The discovery of complete ammonia oxidizers (comammox) refutes the century-old paradigm that nitrification requires the activity of two types of microbes. Determining the distribution and abundance of comammox in various environments is important for revealing the ecology of microbial nitrification within the global nitrogen cycle. In this study, the ubiquity and diversity of comammox were analyzed for samples from different types of environments, including soil, sediment, sludge, and water. The results of a two-step PCR using highly degenerate primers (THDP-PCR) and quantitative real-time PCR (qPCR) supported the relatively high abundance of comammox in nearly half of all samples tested, sometimes even outnumbering canonical ammonia-oxidizing bacteria (AOB). In addition, a relatively high proportion of comammox in tap and coastal water samples was confirmed via analysis of metagenomic data sets in public databases. The diversity of comammox was estimated by comammox-specific partial nested PCR amplification of the ammonia monooxygenase subunit A (amoA) gene, and phylogenetic analysis of comammox AmoA clearly showed a split of clade A into clades A.1 and A.2, with the proportions of clades A.1, A.2, and B differing among the various environmental samples. Moreover, compared to the amoA genes of AOB and ammonia-oxidizing archaea (AOA), the comammox amoA gene exhibited higher diversity indices. The ubiquitous distribution and high diversity of comammox indicate that they are likely overlooked contributors to nitrification in various ecosystems. IMPORTANCE The discovery of complete ammonia oxidizers (comammox), which oxidize ammonia to nitrate via nitrite, refutes the century-old paradigm that nitrification requires the activity of two types of microbes and redefines a key process in the biogeochemical nitrogen cycle. Understanding the functional relationships between comammox and other nitrifiers is important for ecological studies on the nitrogen cycle. Therefore, the diversity and contribution of comammox should be considered during ecological analyses of nitrifying microorganisms. In this study, a ubiquitous and highly diverse distribution of comammox was observed in various environmental samples, similar to the distribution of canonical ammonia-oxidizing bacteria. The proportion of comammox was relatively high in coastal water and sediment samples, whereas it was nearly undetectable in open-ocean samples. The ubiquitous distribution and high diversity of comammox indicate that these microorganisms might be important contributors to nitrification.

Methanogen populations of an intertidal mudflat in the Yangtze River estuary of China were investigated based on the methyl coenzyme M reductase A (mcrA) gene using 454-pyrosequencing and quantitative real-time polymerase chain reaction (qPCR). Samples were collected at six depths from three locations. In the qPCR analyses, a mean depth-wise change of mcrA gene abundance was observed from (1.23 ± 0.13) × 10(7) to (1.16 ± 0.29) × 10(8) per g dried soil, which was inversely correlated with the depletion of sulfate (R(2) = 0.74; α = 0.05) and salinity (R (2) = 0.66; α = 0.05). The copy numbers of mcrA was at least 1 order of magnitude higher than dissimilatory sulfate reductase B (dsrB) genes, likely indicating the importance of methanogenesis at the mudflat. Sequences related to the orders Methanomicrobiales, Methanosarcinales, Methanobacteriales, Methanococcales and the uncultured methanogens; Rice Cluster I (RC-I), Zoige cluster I (ZC-I) and anaerobic methane oxidizing archaeal lineage-1 (ANME-1) were detected. Methanomicrobiales and Methanosarcinales dominated the entire sediment layers, but detectable changes of proportions were observed with depth. The hydrogenotrophic methanogens Methanomicrobiales slightly increased with depth while Methanosarcinales showed the reverse. Chao1 and ACE richness estimators revealed higher diversity of methanogens near the surface (0-10 cm) when compared with the bottom sediments. The near-surface sediments were mainly dominated by the family Methanosarcinaceae (45 %), which has members that can utilize substrates that cannot be used by sulfate-reducing bacteria. Overall, current data indicate that Methanosarcinales and Methanomicrobiales are the most dominant methanogens within the entire depth profile down to 100 cm, with higher abundance and diversity of methanogens in the deeper and upper sediment layers, respectively.

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The effect of plant invasion on the microorganisms of soil sediments is very important for estuary ecology. The community structures of methanogens and sulfate-reducing bacteria (SRB) as a function of Spartina alterniflora invasion in Phragmites australis-vegetated sediments of the Dongtan wetland in the Yangtze River estuary, China, were investigated using 454 pyrosequencing and quantitative real-time PCR (qPCR) of the methyl coenzyme M reductase A (mcrA) and dissimilatory sulfite-reductase (dsrB) genes. Sediment samples were collected from two replicate locations, and each location included three sampling stands each covered by monocultures of P. australis, S. alterniflora and both plants (transition stands), respectively. qPCR analysis revealed higher copy numbers of mcrA genes in sediments from S. alterniflora stands than P. australis stands (5- and 7.5-fold more in the spring and summer, respectively), which is consistent with the higher methane flux rates measured in the S. alterniflora stands (up to 8.01 ± 5.61 mg m−2 h−1). Similar trends were observed for SRB, and they were up to two orders of magnitude higher than the methanogens. Diversity indices indicated a lower diversity of methanogens in the S. alterniflora stands than the P. australis stands. In contrast, insignificant variations were observed in the diversity of SRB with the invasion. Although Methanomicrobiales and Methanococcales, the hydrogenotrophic methanogens, dominated in the salt marsh, Methanomicrobiales displayed a slight increase with the invasion and growth of S. alterniflora, whereas the later responded differently. Methanosarcina, the metabolically diverse methanogens, did not vary with the invasion of, but Methanosaeta, the exclusive acetate utilizers, appeared to increase with S. alterniflora invasion. In SRB, sequences closely related to the families Desulfobacteraceae and Desulfobulbaceae dominated in the salt marsh, although they displayed minimal changes with the S. alterniflora invasion. Approximately 11.3 ± 5.1% of the dsrB gene sequences formed a novel cluster that was reduced upon the invasion. The results showed that in the sediments of tidal salt marsh where S. alterniflora displaced P. australis, the abundances of methanogens and SRB increased, but the community composition of methanogens appeared to be influenced more than did the SRB.

The copper-containing membrane-bound monooxygenase (CuMMO) family comprises key enzymes for methane or ammonia oxidation: particulate methane monooxygenase (PMMO) and ammonia monooxygenase (AMO). To comprehensively amplify CuMMO genes, a two-step PCR strategy was developed using a newly designed tagged highly degenerate primer (THDP; degeneracy = 4608). Designated THDP-PCR, the technique consists of primary CuMMO gene-specific PCR followed by secondary PCR with a tag as a single primer. No significant bias in THDP-PCR amplification was found using various combinations of template mixtures of pmoA and amoA genes, which encode key subunits of the pMMO and AMO enzymes, respectively, from different microbes. THDP-PCR was successfully applied to nine different environmental samples and revealed relatively high contents of complete ammonia oxidation (Comammox)-related bacteria and a novel group of the CuMMO family. The levels of freshwater cluster methanotrophs obtained by THDP-PCR were much higher than those obtained by conventional methanotroph-specific PCR. The THDP-PCR strategy developed in this study can be extended to other functional gene-based community analyses, particularly when the target gene sequences lack regions of high consensus for primer design.

The treatment of advanced triple-negative breast cancer, which failed in first-line or second-line therapy, is a significant challenge. We conducted this retrospective study to explore the efficacy and safety of apatinib and capecitabine as the third-line treatment for advanced triple-negative breast cancer.This retrospective study involved 44 advanced triple-negative breast cancer patients who failed in first-line or second-line therapy in Tangshan People's Hospital from January 2016 to February 2017. Twenty-two patients received apatinib and capecitabine, while 22 patients were treated with capecitabine monotherapy as third-line therapy. The progression-free survival (PFS), objective response rate (ORR), disease control rate (DCR), and adverse events were compared between 2 groups.The apatinib and capecitabine group exhibited a higher PFS than capecitabine group (P = .001). Meanwhile, ORR and DCR in apatinib and capecitabine group were better than in capecitabine group (P = .042; .016). The 2 groups showed no significant difference in adverse events except degree I-II bleeding (P = .021). Both the apatinib and capecitabine and the capecitabine regimens revealed good tolerability.The apatinib and capecitabine regimen can achieve a better efficacy and similar serious adverse events compared with capecitabine regimen as the third-line treatment for advanced triple-negative breast cancer.

Complete ammonia oxidizing (comammox) bacteria play key roles in environmental nitrogen cycling and all belong to the genus Nitrospira, which was originally believed to include only strict nitrite-oxidizing bacteria (sNOB). Thus, differential estimation of sNOB abundance from comammox Nitrospira has become problematic since both contain nitrite oxidoreductase genes that serve as common targets for sNOB detection. Herein, we developed novel comammox Nitrospira clades A- and B-specific primer sets targeting the α-subunit of ammonia monooxygenase gene (amoA) and a sNOB-specific primer set targeting cyanase gene (cynS) for quantitative PCR (qPCR). The high coverage and specificity of these primers had been checked by metagenome and metatranscriptome datasets. Efficient and specific amplification with these primers were demonstrated using various environmental samples. Using the newly designed primers, we successfully estimated the abundances of comammox Nitrospira and sNOB in samples from two chloramination-treated drinking water systems and found that, in most samples, comammox Nitrospira clade A was the dominant Nitrospira, and also served as the primary ammonia oxidizer. Compared with other ammonia oxidizers, comammox Nitrospira took advantage in process water samples in these two drinking water systems. We also demonstrated sNOB can be readily misrepresented with the previous method, calculated by subtracting the comammox abundance from the total Nitrospira, especially when the comammox Nitrospira proportion is relatively high. The new primer sets were successfully applied for the quantification of comammox Nitrospira and sNOB, which may prove useful in understanding the roles of Nitrospira in nitrification in various ecosystems. Importance Nitrospira is a dominant nitrite-oxidizing bacteria in many artificial and natural environments. The discovery of complete ammonia oxidizers in Nitrospira prevents the use of previously identified primers targeting Nitrospira 16S rRNA gene or nitrite oxidoreductase (nxr) gene for differential determination of strict nitrite-oxidizing bacteria in Nitrospira (sNOB) and comammox Nitrospira. We designed three novel primer sets that enabled quantification of comammox Nitrospira clades A and B and sNOB with high coverage, specificity, and accuracy in various environments. With designed primer sets, sNOB and comammox Nitrospira were differentially estimated in drinking water systems, and we found that comammox clade A was overcome sNOB and other ammonia oxidizers in process water samples. Accurate quantification of comammox Nitrospira and sNOB using the newly designed primers will provide essential information for evaluating the contribution of Nitrospira to nitrification in various ecosystems.

Intertidal mudflats are land–sea interaction areas and play important roles in global nutrient cycles. However, a comprehensive understanding of microbial communities in these mudflats remains elusive. In this study, mudflat sediment samples from the Dongtan wetland of Chongming Island, the largest alluvial island in the world, were collected. Using a modified metatranscriptomic method, the depth-wise distributions of potentially active microbial communities were investigated based on small subunit ribosomal RNA (SSU rRNA) sequences. Multiple environmental factors were also measured and analyzed in conjunction with the prokaryotic composition profiles. A prokaryotic diversity analysis based on the metatranscriptome datasets revealed two or threefold higher diversity indices (associated with potentially active microbes participating in biogeochemical processes in Dongtan) compared with the diversity indices based on 16S rRNA gene amplicons. Bacteria were numerically dominant relative to archaea, and the potentially active prokaryotic taxa were mostly assigned to the bacterial phyla Chloroflexi, Acidobacteria, and Bacteroidetes and the classes Delta- and Gamma-proteobacteria, along with the archaeal lineages phylum Bathyarchaeota and the order Thermoplasmatales. The total nitrogen and carbon content of the sediment samples were environmental factors that significantly affected the depth-wise distributions of both bacterial and archaeal communities. Furthermore, the activity of potentially active taxa (including the prevalent order Desulfobacterales and family Anaerolineaceae) appeared to be significantly underestimated by PCR-based methods, notably at the DNA level, and indicates that using normal PCR amplification of DNA limits the study of potential microbial activity. This is the first study of potentially active microbial communities in depth-wise sediments from Dongtan. The improved knowledge of microbial communities in Dongtan provides a foundation for exploring biogeochemical cycling and microbial functions.