT, mTOR, but total PI3K, AKT, mTOR have no change. Inversely, the upregulation of KIFC3 increased the expression of phosphorylated PI3K, AKT and mTOR, total PI3K, AKT, mTOR have no change. In a xenograft mouse model, the depletion of KIFC3 suppressed tumor growth. the increased expression levels of KIFC3 could enhance the proliferation, migration and invasion of CRC cells, and enhance the EMT process through the PI3K/AKT/mTOR pathway. Conclusion: Our study substantiates that KIFC3 can participate in the regulation of CRC progression by which regulates EMT via the PI3K/AKT/mTOR axis.

The appearance of magnetic susceptibility objects in SWI phase depends on object size: Comparison with QSM and CT.

PURPOSE: Tissue magnetic susceptibility sign can potentially be detected on susceptibility weighted imaging (SWI) phase (SW-P). This study aims to investigate its performance for depicting brain susceptibility structures. METHODS: A simulation was performed to depict magnetic susceptibility structures of various geometries on SW-P and quantitative susceptibility mapping (QSM). Brain MRI was performed on 25 subjects using SWI on a 3 T MRI system. QSM was generated from the same data. SW-P and QSM were analyzed according to radiological assessment for depicting globus pallidus nuclei, optic radiation white matter tracts, and lateral ventricular choroid plexus calcifications. In 11 of these subjects, CT was available and correlated with SW-P and QSM to assess their performance in quantifying calcifications in the choroid plexus. RESULTS: In simulation, the appearance of a sphere on SW-P ranged from centric nodule to mixed positive and negative values as the diameter increased. Large cylinders also appeared as mixed positive and negative values. In comparison, QSM correctly depicted the susceptibility distribution of all magnetic structures. On human brain images, SW-P depicted the globus pallidus and optic radiation with mixed positive and negative values, consistent with simulation, and small choroid plexus calcifications as either mixed positive and negative values or as centric nodules; QSM depicted all structures as solid structures with the expected signs. For measuring calcification in the choroid plexus, QSM vs CT linear regression had a higher coefficient of determination compared to SW-P vs CT and SW-P vs QSM. CONCLUSION: Appearance of susceptibility sources on SW-P changes with object size. This problem can be overcome using QSM.

Pollution characteristics, exposure assessment and potential cardiotoxicities of PM2.5-bound benzotriazole and its derivatives in typical Chinese cities.

Benzotriazole and its derivatives (BTRs), classified as high-volume production chemicals, have been widely detected in various environmental media, including the atmosphere, water, soil and dust, as well as organisms. However, studies on the pollution characteristics and health impact of PM2.5 related BTRs are so far limited. This study is the first to demonstrate the regional scale distribution of PM2.5-bound BTRs and their potential cardiotoxicities. Optimized methods of extraction, purification and GC-EI-MS/MS were applied to characterize and analyze PM2.5-bound BTRs from three cities in China during the winter of 2018. The concentration of ∑BTRs in Taiyuan (6.28 ng·m-3) was more than three times that in Shanghai (1.53 ng·m-3) and Guangzhou (1.99 ng·m-3). Benzotriazole (BTR) and 5-methyl-1H-benzotriazole (5TTR) contributed more than 80% of ∑BTRs concentration as the major pollutants among three cities. The correlation analysis indicated that there was a positive correlation between temperature and concentration of BTR and a negative correlation between temperature and concentration of 5TTR. In addition, the risk of BTRs exposure to toddlers should be paid more attention in Taiyuan by the human exposure assessment. Furthermore, toxicity screening by experimental methods indicated that 4-methyl-1H-benzotriazole (4TTR) was the most harmful to cardiomyocytes. The western blot assay showed a ROS-mediated mitochondrial apoptosis signaling pathway was activated after exposure to 4TTR in neonatal rat cardiomyocytes (NRCMs). On the other hand, metabolomics revealed that exposure of 4TTR to NRCMs disturbed mitochondrial energy metabolism by disturbing pantothenate and coenzyme A synthesis pathway. Our study not only clarifies the contamination profiles of PM2.5-bound BTRs in typical Chinese cities but also reveals their cardiotoxicities associated with mitochondrial dysfunction.

A Review of Ammonia-Oxidizing Archaea and Anaerobic Ammonia-Oxidizing Bacteria in the Aquaculture Pond Environment in China.

The excessive ammonia produced in pond aquaculture processes cannot be ignored. In this review, we present the distribution and diversity of ammonia-oxidizing archaea (AOA) and anaerobic ammonia-oxidizing bacteria (AnAOB) in the pond environment. Combined with environmental conditions, we analyze the advantages of AOA and AnAOB in aquaculture water treatment and discuss the current situation of pond water treatment engineering involving these microbes. AOA and AnAOB play an important role in the nitrogen removal process of aquaculture pond water, especially in seasonal low temperatures and anoxic sediment layers. Finally, we prospect the application of bioreactors to purify pond aquaculture water using AOA and AnAOB, in autotrophic nitrogen removal, which can reduce the production of greenhouse gases (such as nitrous oxide) and is conducive to the development of environmentally sustainable pond aquaculture.

Impacts of Type 2 Diabetes on Disease Severity, Therapeutic Effect, and Mortality of Patients With COVID-19.

PURPOSE: Coronavirus disease 2019 (COVID-19) has become a topic of concern worldwide; however, the impacts of type 2 diabetes mellitus (T2DM) on disease severity, therapeutic effect, and mortality of patients with COVID-19 are unclear. METHODS: All consecutive patients with COVID-19 admitted to the Renmin Hospital of Wuhan University from January 11 to February 6, 2020, were included in this study. RESULTS: A total of 663 patients with COVID-19 were included, while 67 patients with T2DM accounted for 10.1% of the total. Compared with patients with COVID-19 without T2DM, those with T2DM were older (aged 66 years vs 57 years; P < 0.001) and had a male predominance (62.7% vs 37.3%; P = 0.019) and higher prevalence of cardiovascular diseases (61.2% vs 20.6%; P < 0.001) and urinary diseases (9% vs 2.5%; P = 0.014). Patients with T2DM were prone to developing severe (58.2% vs 46.3%; P = 0.002) and critical COVID-19 (20.9% vs 13.4%; P = 0.002) and having poor therapeutic effect (76.1% vs 60.4%; P = 0.017). But there was no obvious difference in the mortality between patients with COVID-19 with and without T2DM (4.5% vs 3.7%; P = 0.732). Multivariate logistic regression analysis identified that T2DM was associated with poor therapeutic effect in patients with COVID-19 (odd ratio [OR] 2.99; 95% confidence interval [CI], 1.07-8.66; P = 0.04). Moreover, having a severe and critical COVID-19 condition (OR 3.27; 95% CI, 1.02-9.00; P = 0.029) and decreased lymphocytes (OR 1.59; 95% CI, 1.10-2.34; P = 0.016) were independent risk factors associated with poor therapeutic effect in patients with COVID-19 with T2DM. CONCLUSIONS: T2DM influenced the disease severity and therapeutic effect and was one of the independent risk factors for poor therapeutic effect in patients with COVID-19.

Do underlying cardiovascular diseases have any impact on hospitalised patients with COVID-19?

OBJECTIVES: An outbreak of the highly contagious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has sickened thousands of people in China. The purpose of this study was to explore the early clinical characteristics of COVID-19 patients with cardiovascular disease (CVD). METHODS: This is a retrospective analysis of patients with COVID-19 from a single centre. All patients underwent real-time reverse transcription PCR for SARS-CoV-2 on admission. Demographic and clinical factors and laboratory data were reviewed and collected to evaluate for significant associations. RESULTS: The study included 541 patients with COVID-19. A total of 144 (26.6%) patients had a history of CVD. The mortality of patients with CVD reached 22.2%, which was higher than that of the overall population of this study (9.8%). Patients with CVD were also more likely to develop liver function abnormality, elevated blood creatinine and lactic dehydrogenase (p<0.05). Symptoms of sputum production were more common in patients with CVD (p=0.026). Lymphocytes, haemoglobin and albumin below the normal range were pervasive in the CVD group (p<0.05). The proportion of critically ill patients in the CVD group (27.8%) was significantly higher than that in the non-CVD group (8.8%). Multivariable logistic regression analysis revealed that CVD (OR: 2.735 (95% CI 1.495 to 5.003), p=0.001) was associated with critical COVID-19 condition, while patients with coronary heart disease were less likely to reach recovery standards (OR: 0.331 (95% CI 0.125 to 0.880), p=0.027). CONCLUSIONS: Considering the high prevalence of CVD, a thorough CVD assessment at diagnosis and early intervention are recommended in COVID-19 patients with CVD. Patients with CVD are more vulnerable to deterioration.

Glandular orientation and shape determined by computational pathology could identify aggressive tumor for early colon carcinoma: a triple-center study.

BACKGROUND: Identifying the early-stage colon adenocarcinoma (ECA) patients who have lower risk cancer vs. the higher risk cancer could improve disease prognosis. Our study aimed to explore whether the glandular morphological features determined by computational pathology could identify high risk cancer in ECA via H&E images digitally. METHODS: 532 ECA patients retrospectively from 2 independent data centers, as well as 113 from The Cancer Genome Atlas (TCGA), were enrolled in this study. Four tissue microarrays (TMAs) were constructed across ECA hematoxylin and eosin (H&E) stained slides. 797 quantitative glandular morphometric features were extracted and 5 most prognostic features were identified using minimum redundancy maximum relevance to construct an image classifier. The image classifier was evaluated on D2/D3 = 223, D4 = 46, D5 = 113. The expression of Ki67 and serum CEA levels were scored on D3, aiming to explore the correlations between image classifier and immunohistochemistry data and serum CEA levels. The roles of clinicopathological data and ECAHBC were evaluated by univariate and multivariate analyses for prognostic value. RESULTS: The image classifier could predict ECA recurrence (accuracy of 88.1%). ECA histomorphometric-based image classifier (ECAHBC) was an independent prognostic factor for poorer disease-specific survival [DSS, (HR = 9.65, 95% CI 2.15-43.12, P = 0.003)]. Significant correlations were observed between ECAHBC-positive patients and positivity of Ki67 labeling index (Ki67Li) and serum CEA. CONCLUSION: Glandular orientation and shape could predict the high risk cancer in ECA and contribute to precision oncology. Computational pathology is emerging as a viable and objective means of identifying predictive biomarkers for cancer patients.

Culture of attached and suspended Rhodopseudomonas faecalis in the presence of decomposing fish feed.

An approach to culturing attached and suspended forms of Rhodopseudomonas faecalis by using compound fish feed with tap water in transparent containers is reported in this study. The ratio of fish feed to tap water was 14.3-50.8 g/L, and no other inoculum or substances were added during the culture process. When the ratio of fish feed to tap water was 14.3 g/L, the highest total nitrogen, total phosphorus, and total dissolved carbon content recorded in the water in the containers were approximately 730 mg/L, 356 mg/L, and 1,620 mg/L, respectively, during the process of feed decay. Comamonas, Rhodopseudomonas, and Clostridium successively dominated during the culture process. Rhodopseudomonas was the most common dominant genus in both the attached and suspended forms when the water was dark red, and the relative operational taxonomic unit abundance reached 80-89% and 24.8%, respectively. The dominant species was R. faecalis. The maximum thickness of attached bacteria and the biomass of attached Rhodopseudomonas reached up to 0.56 mm and 7.5 mg/cm2 , respectively. This study provides a method for the mass culture of Rhodopseudomonas by using the fermentation of aquatic compound fish feed.

Effects of portable solar water quality control machines on aquaculture ponds.

The effects of a portable solar water quality control machine (PSWM) on water quality and sediment of aquaculture ponds were studied in bream aquaculture ponds in Shanghai, China. PSWM operation reduced the temperature and dissolved oxygen (DO) differences between upper and lower water levels. Concentrations of NH4+-N, NO2--N, TN, TP, COD and TSS increased rapidly and reached maximums at 12 h. The density and biomass of phytoplankton and levels of chlorophyll a reached maximums after 40 h of PSWM operation. In a 165-day study, the mean concentrations of NH4+-N, NO2--N and the available phosphorous (AP) in the PSWM ponds were significantly lower than in the control ponds, but the TP was significantly greater than the control ponds. Compared with the test began, the thickness of the sediment in PSWM ponds declined by 12.4 ± 4.3 cm, the control ponds increased by 5.0 ± 2.3 cm and the TN and AP levels in sediment significantly declined. PSWM treatment increased the production of bream and silver carp by 30 and 25%, respectively, and the feed coefficient was reduced by 24.2%. Use of PSWM in bream aquaculture ponds improved water quality, reduced sediment, reduced aquaculture pollution emissions and increased production.

[Community Characteristics of ANAMMOX Bacteria in Subsurface Flow Constructed Wetland (SSFCW) for Processing of Aquaculture Waster Water].

Anaerobic ammonium oxidation (ANAMMOX) is one of the important functions in waste water treatment by subsurface flow constructed wetland (SSFCW), however, there are few studies on ANAMMOX in SSFCW environment at present. The community characteristics of ANAMMOX in the SSFCW of processing aquaculture waste water were explored in this study. In order to analyze the structure, diversity and abundance of ANAMMOX bacteria, several 16S rRNA clone libraries were constructed and real-time PCR targeting specific 16S rRNA genes together with diversity analysis was adopted. The obtained results showed that the SSFCW identified a total of three unknown clusters and two known clusters including Candidatus brocadia and Candidatus kuenenia. The dominant cluster was Candidatus brocadia. The highest diversity levels of ANAMMOX bacteria occurred in autumn (H', 1.21), while the lowest in spring (H', 0.64). The abundance of ANAMMOX bacteria in SSFCW environment ranged from 8.0 x 10(4) to 9.4 x 10(6) copies x g(-1) of fresh weight and the copy number of total bacterial 16S rRNA genes ranged from 7.3 x 10(9) to 9.1 x 10(10) copies x g(-1) of fresh weight during culture cycle. There were significant differences in the ANAMMOX bacteria abundances of different stratum and seasons in SSFCW environment, but the differences in total bacterial abundances were not obvious. In addition, the differences in ANAMMOX bacteria abundances in different stratum and seasons in SSFCW environment were irregular in different culture cycle. According to the distribution characteristics of ANAMMOX bacteria in the wetland, the denitrification effect of SSFCW could be improved by changing the supplying manners of aquaculture wastewater and adjusting the structure of wetland. The research results will provide reference for further optimizing the SSFCW and improving the efficiency of purification.

Design and Application of a Solar Mobile Pond Aquaculture Water Quality-Regulation Machine Based in Bream Pond Aquaculture.

Bream pond aquaculture plays a very important role in China's aquaculture industry and is the main source of aquatic products. To regulate and control pond water quality and sediment, a movable solar pond aquaculture water quality regulation machine (SMWM) was designed and used. This machine is solar-powered and moves on water, and its primary components are a solar power supply device, a sediment lifting device, a mechanism for walking on the water's surface and a control system. The solar power supply device provides power for the machine, and the water walking mechanism drives the machine's motion on the water. The sediment lifting device orbits the main section of the machine and affects a large area of the pond. Tests of the machine's mechanical properties revealed that the minimum illumination necessary for the SMWM to function is 13,000 Lx and that its stable speed on the water is 0.02-0.03 m/s. For an illumination of 13,000-52,500 Lx, the sediment lifting device runs at 0.13-0.35 m/s, and its water delivery capacity is 110-208 m(3)/h. The sediment lifting device is able to fold away, and the angle of the suction chamber can be adjusted, making the machine work well in ponds at different water depths from 0.5 m to 2 m. The optimal distance from the sediment lifting device to the bottom of the pond is 10-15 cm. In addition, adjusting the length of the connecting rod and the direction of the traction rope allows the SMWM to work in a pond water area greater than 80%. The analysis of water quality in Wuchang bream (Parabramis pekinensis) and silver carp (Hypophthalmichthys molitrix) culture ponds using the SMWM resulted in decreased NH3(+)-N and available phosphorus concentrations and increased TP concentrations. The TN content and the amount of available phosphorus in the sediment were reduced. In addition, the fish production showed that the SMWM enhanced the yields of Wuchang bream and silver carp by more than 30% and 24%, respectively. These results indicate that the SMWM may be suitable for Wuchang bream pond aquaculture in China and that it can be used in pond aquaculture for regulating and controlling water quality.

Vertical Segregation and Phylogenetic Characterization of Ammonia-Oxidizing Bacteria and Archaea in the Sediment of a Freshwater Aquaculture Pond.

Pond aquaculture is the major freshwater aquaculture method in China. Ammonia-oxidizing communities inhabiting pond sediments play an important role in controlling culture water quality. However, the distribution and activities of ammonia-oxidizing microbial communities along sediment profiles are poorly understood in this specific environment. Vertical variations in the abundance, transcription, potential ammonia oxidizing rate, and community composition of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) in sediment samples (0-50 cm depth) collected from a freshwater aquaculture pond were investigated. The concentrations of the AOA amoA gene were higher than those of the AOB by an order of magnitude, which suggested that AOA, as opposed to AOB, were the numerically predominant ammonia-oxidizing organisms in the surface sediment. This could be attributed to the fact that AOA are more resistant to low levels of dissolved oxygen. However, the concentrations of the AOB amoA mRNA were higher than those of the AOA by 2.5- to 39.9-fold in surface sediments (0-10 cm depth), which suggests that the oxidation of ammonia was mainly performed by AOB in the surface sediments, and by AOA in the deeper sediments, where only AOA could be detected. Clone libraries of AOA and AOB amoA sequences indicated that the diversity of AOA and AOB decreased with increasing depth. The AOB community consisted of two groups: the Nitrosospira and Nitrosomonas clusters, and Nitrosomonas were predominant in the freshwater pond sediment. All AOA amoA gene sequences in the 0-2 cm deep sediment were grouped into the Nitrososphaera cluster, while other AOA sequences in deeper sediments (10-15 and 20-25 cm depths) were grouped into the Nitrosopumilus cluster.