Intravenous immunoglobulin for treatment of hospitalized COVID-19 patients: an evidence mapping and meta-analysis.

BACKGROUND: The clinical efficacy and safety of intravenous immunoglobulin (IVIg) treatment for COVID-19 remain controversial. This study aimed to map the current status and gaps of available evidence, and conduct a meta-analysis to further investigate the benefit of IVIg in COVID-19 patients. METHODS: Electronic databases were searched for systematic reviews/meta-analyses (SR/MAs), primary studies with control groups, reporting on the use of IVIg in patients with COVID-19. A random-effects meta-analysis with subgroup analyses regarding study design and patient disease severity was performed. Our outcomes of interest determined by the evidence mapping, were mortality, length of hospitalization (days), length of intensive care unit (ICU) stay (days), number of patients requiring mechanical ventilation, and adverse events. RESULTS: We included 34 studies (12 SR/MAs, 8 prospective and 14 retrospective studies). A total of 5571 hospitalized patients were involved in 22 primary studies. Random-effects meta-analyses of very low to moderate evidence showed that there was little or no difference between IVIg and standard care or placebo in reducing mortality (relative risk [RR] 0.91; 95% CI 0.78-1.06; risk difference [RD] 3.3% fewer), length of hospital (mean difference [MD] 0.37; 95% CI - 2.56, 3.31) and ICU (MD 0.36; 95% CI - 0.81, 1.53) stays, mechanical ventilation use (RR 0.92; 95% CI 0.68-1.24; RD 2.8% fewer), and adverse events (RR 0.98; 95% CI 0.84-1.14; RD 0.5% fewer) of patients with COVID-19. Sensitivity analysis using a fixed-effects model indicated that IVIg may reduce mortality (RR 0.76; 95% CI 0.60-0.97), and increase length of hospital stay (MD 0.68; 95% CI 0.09-1.28). CONCLUSION: Very low to moderate certainty of evidence indicated IVIg may not improve the clinical outcomes of hospitalized patients with COVID-19. Given the discrepancy between the random- and fixed-effects model results, further large-scale and well-designed RCTs are warranted.

Pretreatment with Methylene Blue Protects Against Acute Seizure and Oxidative Stress in a Kainic Acid-Induced Status Epilepticus Model.

BACKGROUND The aim of the present study was to investigate the potential anticonvulsant effect of methylene blue (MB) in a kainic acid (KA)-induced status epilepticus (SE) model. The effects of MB on levels of oxidative stress and glutamate (Glu) also were explored. MATERIAL AND METHODS Sixty C57BL/6 mice were randomly divided into 5 equal-sized groups: (1) controls; (2) KA; (3) MB 0.5 mg/kg+KA; (4) MB 1 mg/kg+KA; and (5) vehicle+KA. The SE model was established by intra-amygdala microinjection of KA. Behavioral observations and simultaneous electroencephalographic records of the seizures in different groups were analyzed to determine the potential anticonvulsant effect of MB. The influences of MB on oxidative stress markers and glutamate were also detected to explore the possible mechanism. RESULTS MB afforded clear protection against KA-induced acute seizure, as measured by the delayed latency of onset of generalized seizures and SE, decreased percentage of SE, and increased survival rate in mice with acute epilepsy. MB markedly increased the latency to first onset of epileptiform activity and decreased the average duration of epileptiform events, as well as the percentage of time during which the epileptiform activity occurred. Administration of MB prevented KA-induced deterioration of oxidative stress markers and Glu. CONCLUSIONS MB is protective against acute seizure in SE. This beneficial effect may be at least partially related to its potent antioxidant ability and influence on Glu level.

Distribution of ammonia-oxidizing archaea and bacteria along an engineered coastal ecosystem in subtropical China.

Ammonia-oxidizing archaea (AOA) and bacteria (AOB) are the crucial players in nitrogen cycle. Both AOA and AOB were examined along a gradient of human activity in a coastal ecosystem from intertidal zone, grassland, and Casuarina equisetifolia forest to farmland. Results showed that the farmland soils had noticeably higher nitrate-N, available P than soils in the other three sites. Generally, AOA and AOB community structures varied across sites. The farmland mainly had Nitrosotalea-like AOA, intertidal zone was dominated by Nitrosopumilus AOA, while grassland and C. equisetifolia forest primarily harbored Nitrososphaera-like AOA. The farmland and C. equisetifolia forest owned Nitrosospira-like AOB, intertidal zone possessed Nitrosomonas-like AOB, and no AOB was detected in the grassland. AOA abundance was significantly greater than AOB in this coastal ecosystem (p < 0.05, n = 8). AOB diversity and abundance in the farmland were significantly higher than those in the other three sites (p < 0.05, n = 2). The biodiversity and abundance of AOA were not significantly correlated with any soil property (p < 0.05, n = 8). However, the diversity of AOB was significantly correlated with pH, available P and total P (p < 0.05, n = 6). The abundance of AOB was significantly correlated with pH, nitrite, available N, available P and total P (p < 0.05, n = 6). This study suggested that the community structures of AOA and AOB vary in the different parts in the bio-engineered coastal ecosystem and agricultural activity appears to influence these nitrifiers.

VEGF-C/VEGFR-3/iNOS Signaling in Osteosarcoma MG63 Cells Mediates Stimulatory Effects on Human Umbilical Vein Endothelial Cell Proliferation.

Objective To assess the effects of vascular endothelial growth factor-C (VEGF-C)/vascular endothelial growth factor receptor-3 (VEGFR-3) signaling on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in human osteosarcoma MG63 cells and the subsequent impact on the proliferation of human umbilical vein endothelial cells (HUVECs). MethodsMG63 cells were treated with VEGF-C alone (VEGF-C group), VEGF-C + iNOS inhibitor aminoguanidine (AG; AG group), and VEGF-C + VEGFR-3 inhibitor MAZ51 (MAZ51 group); untreated MG63 cells were used as controls. NO production was evaluated by a colorimetric method involving nitrate reductase. Meanwhile, mRNA and protein levels of iNOS were examined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. To explore the effect of VEGF-C/VEGFR-3/iNOS signaling of MG63 cells on proliferation of HUVECs, we set up six groups: HUVECs, HUVECs+MG63, HUVECs+VEGF-C, HUVECs+MG63+VEGF-C, HUVECs+MG63+VEGF-C+AG, and HUVECs+MG63+VEGF-C+MAZ51 groups. The proliferation of HUVEC cells was assessed by Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay, and proliferating cell nuclear antigen (PCNA) expression quantitation. ResultsVEGF-C treatment enhanced iNOS expression at both gene and protein levels (mRNA: LSD-t=4.152, P<0.01; protein: LSD-t=3.486, P<0.01) and increased NO release of MG63 cells (LSD-t=3.774, P<0.01); treatment with either AG or MAZ51 decreased these effects (mRNA: LSD-t=9.183, P<0.001; LSD-t=8.639, P<0.001; protein: LSD-t=5.170, P<0.001; LSD-t=7.255, P<0.001; NO production:LSD-t=10.326, P<0.001; LSD-t=10.540, P<0.001). Interestingly, co-incubation of HUVECs with MG63 cells and/or VEGF-C significantly promoted HUVEC proliferation (EdU: LSD-t=5.374, P<0.001; LSD-t=2.984, P<0.05; LSD-t=8.526, P<0.001; PCNA: LSD-t=9.267, P<0.001; LSD-t=5.515, P<0.001; LSD-t=14.873, P<0.001).The proliferation effects of HUVEC induced by MG63 cells and VEGF-C attenuated by the treatment of AG (EdU: LSD-t=10.770, P<0.001; PCNA: LSD-t=19.940, P<0.001) or MAZ51 (EdU: LSD-t=6.950, P<0.001; PCNA: LSD-t=14.001, P<0.001). ConclusionIn human osteosarcoma MG63 cells, activation of VEGFR-3 by VEGF-C promotes iNOS expression and NO production, which subsequently induces HUVEC proliferation.

Vitamin D suppress the production of vascular endothelial growth factor in mast cell by inhibiting PI3K/Akt/p38 MAPK/HIF-1α pathway in chronic spontaneous urticaria.

Mast cells play a significant role in urticaria pathogenesis. It's evidenced that vitamin D has positive impact in chronic spontaneous urticaria (CSU) recently, but underlying mechanisms remain unclear. In this study, isobaric tag for relative and absolute quantification-liquid chromatography-mass spectrometer/mass spectrometer was used to detect the expression of proteins in sera of CSU patients and healthy subjects. Thirty-one differentially expressed proteins were identified, in which vitamin D binding protein (VDBP) was higher in CSU patients than that in healthy subjects after verification. Our results indicated that sera of CSU patients induced the production of vascular endothelial growth factor (VEGF) in mast cells through PI3K/Akt/p38 MAPK/HIF-1α axis in an IgE-depended way, and 25(OH)D3 suppressed the expression of VEGF by inhibiting this signaling pathway axis in this process. Collectively, these results suggest VDBP to be a potential biomarker and propose a potential mechanism of benefit for vitamin D therapy in CSU.

Case report of cryptogenic multifocal ulcerous stenosing enteritis (CMUSE): a rare disease may contribute to endoscopy-capsule retention in the small intestine.

BACKGROUND: CMUSE is a rare disease whose diagnosis remains difficult because the lesion is confined to the small bowel. CASE PRESENTATION: Here, we present a case of 43-year-old female patient suffered chronic abdominal pain for 20 years, and finally diagnosed with CMUSE. Capsule endoscopy was performed when general endoscopic investigation failed to find the lesion, but the capsule was stranded in the small intestine. Moreover, capsule retention results in acute intestinal obstruction. Thus, surgery was performed and CMUSE was confirmed. The patient was recovered after partial small intestine resection. CONCLUSIONS: Capsule retention occurred in nearly 60% of patients with CMUSE. Capsule endoscopy should be avoided when the patient is suspected of CMUSE, especially with severe anemia and radiologic finding in the ileum.

Correction to: Case report of cryptogenic multifocal ulcerous stenosing enteritis (CMUSE): a rare isease may contribute to endoscopy-capsule retention in the small intestine.

Following publication of the original article [1], the author reported the wrong version of Table 1 has been published. The word of 'Capsule' was mistakenly written as 'Capusle'.

Functional dominance and community compositions of ammonia-oxidizing archaea in extremely acidic soils of natural forests.

Extremely acidic soils of natural forests in Nanling National Nature Reserve have been previously investigated and revisited in two successive years to reveal the active ammonia oxidizers. Ammonia-oxidizing archaea (AOA) rather than ammonia-oxidizing bacteria (AOB) were found more functionally important in the extremely acidic soils of the natural forests in Nanling National Nature Reserve. The relative abundances of Nitrosotalea, Nitrososphaera sister group, and Nitrososphaera lineages recovered by ammonia monooxygenase subunit A (amoA) transcripts were reassessed and compared to AOA communities formerly detected by genomic DNA. Nitrosotalea, previously found the most abundant AOA, were the second-most-active lineage after Nitrososphaera sister group. Our field study results, therefore, propose the acidophilic AOA, Nitrosotalea, can better reside in extremely acidic soils while they may not contribute to nitrification proportionately according to their abundances or they are less functionally active. In contrast, the functional importance of Nitrososphaera sister group may be previously underestimated and the functional dominance further extends their ecological distribution as little has been reported. Nitrososphaera gargensis-like AOA, the third abundant lineage, were more active in summer. The analyses of AOA community composition and its correlation with environmental parameters support the previous observations of the potential impact of organic matter on AOA composition. Al3+, however, did not show a strong adverse correlation with the abundances of functional AOA unlike in the DNA-based study. The new data further emphasize the functional dominance of AOA in extremely acidic soils, and unveil the relative contributions of AOA lineages to nitrification and their community transitions under the environmental influences.

Abundance and Diversity of Aerobic/Anaerobic Ammonia/Ammonium-Oxidizing Microorganisms in an Ammonium-Rich Aquitard in the Pearl River Delta of South China.

Natural occurring groundwater with abnormally high ammonium concentrations was discovered in the aquifer-aquitard system in the Pearl River Delta, South China. The community composition and abundance of aerobic/anaerobic ammonia/ammonium-oxidizing microorganisms (AOM) in the aquitard were investigated in this study. The alpha subunit of ammonia monooxygenase gene (amoA) was used as the biomarker for the detection of aerobic ammonia-oxidizing archaea (AOA) and bacteria (AOB), and also partial 16S rRNA gene for Plantomycetes and anaerobic ammonium-oxidizing (anammox) bacteria. Phylogenetic analysis showed that AOA in this aquitard were affiliated with those from water columns and wastewater treatment plants; and AOB were dominated by sequences among the Nitrosomonas marina/Nitrosomonas oligotropha lineage, which were affiliated with environmental sequences from coastal eutrophic bay and subtropical estuary. The richness and diversity of both AOA and AOB communities had very little variations with the depth. Candidatus Scalindua-related sequences dominated the anammox bacterial community. AOB amoA gene abundances were always higher than those of AOA at different depths in this aquitard. The Pearson moment correlation analysis showed that AOA amoA gene abundance positively correlated with pH and ammonium concentration, whereas AOB amoA gene abundance negatively correlated with C/N ratio. This is the first report that highlights the presence with low diversity of AOM communities in natural aquitard of rich ammonium.

Effects of reforestation on ammonia-oxidizing microbial community composition and abundance in subtropical acidic forest soils.

Forest ecosystems have great ecological values in mitigation of climate change and protection of biodiversity of flora and fauna; re-forestry is commonly used to enhance the sequestration of atmospheric CO2 into forest storage biomass. Therefore, seasonal and spatial dynamics of the major microbial players in nitrification, ammonia-oxidizing archaea (AOA) and bacteria (AOB), in acidic soils of young and matured revegetated forests were investigated to elucidate the changes of microbial communities during forest restoration, and compared to delineate the patterns of community shifts under the influences of environmental factors. AOA were more abundant than AOB in both young and matured revegetated forest soils in both summer and winter seasons. In summer, however, the abundance of amoA-AOA decreased remarkably (p < 0.01), ranging from 1.90 (± 0.07) × 108 copies per gram dry soil in matured forest to 5.04 (± 0.43) × 108 copies per gram dry soil in young forest, and amoA-AOB was below detection limits to obtain any meaningful values. Moreover, exchangeable Al3+ and organic matter were found to regulate the physiologically functional nitrifiers, especially AOA abundance in acidic forest soils. AOB community in winter showed stronger correlation with the restoration status of revegetated forests and AOA community dominated by Nitrosotalea devanaterra, in contrast, was more sensitive to the seasonal and spatial variations of environmental factors. These results enrich the current knowledge of nitrification during re-forestry and provide valuable information to developmental status of revegetated forests for management through microbial analysis.

A More Comprehensive Community of Ammonia-Oxidizing Archaea (AOA) Revealed by Genomic DNA and RNA Analyses of amoA Gene in Subtropical Acidic Forest Soils.

Ammonia-oxidizing bacteria (AOB) and archaea (AOA) are the main nitrifiers which are well studied in natural environments, and AOA frequently outnumber AOB by orders especially in acidic conditions, making AOA the most promising ammonia oxidizers. The phylogeny of AOA revealed in related studies, however, often varied and hardly reach a consensus on functional phylotypes. The objective of this study was to compare ammonia-oxidizing communities by amoA gene and transcript based on both genomic DNA and RNA in extremely acidic forest soils (pH <4.5). Our results support the numerical and functional dominance of AOA over AOB in acidic soils as bacterial amoA gene and transcript were both under detection limits and archaeal amoA, in contrast, were abundant and responded to the fluctuations of environmental factors. Organic matter from tree residues was proposed as the main source of microbial available nitrogen, and the potential co-precipitation of dissolved organic matter (DOM) with soluble Al3+ species in acidic soil matrix may further restrict the amount of nitrogen sources required by AOB besides NH3/NH4+ equilibrium. Although AOA were better adapted to oligotrophic environments, they were susceptible to the toxicity of exchangeable Al3+. Phylotypes affiliated to Nitrososphaera, Nitrososphaera sister group, and Nitrosotalea were detected by amoA gene and transcript. Nitrosotalea devantaerra and Nitrososphaera sister group were the major AOA. Compared to the genomic DNA data, higher relative abundances of Nitrososphaera and Nitrososphaera sister group were recognized in amoA transcript inferred AOA communities, where Nitrosotalea relative abundance was found lower, implying the functional activities of Nitrososphaera sister group and Nitrososphaera were easily underestimated and Nitrosotalea did not attribute proportionally to nitrification in extremely acidic soils. Further comparison of the different AOA community compositions and relative abundance of each phylotypes revealed by amoA genes and transcripts make it possible to identify the functional AOA species and assess their ecological role in extremely acidic soils.

A comparison of denitrifying bacterial community structures and abundance in acidic soils between natural forest and re-vegetated forest of Nanling Nature Reserve in southern China.

Denitrification plays a key role in converting reactive nitrogen species to dinitrogen gas back into the atmosphere to maintain the equilibrium of nitrogen cycling in ecosystems. In this study, functional genes of nirK and nosZ were used to detect the community structure and abundance of denitrifying microorganisms in acidic forest soils in southern China. Three sets of factors were considered for a comparison among 5 forests, including forest types (natural vs. re-vegetated), depths (surface layer vs. lower layer) and seasons (winter vs. summer). The community of nirK gene detected from these acidic forest soils was closely related to Proteobacteria especially α-Proteobacteria and uncultured soil sequences, while that of nosZ gene was affiliated with the α-, ß- and γ-Proteobacteria. Higher diversity of denitrifiers was observed in re-vegetated forest soils than natural ones. Not only the community but also the abundance showed significant differences between forest types as well as depths. The abundance of denitrifiers ranged from 105 to 107 gene copies g-1 dry soil in this study. For nirK gene, the abundance was much higher in the lower layer than surface layer in both forest types, and the differences between winter and summer in natural forest soils were higher than those in re-vegetated forest soils. The abundance of nosZ and nirK genes showed a similar trend in natural forest, but the former was higher in matured forest than re-vegetated forest. This study provided a direct comparison on community composition and abundance of denitrifying bacteria in natural and re-vegetated acidic forest soils to allow further assessment of the nitrogen cycling.

Co-occurrence of nitrite-dependent anaerobic ammonium and methane oxidation processes in subtropical acidic forest soils.

Anaerobic ammonium oxidation (anammox) and nitrite-dependent anaerobic methane oxidation (n-damo) are two new processes of recent discoveries linking the microbial nitrogen and carbon cycles. In this study, 16S ribosomal RNA (rRNA) gene of anammox bacteria and pmoA gene of n-damo bacteria were used to investigate their distribution and diversity in natural acidic and re-vegetated forest soils. The 16S rRNA gene sequences retrieved featured at least three species in two genera known anammox bacteria, namely Candidatus Brocadia anammoxidans, Candidatus Brocadia fulgida, and Candidatus Kuenenia stuttgartiensis while the pmoA gene amplified was affiliated with two species of known n-damo bacteria Candidatus Methylomirabilis oxyfera and a newly established Candidatus Methylomirabilis sp. According to the results, the diversity of anammox bacteria in natural forests was lower than in re-vegetated forests, but no significant difference was observed in n-damo community between them. Quantitative real-time PCR showed that both anammox and n-damo bacteria were more abundant in the lower layer (10-20 cm) than the surface layer (0-5 cm). The abundance of anammox bacteria varied from 2.21 × 10(5) to 3.90 × 10(6) gene copies per gram dry soil, and n-damo bacteria quantities were between 1.69 × 10(5) and 5.07 × 10(6) gene copies per gram dry soil in the two different layers. Both anammox and n-damo bacteria are reported for the first time to co-occur in acidic forest soil in this study, providing a more comprehensive information on more defined microbial processes contributing to C and N cycles in the ecosystems.

Dominance of ammonia-oxidizing archaea community induced by land use change from Masson pine to eucalypt plantation in subtropical China.

A considerable proportion of Masson pine forests have been converted into eucalypt plantations in the last 30 years in Guangdong Province, subtropical China, for economic reasons, which may affect the ammonia-oxidizing archaea (AOA) community and the process of ammonia transformation. In order to determine the effects of forest conversion on AOA community, AOA communities in a Masson pine (Pinus massoniana) plantation and a eucalypt (Eucalyptus urophylla) plantation, which was converted from the Masson pine, were compared. Results showed that the land use change from the Masson pine to the eucalypt plantation decreased soil nutrient levels. A significant decrease of the potential nitrification rates (PNR) was also observed after the forest conversion (p < 5 %, n = 6). AOA were the only ammonia oxidizers in both plantations (no ammonia-oxidizing bacteria were detected). The detected AOA are affiliated with the genera Nitrosotalea and Nitrososphaera. A decrease of AOA abundance and an increase of the diversity were evident with the plantation conversion in the surface layer. AOA amoA gene diversity was negatively correlated with organic C and total N, respectively (p < 0.05, n = 12). AOA amoA gene abundance was negatively correlated with NH4 (+) and available P, respectively (p < 0.05, n = 12). However, AOA abundance was positively correlated with PNR, but not significantly (p < 0.05, n = 6), indicating AOA community change was only a partial reason for the decrease of PNR.

Differential distribution patterns of ammonia-oxidizing archaea and bacteria in acidic soils of Nanling National Nature Reserve forests in subtropical China.

In addition to ammonia-oxidizing bacteria (AOB) the more recently discovered ammonia-oxidizing archaea (AOA) can also oxidize ammonia, but little is known about AOA community structure and abundance in subtropical forest soils. In this study, both AOA and AOB were investigated with molecular techniques in eight types of forests at surface soils (0-2 cm) and deep layers (18-20 cm) in Nanling National Nature Reserve in subtropical China. The results showed that the forest soils, all acidic (pH 4.24-5.10), harbored a wide range of AOA phylotypes, including the genera Nitrosotalea, Nitrososphaera, and another 6 clusters, one of which was reported for the first time. For AOB, only members of Nitrosospira were retrieved. Moreover, the abundance of the ammonia monooxygenase gene (amoA) from AOA dominated over AOB in most soil samples (13/16). Soil depth, rather than forest type, was an important factor shaping the community structure of AOA and AOB. The distribution patterns of AOA and AOB in soil layers were reversed: AOA diversity and abundances in the deep layers were higher than those in the surface layers; on the contrary, AOB diversity and abundances in the deep layers were lower than those in the surface layers. Interestingly, the diversity of AOA was positively correlated with pH, but negatively correlated with organic carbon, total nitrogen and total phosphorus, and the abundance of AOA was negatively correlated with available phosphorus. Our results demonstrated that AOA and AOB were differentially distributed in acidic soils in subtropical forests and affected differently by soil characteristics.

The synthesis of novel taxoids for oral administration.

A group of novel taxoids, with modifications at C-7, C-10, C-3' and C-14 positions of paclitaxel, was synthesized in order to improve their biological profile by decreasing their affinity with P-glycoprotein (P-gp) and increasing cellular permeability. Most of the new taxoids demonstrated the similar potent cytotoxic activities in MCF-7 human tumor cell line as paclitaxel in vitro. In the permeability assay with monolayers of Caco-2 cells, most of the compounds demonstrated an increased trans-cellular transport in A-to-B direction in comparison with paclitaxel. Among them the compounds T-13, T-15 and T-26 showed the highest permeability, and with efflux ratios better than that of ortataxel. The interaction of the compounds T-13 and T-26 with P-gp was evaluated using Madin-Darby canine kidney (MDCK)-multidrug resistance-1(MDR1) and MDCK-wild-type (WT). The results indicated that T-13 and T-26 were poor substrates for P-gp and possessed inhibiting effects of P-gp mediated efflux. It was thus clear that simultaneous modifications at the C-7, C-10 and C-3' positions of paclitaxel significantly impaired its interactions with P-gp and interfered with P-gp mediated efflux.

Effects of allylthiourea, salinity, and pH on ammonia/ammonium-oxidizing prokaryotes in mangrove sediment incubated in laboratory microcosms.

Anaerobic ammonium-oxidizing (anammox) bacteria, aerobic ammonia-oxidizing archaea (AOA) and bacteria (AOB) are three groups of ammonia/ammonium-oxidizing prokaryotes (AOPs) involved in the biochemical nitrogen cycling. In this study, the effects of allylthiourea (ATU), pH, and salinity on these three groups from mangrove sediment were investigated through microcosm incubation in laboratory. ATU treatments (50, 100, and 500 mg L(-1)) obviously affected the community structure of anammox bacteria and AOB, but only slightly for AOA. ATU began to inhibit anammox bacteria growth slightly from day 10, but had an obvious inhibition on AOA growth from the starting of the study. At 100 mg L(-1) of ATU or higher, AOB growth was inhibited, but only lasted for 5 days. The pH treatments showed that acidic condition (pH 5) had a slight effect on the community structure of anammox bacteria and AOA, but an obvious effect on AOB. Acidic condition promoted the growth of all groups of AOPs in different extent, but alkaline condition (pH 9) had a weak effect on AOB community structure and a strong effect on both anammox bacteria and AOA. Alkaline condition obviously inhibited anammox bacteria growth, slightly promoted AOA, and slightly promoted AOB in the first 20 days, but inhibited afterward. Salinity treatment showed that higher salinity (20 and 40 ‰) resulted in higher anammox bacteria diversity, and both AOA and AOB might have species specificity to salinity. High salinity promoted the growth of both anammox bacteria and AOB, inhibited AOA between 5 and 10 days, but promoted afterward. The results help to understand the role of these microbial groups in biogeochemical nitrogen cycling and their responses to the changing environments.

Differential responses of ammonia/ammonium-oxidizing microorganisms in mangrove sediment to amendment of acetate and leaf litter.

The effects of acetate and leaf litter powder on ammonia/ammonium-oxidizing microorganisms (AOMs) in mangrove sediment were investigated in a laboratory incubation study for a period of 60 days. The results showed that different AOMs responded differently to the addition of acetate and leaf litter. A higher diversity of anaerobic ammonium-oxidizing (anammox) bacteria was observed when acetate or leaf litter was added than the control. However, acetate and leaf litter generally inhibited the growth of anammox bacteria despite that leaf litter promoted their growth in the first 5 days. The inhibitory effects on anammox bacteria were more pronounced by acetate than by leaf litter. Neither acetate nor leaf litter affected ammonia-oxidizing archaea (AOA) community structures, but promoted their growth. For ammonia-oxidizing bacteria (AOB), the addition of acetate or leaf litter resulted in changes of community structures and promoted their growth in the early phase of the incubation. In addition, the promoting effects by leaf litter on AOB growth were more obvious than acetate. These results indicated that organic substances affect AOM community structures and abundances. The study suggests that leaf litter has an important influence on the community structures and abundances of AOMs in mangrove sediment and affects the nitrogen cycle in such ecosystem.

Improvement of DGGE analysis by modifications of PCR protocols for analysis of microbial community members with low abundance.

Denaturing gradient gel electrophoresis (DGGE) is a powerful technique to reveal the community structures and composition of microorganisms in complex natural environments and samples. However, positive and reproducible polymerase chain reaction (PCR) products, which are difficult to acquire for some specific samples due to low abundance of the target microorganisms, significantly impair the effective applications of DGGE. Thus, nested PCR is often introduced to generate positive PCR products from the complex samples, but one problem is also introduced: The total number of thermocycling in nested PCR is usually unacceptably high, which results in skewed community structures by generation of random or mismatched PCR products on the DGGE gel, and this was demonstrated in this study. Furthermore, nested PCR could not resolve the uneven representative issue with PCR products of complex samples with unequal richness of microbial population. In order to solve the two problems in nested PCR, the general protocol was modified and improved in this study. Firstly, a general PCR procedure was used to amplify the target genes with the PCR primers without any guanine cytosine (GC) clamp, and then, the resultant PCR products were purified and diluted to 0.01 µg ml(-1). Subsequently, the diluted PCR products were utilized as templates to amplify again with the same PCR primers with the GC clamp for 17 cycles, and the products were finally subjected to DGGE analysis. We demonstrated that this is a much more reliable approach to obtain a high quality DGGE profile with high reproducibility. Thus, we recommend the adoption of this improved protocol in analyzing microorganisms of low abundance in complex samples when applying the DGGE fingerprinting technique to avoid biased results.

Distribution patterns of ammonia-oxidizing bacteria and anammox bacteria in the freshwater marsh of Honghe wetland in Northeast China.

Community characteristics of aerobic ammonia-oxidizing bacteria (AOB) and anaerobic ammonium-oxidizing (anammox) bacteria in Honghe freshwater marsh, a Ramsar-designated wetland in Northeast China, were analyzed in this study. Samples were collected from surface and low layers of sediments in the Experimental, Buffer, and Core Zones in the reserve. Community structures of AOB were investigated using both 16S rRNA and amoA (encoding for the α-subunit of the ammonia monooxygenase) genes. Majority of both 16S rRNA and amoA gene-PCR amplified sequences obtained from the samples in the three zones affiliated with Nitrosospira, which agreed with other wetland studies. A relatively high richness of ß-AOB amoA gene detected in the freshwater marsh might suggest minimal external pressure was experienced, providing a suitable habitat for ß-AOB communities. Anammox bacteria communities were assessed using both 16S rRNA and hzo (encoding for hydrazine oxidoreductase) genes. However, PCR amplification of the hzo gene in all samples failed, suggesting that the utilization of hzo biomarker for detecting anammox bacteria in freshwater marsh might have serious limitations. Results with 16S rRNA gene showed that Candidatus Kuenenia was detected in only the Experimental Zone, whereas Ca. Scalindua including different lineages was observed in both the Buffer and Experimental Zones but not the Core Zone. These results indicated that both AOB and anammox bacteria have specific distribution patterns in the ecosystem corresponding to the extent of anthropogenic impact.