Response of denitrification microbiome to the nitrogen flux in three Gorges reservoir (TGR) sediments during two seasonal water fluctuation events.

Three Gorges Reservoir (TGR) water fluctuation creates high water level (HWL) and low water level (LWL) condition in TGR aquatic ecosystem. HWL fluies significant nutrients, mainly introducing carbon and nitrogen into the ecosystem. The nitrogen input is a concern for water quality management of TGR since the possible eutrophication caused by nitrogen spike. Sediment denitrification is widely recognized as the dominant nitrogen removal process in freshwater ecosystem. Therefore, the response of TGR sediments microbiome to the input nitrogen flucatution is crucial for both nitrogen balance and the eutrophication status of the ecosystem. Using high throughout sequencing of 16S rRNA gene and the predicted denitrification enzyme, and qualitative PCR of denitrification functional genes, we investigated how TGR sediments denitrification microbiome respond to the input nitrogen flux during two seasonal water fluctuation events. Concomitant to expected input carbon and nitrogen, we observed distinct microbial community structure and denitrification microbiota in HWL and LWL, and also in seasonal sampling events. Sediments pH, total nitrogen and nitrate were the significant impact factors in shaping the microbial community structure. Important denitrification microbiota (e.g., Saprospiraceae, Gemmatimonadaceae, Pseudomonas) are the main taxa of the microbial community and also showed water level and seasonal variation. The relative abundance of denitrification enzyme (nar, nir, nor, nos) and function genes (nirS, nirK, nosZ) were higher in LWL than HWL. Denitrification enzyme were significantly (p < 0.05) correlated with the nitrate concentration. In addition, the relative abundance of denitrification enzyme and function genes increased during the transition from 2014 HWL to 2015 LWL. Results suggested that TGR sediments denitrification is nitrate concentration dependent. The denitrification microbiome is initially inhibited due to high nitrate input, then they developed denitrification ability in response to high nitrate concentration.

Microbial Mediation of Carbon, Nitrogen, and Sulfur Cycles During Solid Waste Decomposition.

Landfills are a unique "terrestrial ecosystem" and serve as a significant carbon sink. Microorganisms convert biodegradable substances in municipal solid waste (MSW) to CH4, CO2, and microbial biomass, consisting of the carbon cycling in landfills. Microbial-mediated N and S cycles are also the important biogeochemical process during MSW decomposition, resulting in N2O and H2S emission, respectively. Meanwhile, microbial-mediated N and S cycles affect carbon cycling. How microbial community structure and function respond to C, N, and S cycling during solid waste decomposition, however, are not well-characterized. Here, we show the response of bacterial and archaeal community structure and functions to C, N, and S cycling during solid waste decomposition in a long-term (265 days) operation laboratory-scale bioreactor through 16S rRNA-based pyrosequencing and metagenomics analysis. Bacterial and archaeal community composition varied during solid waste decomposition. Aerobic respiration was the main pathway for CO2 emission, while anaerobic C fixation was the main pathway in carbon fixation. Methanogenesis and denitrification increased during solid waste decomposition, suggesting increasing CH4 and N2O emission. In contract, fermentation decreased along solid waste decomposition. Interestingly, Clostridiales were abundant and showed potential for several pathways in C, N, and S cycling. Archaea were involved in many pathways of C and N cycles. There is a shift between bacteria and archaea involvement in N2 fixation along solid waste decomposition that bacteria Clostridiales and Bacteroidales were initially dominant and then Methanosarcinales increased and became dominant in methanogenic phase. These results provide extensive microbial mediation of C, N, and S cycling profiles during solid waste decomposition.

Chronic Exposure to Environmentally Relevant Concentrations of Tetracycline Perturbs Gut Homeostasis in Zebrafish.

The prevalence of residual antibiotics is a global threat to human health. Less is known about the potential health effects of residual antibiotics in freshwater systems. Here, zebrafish were used to explore chronic effects of environmentally relevant concentrations (ERCs) of tetracycline (TC). Although chronic exposure to TC did not significantly alter the body weight of adult zebrafish, the exposed zebrafish parents exhibited substantial changes in gut microbiota composition and a reduced gut-weight ratio. Notably, male fish exposed to TC showed a significant decline of critical intestinal function-related metabolites (i.e., triglycerides, glucose, and free fat acid), while this was not observed in females, resulting in sex-dependency. The gut microbial composition of chronically exposed zebrafish parents changed substantially, but the disruption was not transferred to their respective offspring without exposure. However, the perturbation of insulin-related signaling pathways caused by TC exposure was not attenuated in the zebrafish offspring after removal of TC exposure. Taken together, our findings suggest that chronic exposure to TC disturbs gut microbial communities and metabolism and exerts chronic effects on the insulin/IGF-1 mediated signaling cascades, implying that exposure to antibiotics not only leads to the selection of resistant microbes but also poses long-term deleterious health risks for the next generation.

Antibiotic resistance genes in landfill leachates from seven municipal solid waste landfills: Seasonal variations, hosts, and risk assessment.

Landfills are reservoir of antibiotics and antibiotic resistance. Antibiotic resistance would transport to the environment through landfill leachate, posing threaten to the environment. However, long term monitoring on antibiotic resistance genes in landfill leachate transportation is limited. Furthermore, antibiotic resistance gene hosts and their risk assessment are lacking. In this study, we investigated the seasonal variation of ARGs sulI, tetO and tetW in seven Chinese municipal solid waste landfill leachates over two years (2017-2018) by quantitative polymerase chain reaction. We also evaluated the associated bacterial hosts and their risk levels based on metagenomics and omics-based framework for assessing the health risk of antimicrobial resistance genes, respectively. Because sulI, tetO and tetW are abundant and the most frequently detected ARGs in global landfill system, they are selected as target ARGs. Results showed that the relative content of target ARGs in 2017 was 100 times higher than that in 2018, suggesting ARGs attenuation. The hosts of sulI were phyla of Lentisphaerae and Proteobacteria, whereas the hosts of tetO and tetW were Bacteroidetes and Firmicutes. Remarkably, the host species include pathogenic bacterium (Salmonella enterica, Labilibaculum filiforme, Bacteroidales bacterium, Anaeromassilibacillus senegalensis, and Pseudochrobactrum sp. B5). ARGs tetO and tetW belong to the Rank II level with characters of enrichment in the human-associated environment and gene mobility, and sulI ranked as Rank VI. In addition, among 1210 known ARGs in the landfill leachate, 78 ARGs belonged to risk Rank I (enrichment in human-associated environment, gene mobility and pathogenicity), demonstrating high health risk of landfill system. These results demonstrate that antibiotic resistance in landfill and landfill leachate have high health risk and the kind of ARGs with high abundance in human-associated environment, gene mobility and pathogenicity should be paid more attention.

Microbial mercury methylation potential in a large-scale municipal solid waste landfill, China.

Landfills harbor ideal conditions for microbial mercury methylation. However, the levels and distribution of mercury (Hg) and methylmercury (MeHg), potential microbial Hg methylation, and their linkage within landfills are largely unknown. In the present study, total mercury (THg), MeHg, the Hg methylation gene (hgcA) and mer operon were quantified in 30 waste samples from different depths (0-30 m) at 5 locations within a large-scale landfill in China. The average concentrations of THg and MeHg in the solid waste samples were 1422.91 ng/g and 3.15 ng/g, respectively. THg (up to 14405.29 ng/g) and MeHg (up to 10.42 ng/g) have high concentrations in the middle part (10-15 m) along the depth profiles. The concentration of THg was strongly positively (both p < 0.05) correlated with the MeHg concentration and the relative abundance of hgcA, indicating that the THg concentration can play an important role in microbial Hg methylation. The hgcA genes were detected in most samples and mer operon were detected in all samples. Combined hgcA qPCR and metagenomics data showed that Archaea Methanofollis may mainly account for Hg methylation within landfills. These findings provide fundamental knowledge on Hg cycles in landfills.

Sulfonamide and tetracycline in landfill leachates from seven municipal solid waste (MSW) landfills: Seasonal variation and risk assessment.

Antibiotics have received increased attention as emerging contaminants due to their toxicity and potential risk. Landfills serve as one of the important reservoirs of antibiotics. The antibiotics in landfills leaching to nearby environment by leachate may threat ecosystem health. The present study aimed to evaluate the levels of tetracyclines (TCs) and sulfonamides (SAs) in seven Chinese Municipal Solid Waste (MSW) landfill leachates over two years (2017-2018). Seven target antibiotics, TC, oxytetracycline (OTC), doxycycline (DXC), sulfonamide sulfadiazine (SD), sulfamerazine (SM), sulfamethazine (SMX), and sulfamethoxazole (SMT), were detected in 56 landfill leachate samples. Among these, SMT had the highest mean concentration at 654 ng/L (n = 45), followed by OTC (219.58 ng/L, n = 47), and SD (209.98 ng/L, n = 49). The temporal trend showed that antibiotic concentrations were higher in 2017 than in 2018. Furthermore, physicochemical properties were significantly correlated with SAs (p < 0.05), whereas no significant correlation was found for TCs. Seasonal variation analysis revealed that antibiotic levels were higher in spring and winter compared to summer and fall seasons, which might be attributed to the higher waterfall levels in these seasons. Risk assessment revealed that SAs (SM, SMX, SMT) are associated with high risk, and the RQs follow the order of: SMX > SMT > SM. In contrast, TCs had insignificant risk. The findings of this two-year comprehensive monitoring project have produced positive results regarding antibiotic pollution at landfill sites, which can be applied to antibiotics management in landfill and further ensure public health.

Antibiotics and antibiotic resistance genes in landfills: A review.

Landfill are important reservoirs of antibiotics and antibiotic resistance genes (ARGs). They harbor diverse contaminants, such as heavy metals and persistent organic chemicals, complex microbial consortia, and anaerobic degradation processes, which facilitate the occurrence, development, and transfer of ARGs and antibiotic resistant bacteria (ARB). The main concern is that antibiotics and developed ARGs and ARB may transfer to the local environment via leachate and landfill leakage. In this paper, we provide an overview of established studies on antibiotics and ARGs in landfills, summarize the origins and distribution of antibiotics and ARGs, discuss the linkages among various antibiotics, ARGs, and bacterial communities as well as the influencing factors of ARGs, and evaluate the current treatment processes of antibiotics and ARGs. Finally, future research is proposed to fill the current knowledge gaps, which include mechanisms for the development and transmission of antibiotic resistance, as well as efficient treatment approaches for antibiotic resistance.

Metagenomic analysis of antibiotic resistance genes (ARGs) during refuse decomposition.

Landfill is important reservoirs of residual antibiotics and antibiotic resistance genes (ARGs), but the mechanism of landfill application influence on antibiotic resistance remains unclear. Although refuse decomposition plays a crucial role in landfill stabilization, its impact on the antibiotic resistance has not been well characterized. To better understand the impact, we studied the dynamics of ARGs and the bacterial community composition during refuse decomposition in a bench-scale bioreactor after long term operation (265d) based on metagenomics analysis. The total abundances of ARGs increased from 431.0ppm in the initial aerobic phase (AP) to 643.9ppm in the later methanogenic phase (MP) during refuse decomposition, suggesting that application of landfill for municipal solid waste (MSW) treatment may elevate the level of ARGs. A shift from drug-specific (bacitracin, tetracycline and sulfonamide) resistance to multidrug resistance was observed during the refuse decomposition and was driven by a shift of potential bacteria hosts. The elevated abundance of Pseudomonas mainly contributed to the increasing abundance of multidrug ARGs (mexF and mexW). Accordingly, the percentage of ARGs encoding an efflux pump increased during refuse decomposition, suggesting that potential bacteria hosts developed this mechanism to adapt to the carbon and energy shortage when biodegradable substances were depleted. Overall, our findings indicate that the use of landfill for MSW treatment increased antibiotic resistance, and demonstrate the need for a comprehensive investigation of antibiotic resistance in landfill.

Increasing atmospheric deposition nitrogen and ammonium reduced microbial activity and changed the bacterial community composition of red paddy soil.

Atmospheric deposition nitrogen (ADN) increases the N content in soil and subsequently impacts microbial activity of soil. However, the effects of ADN on paddy soil microbial activity have not been well characterized. In this study, we studied how red paddy soil microbial activity responses to different contents of ADN through a 10-months ADN simulation on well managed pot experiments. Results showed that all tested contents of ADN fluxes (27, 55, and 82kgNha-1 when its ratio of NH4+/NO3--N (RN) was 2:1) enhanced the soil enzyme activity and microbial biomass carbon and nitrogen and 27kgNha-1 ADN had maximum effects while comparing with the fertilizer treatment. Generally, increasing of both ADN flux and RN (1:2, 1:1 and 2:1 with the ADN flux of 55kgNha-1) had similar reduced effects on microbial activity. Furthermore, both ADN flux and RN significantly reduced soil bacterial alpha diversity (p<0.05) and altered bacterial community structure (e.g., the relative abundances of genera Dyella and Rhodoblastus affiliated to Proteobacteria increased). Redundancy analysis demonstrated that ADN flux and RN were the main drivers in shaping paddy soil bacteria community. Overall, the results have indicated that increasing ADN flux and ammonium reduced soil microbial activity and changed the soil bacterial community. The finding highlights how paddy soil microbial community response to ADN and provides information for N management in paddy soil.

Methanogen communities in a municipal landfill complex in China.

Landfills are significant global sources of atmospheric methane, but little is known about the ecology and community structure of methanogens in these sites. Here, we investigated the methanogen community based on methyl coenzyme M reductase A gene amplicons in the vertical profiles of three different sites at a municipal landfill complex in China. Links between methanogen communities and refuse properties were explored using multivariate analysis. Clone library results showed that most clones (92%) were related to the hydrogenotrophic methanogens, Methanomicrobiales. Almost all of the Methanomicrobiales clones retrieved in this study are members of the genus Methanoculleus Eight clones were affiliated with the genus Methanofollis The remaining clones were clustered within the genus Methanosarcina Terminal restriction fragment length polymorphism profiles showed that the landfill was predominated by 22 taxa, making up 69%-96% of the community. Of these, a single taxon comprised 36%-65% of the communities across all sites and depths. Principal components analysis separated the methanogen community into three groups, irrespective of site or depth. Redundancy analysis suggested that total phosphorus and pH play roles in structuring methanogen communities in landfills.

A novel heterotrophic nitrifying and aerobic denitrifying bacterium, Zobellella taiwanensis DN-7, can remove high-strength ammonium.

A novel heterotrophic bacterium capable of heterotrophic nitrification and aerobic denitrification was isolated from ammonium contaminated landfill leachate and physiochemical and phylogenetically identified as Zobellella taiwanensis DN-7. DN-7 converted nitrate, nitrate, and ammonium to N2 as the primary end product. Single factor experiments suggested that the optimal conditions for ammonium removal were trisodium citrate as carbon source, C/N ratio 8, pH 8.0-10.0, salinity less than 3 %, temperature 30 °C, and rotation speed more than 150 rpm. Specifically, DN-7 could remove 1000.0 and 2000.0 mg/L NH4 (+)-N completely within 96 and 216 h, with maximum removal rates of 19.6 and 17.3 mg L(-1) h(-1), respectively. These results demonstrated that DN-7 is a promising candidate for application of high-strength ammonium wastewater treatments.

Depressive-like behavior observed with a minimal loss of locus coeruleus (LC) neurons following administration of 6-hydroxydopamine is associated with electrophysiological changes and reversed with precursors of norepinephrine.

Depression is a common co-morbid condition most often observed in subjects with mild cognitive impairment (MCI) and during the early stages of Alzheimer's disease (AD). Dysfunction of the central noradrenergic nervous system is an important component in depression. In AD, locus coeruleus (LC) noradrenergic neurons are significantly reduced pathologically and the reduction of LC neurons is hypothesized to begin very early in the progression of the disorder; however, it is not known if dysfunction of the noradrenergic system due to early LC neuronal loss is involved in mediating depression in early AD. Therefore, the purpose of this study was to determine in an animal model if a loss of noradrenergic LC neurons results in depressive-like behavior. The LC noradrenergic neuronal population was reduced by the bilateral administration of the neurotoxin 6-hydroxydopamine (6-OHDA) directly into the LC. Forced swim test (FST) was performed three weeks after the administration of 6-OHDA (5, 10 and 14 µg/µl), animals administered the 5 µg/µl of 6-OHDA demonstrated a significant increase in immobility, indicating depressive-like behavior. This increase in immobility at the 5 µg/µl dose was observed with a minimal loss of LC noradrenergic neurons as compared to LC neuronal loss observed at 10 and 14 µg/µl dose. A significant positive correlation between the number of surviving LC neurons after 6-OHDA and FST immobile time was observed, suggesting that in animals with a minimal loss of LC neurons (or a greater number of surviving LC neurons) following 6-OHDA demonstrated depressive-like behavior. As the 6-OHDA-induced loss of LC neurons is increased, the time spent immobile is reduced. Depressive-like behavior was also observed with the 5 µg/µl dose of 6-OHDA with a second behavior test, sucrose consumption. FST increased immobility following 6-OHDA (5 µg/µl) was reversed by the administration of a single dose of L-1-3-4-dihydroxyphenylalanine (DOPA) or l-threo-3,4-dihydroxyphenylserine (DOPS) prior to behavioral assessment. Surviving LC neurons 3 weeks after 6-OHDA (5 µg/µl) demonstrated compensatory changes of increased firing frequency, a more irregular firing pattern, and a higher percentage of cells firing in bursts. These results indicate that depressive-like behavior in mice is observed following the administration of 6-OHDA and the loss of LC noradrenergic neurons; however, the depressive-like behavior correlates positively with the number of surviving LC neurons with 6-OHDA administration. This data suggests the depression observed in MCI subjects and in the early stages of AD may due to the hypothesized early, minimal loss of LC neurons with remaining LC neurons being more active than normal.

Composition of bacterial and archaeal communities during landfill refuse decomposition processes.

Little is known about the archaeal and the bacterial diversities in a landfill during different phases of decomposition. In this study, the archaeal and the bacterial diversities of Laogang landfill (Shanghai, China) at two different decomposition phases (i.e., initial methanogenic phase (IMP) and stable methanogenic phase (SMP)), were culture-independently examined using PCR-based 454 pyrosequencing. A total of 47,753 sequences of 16S rRNA genes were retrieved from 69,954 reads and analyzed to evaluate the diversities of the archaeal and bacterial communities. The most predominant types of archaea were hydrogenotrophic Methanomicrobiales, and of bacteria were Proteobacteria, Firmicutes, and Bacteroidetes. As might be expected, their abundances varied at decomposition phases. Archaea Methanomicrobiales accounts for 97.6% of total archaeal population abundance in IMP and about 57.6% in SMP. The abundance of archaeal genus Halobacteriale was 0.1% in IMP and was 20.3% in the SMP. The abundance of Firmicutes was 21.3% in IMP and was 4.3% in SMP. The abundance of Bacteroidetes represented 11.5% of total bacterial in IMP and was dominant (49.4%) in SMP. Both the IMP and SMP had unique cellulolytic bacteria compositions. IMP consisted of members of Bacillus, Fibrobacter, and Eubacterium, while SMP harbored groups of Microbacterium. Both phases had Clostridium with different abundance, 4-5 folds higher in SMP.

Bacterial community diversity in municipal waste landfill sites.

Little is known about the bacterial diversity of landfills and how environmental factors impact the diversity. In this study, PCR-based 454 pyrosequencing was used to investigate the bacterial communities of ten landfill leachate samples from five landfill sites in China. A total of 137 K useable sequences from the V3-V6 regions of the 16S rRNA gene were retrieved from 205 K reads. These sequences revealed the presence of a large number of operational taxonomic units (OTUs) in the landfills (709-1599 OTUs per sample). The most predominant bacterial representatives in the landfills investigated, regardless of geographic area, included Gammaproteobacteria, Firmicutes, and Bacteroidetes. The phyla Fusobacteria and Tenericutes were also found for the first time to be predominant in the landfills. The phylum Fusobacteria predominated (51.5 and 48.8%) in two semi-arid landfills, and the phylum Tenericutes dominated (30.6%) at one humid, subtropical landfill. Further, a large number of Pseudomonas was detected in most samples, comprising the dominant group and accounting for 40.9 to 92.4% of the total abundance. Principal component analysis (PCA) and cluster analysis based on OTU abundance showed that the abundant taxa separated the bacterial community. Canonical correlation analysis (CCA) suggested that precipitation and landfilling age significantly impact on the bacterial community structure. The bacterial community function (e.g., cellulolytic bacteria, sulfate-reducing bacteria (SRB), sulfate-oxidizing bacteria, and xenobiotic organic compound (XOC)-degrading bacteria) was also diverse, but the pattern is unclear.

Occurrence and prevalence of antibiotic resistance in landfill leachate.

Antibiotic resistance (AR) is extensively present in various environments, posing emerging threat to public and environmental health. Landfill receives unused and unwanted antibiotics through household waste and AR within waste (e.g., activated sludge and illegal clinical waste) and is supposed to serve as an important AR reservoir. In this study, we used culture-dependent methods and quantitative molecular techniques to detect and quantify antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in 12 landfill leachate samples from six geographic different landfills, China. Five tested ARGs (tetO, tetW, bla(TEM), sulI, and sulII) and seven kinds of antibiotic-resistant heterotrophic ARB were extensively detected in all samples, demonstrating their occurrence in landfill. The detected high ratio (10(-2) to 10(-5)) of ARGs to 16S ribosomal RNA (rRNA) gene copies implied that ARGs are prevalent in landfill. Correlation analysis showed that ARGs (tetO, tetW, sulI, and sulII) significantly correlated to ambient bacterial 16S rRNA gene copies, suggesting that the abundance of bacteria in landfill leachate may play an important role in the horizontal spread of ARGs.

Archaeal community diversity in municipal waste landfill sites.

Despite the pivotal role of archaea in methane production in landfills, the identity, ecology, and functional diversity of these microorganisms and their link to environmental factors remain largely unknown. We collected 11 landfill leachate samples from six geographically distinct landfills of different ages in China and analyzed the archaeal community by bar-coded 454 pyrosequencing. We retrieved 121,797 sequences from a total of 167,583 sequences (average length of 464 bp). The archaeal community was geographically structured, and nonabundant taxa primarily contributed to the observed dissimilarities. Canonical correlation analysis (CCA) suggested that the total phosphorous (TP), nitrate, and conductivity are important drivers for shaping the archaeal community. The hydrogenotrophic methanogens Methanomicrobiales and Methanobacteriales greatly dominated 9 of 11 samples, ranging from 83.7 to 99.5 %. These methanogens also dominated the remaining two samples, accounting for 70.3 and 58.8 %, respectively. Interestingly, for all of the studied Chinese landfills, 16S rRNA analysis indicated the predominance of hydrogenotrophic methanogens.

Investigation of microbial community structure of a shallow lake after one season copper sulfate algaecide treatment.

In present work we described, for the first time, the phylogenic structure of the microbial community in a shallow freshwater lake (Hawk Island Lake, located in the Lower Peninsula of the State of Michigan, U.S.A.) after one season (four times during May to August 2007) of CuSO4 treatment for algae growth control. The microbial community structure was characterized by terminal restriction fragment length polymorphism (TRFLP), clone library and 454 pyrosequencing. The similar structure of water chemistry measured across three sampling sites suggested that the lake was well mixed. The concentration of chlorophyll a (chl-a) and turbidity was low, 3.35 ± 1.62 µg/L and 2.5 ± 1.9 NTU, respectively, implying that photosynthesis was suppressed. TRFLP profiles showed that the lake was dominated by 16 terminal fragments (TFs), accounting for 85.5-92.6% abundance. Analysis of similarity (ANOSIM) showed that the difference in microbial community structure between upper and lower depths of the water column was not significant (P=0.101). These results suggested that the microbial community structure within the lake was similar. Clone library and 454 pyrosequencing indicated that the lake was dominated by freshwater phyla, Proteobacteria, Bacteroides, and Actinobacteria. Moreover, the large number of unclassified bacteria (27.4% of total 2090,454 sequences) suggested a complex microbial community structure in the lake.

[Advances in molecular mechanism of bacterial reduction of hexavalent chromium].

Cr(VI) has been causing serious environmental pollution due to its carcinogenicity, teratogenicity and strong migration. Reduction of Cr( VI) to Cr(III), a precipitation that is much less toxic, is an efficient strategy to control Cr pollution. Within the strategy, bacterial reduction of Cr(VI) to Cr(III) has been considered as one of the best bioremediation methods because of its efficiency, environment friendly, and low cost; however, the molecular mechanism remains large unknown. This review summarizes Cr(VI) reduction bacterial species and its application in pollution control, elaborates the pathways of Cr( VI) reduction and functional proteins involved, concludes the molecular mechanism of baterial reduction Cr(VI), and discusses the orientation of the future research.

Functional contributions of N- and O-glycans to L-selectin ligands in murine and human lymphoid organs.

L-selectin initiates lymphocyte interactions with high endothelial venules (HEVs) of lymphoid organs through binding to ligands with specific glycosylation modifications. 6-Sulfo sLe(x), a sulfated carbohydrate determinant for L-selectin, is carried on core 2 and extended core 1 O-glycans of HEV-expressed glycoproteins. The MECA-79 monoclonal antibody recognizes sulfated extended core 1 O-glycans and partially blocks lymphocyte-HEV interactions in lymphoid organs. Recent evidence has identified the contribution of 6-sulfo sLe(x) carried on N-glycans to lymphocyte homing in mice. Here, we characterize CL40, a novel IgG monoclonal antibody. CL40 equaled or surpassed MECA-79 as a histochemical staining reagent for HEVs and HEV-like vessels in mouse and human. Using synthetic carbohydrates, we found that CL40 bound to 6-sulfo sLe(x) structures, on both core 2 and extended core 1 structures, with an absolute dependency on 6-O-sulfation. Using transfected CHO cells and gene-targeted mice, we observed that CL40 bound its epitope on both N-glycans and O-glycans. Consistent with its broader glycan-binding, CL40 was superior to MECA-79 in blocking lymphocyte-HEV interactions in both wild-type mice and mice deficient in forming O-glycans. This superiority was more marked in human, as CL40 completely blocked lymphocyte binding to tonsillar HEVs, whereas MECA-79 inhibited only 60%. These findings extend the evidence for the importance of N-glycans in lymphocyte homing in mouse and indicate that this dependency also applies to human lymphoid organs.