Identification and characterization of a novel hydroxylamine oxidase, DnfA, that catalyzes the oxidation of hydroxylamine to N2.

Nitrogen (N2) gas in the atmosphere is partially replenished by microbial denitrification of ammonia. Recent study has shown that Alcaligenes ammonioxydans oxidizes ammonia to dinitrogen via a process featuring the intermediate hydroxylamine, termed "Dirammox" (direct ammonia oxidation). However, the unique biochemistry of this process remains unknown. Here, we report an enzyme involved in Dirammox that catalyzes the conversion of hydroxylamine to N2. We tested previously annotated proteins involved in redox reactions, DnfA, DnfB, and DnfC, to determine their ability to catalyze the oxidation of ammonia or hydroxylamine. Our results showed that none of these proteins bound to ammonia or catalyzed its oxidation; however, we did find DnfA bound to hydroxylamine. Further experiments demonstrated that, in the presence of NADH and FAD, DnfA catalyzed the conversion of 15N-labeled hydroxylamine to 15N2. This conversion did not happen under oxygen (O2)-free conditions. Thus, we concluded that DnfA encodes a hydroxylamine oxidase. We demonstrate that DnfA is not homologous to any known hydroxylamine oxidoreductases and contains a diiron center, which was shown to be involved in catalysis via electron paramagnetic resonance experiments. Furthermore, enzyme kinetics of DnfA were assayed, revealing a Km of 92.9 ± 3.0 µM for hydroxylamine and a kcat of 0.028 ± 0.001 s-1. Finally, we show that DnfA was localized in the cytoplasm and periplasm as well as in tubular membrane invaginations in HO-1 cells. To the best of our knowledge, we conclude that DnfA is the first enzyme discovered that catalyzes oxidation of hydroxylamine to N2.

The sulfonamide-resistance dihydropteroate synthase gene is crucial for efficient biodegradation of sulfamethoxazole by Paenarthrobacter species.

Sulfonamide antibiotics (SAs) are serious pollutants to ecosystems and environments. Previous studies showed that microbial degradation of SAs such as sulfamethoxazole (SMX) proceeds via a sad-encoded oxidative pathway, while the sulfonamide-resistant dihydropteroate synthase gene, sul, is responsible for SA resistance. However, the co-occurrence of sad and sul genes, as well as how the sul gene affects SMX degradation, was not explored. In this study, two SMX-degrading bacterial strains, SD-1 and SD-2, were cultivated from an SMX-degrading enrichment. Both strains were Paenarthrobacter species and were phylogenetically identical; however, they showed different SMX degradation activities. Specifically, strain SD-1 utilized SMX as the sole carbon and energy source for growth and was a highly efficient SMX degrader, while SD-2 did could not use SMX as a sole carbon or energy source and showed limited SMX degradation when an additional carbon source was supplied. Genome annotation, growth, enzymatic activity tests, and metabolite detection revealed that strains SD-1 and SD-2 shared a sad-encoded oxidative pathway for SMX degradation and a pathway of protocatechuate degradation. A new sulfonamide-resistant dihydropteroate synthase gene, sul918, was identified in strain SD-1, but not in SD-2. Moreover, the lack of sul918 resulted in low SMX degradation activity in strain SD-2. Genome data mining revealed the co-occurrence of sad and sul genes in efficient SMX-degrading Paenarthrobacter strains. We propose that the co-occurrence of sulfonamide-resistant dihydropteroate synthase and sad genes is crucial for efficient SMX biodegradation. KEY POINTS: • Two sulfamethoxazole-degrading strains with distinct degrading activity, Paenarthrobacter sp. SD-1 and Paenarthrobacter sp. SD-2, were isolated and identified. • Strains SD-1 and SD-2 shared a sad-encoded oxidative pathway for SMX degradation. • A new plasmid-borne SMX resistance gene (sul918) of strain SD-1 plays a crucial role in SMX degradation efficiency.

Alkaliphilus flagellatus sp. nov., Butyricicoccus intestinisimiae sp. nov., Clostridium mobile sp. nov., Clostridium simiarum sp. nov., Dysosmobacter acutus sp. nov., Paenibacillus brevis sp. nov., Peptoniphilus ovalis sp. nov. and Tissierella simiarum sp. nov., isolated from monkey faeces.

Non-human primates harbour diverse microbiomes in their guts. As a part of the China Microbiome Initiatives, we cultivated and characterized the gut microbiome of cynomolgus monkeys (Macaca fascicularis). In this report, we communicate the characterization and taxonomy of eight bacterial strains that were obtained from faecal samples of captive cynomolgus monkeys. The results revealed that they represented eight novel bacterial species. The proposed names of the eight novel species are Alkaliphilus flagellatus (type strain MSJ-5T=CGMCC 1.45007T=KCTC 15974T), Butyricicoccus intestinisimiae MSJd-7T (MSJd-7T=CGMCC 1.45013T=KCTC 25112T), Clostridium mobile (MSJ-11T=CGMCC 1.45009T=KCTC 25065T), Clostridium simiarum (MSJ-4T=CGMCC 1.45006T=KCTC 15975T), Dysosmobacter acutus (MSJ-2T=CGMCC 1.32896T=KCTC 15976T), Paenibacillus brevis MSJ-6T (MSJ-6T=CGMCC 1.45008T=KCTC 15973T), Peptoniphilus ovalis (MSJ-1T=CGMCC 1.31770T=KCTC 15977T) and Tissierella simiarum (MSJ-40T=CGMCC 1.45012T=KCTC 25071T).

MiR-199a-3p Overexpression Suppressed Cell Proliferation and Sensitized Chronic Myeloid Leukaemia Cells to Imatinib by Inhibiting mTOR Signalling.

INTRODUCTION: Chronic myeloid leukaemia (CML) is a myeloproliferative neoplasm characterized by constitutive activity of the tyrosine kinase BCR-ABL1. Drug resistance remains one of the major challenges in CML therapy. MicroRNA (miR)-199a-3p plays an important role in many tumours but has rarely been investigated in CML. We aimed to analyse the role and mechanism of miR-199a-3p in regulating imatinib resistance in CML. METHODS: The expression of miR-199a-3p and mammalian target of rapamycin (mTOR) in the serum of CML patients and CML cells was examined by quantitative real-time polymerase chain reaction. The levels of apoptosis-related proteins were determined using western blot. The relative cell survival rate and cell proliferation were determined using a CCK-8 assay and a bromodeoxyuridine (BrdU) assay, respectively. Cell cycle and apoptosis were analysed using flow cytometry. Moreover, a dual-luciferase reporter assay was performed to verify the correlation between miR-199a-3p and mTOR. RESULTS: MiR-199a-3p was downregulated in the serum of CML patients and in CML cells, while mTOR was upregulated. Both miR-199a-3p overexpression and mTOR silencing inhibited CML cell proliferation, promoted CML cell apoptosis, and sensitized these cells to imatinib. mTOR silencing reversed the promoting effect of miR-199a-3p inhibition on the proliferation of CML cells and the inhibitory effects on cell apoptosis and sensitivity to imatinib. MiR-199a-3p directly targeted mTOR. CONCLUSION: MiR-199a-3p suppressed cell propagation, facilitated apoptosis of CML cells, and sensitized CML cells to imatinib by downregulating mTOR signalling.

Novel Alcaligenes ammonioxydans sp. nov. from wastewater treatment sludge oxidizes ammonia to N2 with a previously unknown pathway.

Heterotrophic nitrifiers are able to oxidize and remove ammonia from nitrogen-rich wastewaters but the genetic elements of heterotrophic ammonia oxidation are poorly understood. Here, we isolated and identified a novel heterotrophic nitrifier, Alcaligenes ammonioxydans sp. nov. strain HO-1, oxidizing ammonia to hydroxylamine and ending in the production of N2 gas. Genome analysis revealed that strain HO-1 encoded a complete denitrification pathway but lacks any genes coding for homologous to known ammonia monooxygenases or hydroxylamine oxidoreductases. Our results demonstrated strain HO-1 denitrified nitrite (not nitrate) to N2 and N2 O at anaerobic and aerobic conditions respectively. Further experiments demonstrated that inhibition of aerobic denitrification did not stop ammonia oxidation and N2 production. A gene cluster (dnfT1RT2ABCD) was cloned from strain HO-1 and enabled E. coli accumulated hydroxylamine. Sub-cloning showed that genetic cluster dnfAB or dnfABC already enabled E. coli cells to produce hydroxylamine and further to 15 N2 from (15 NH4 )2 SO4 . Transcriptome analysis revealed these three genes dnfA, dnfB and dnfC were significantly upregulated in response to ammonia stimulation. Taken together, we concluded that strain HO-1 has a novel dnf genetic cluster for ammonia oxidation and this dnf genetic cluster encoded a previously unknown pathway of direct ammonia oxidation (Dirammox) to N2 .

Bacteria and Metabolic Potential in Karst Caves Revealed by Intensive Bacterial Cultivation and Genome Assembly.

Karst caves are widely distributed subsurface systems, and the microbiomes therein are proposed to be the driving force for cave evolution and biogeochemical cycling. In past years, culture-independent studies on the microbiomes of cave systems have been conducted, yet intensive microbial cultivation is still needed to validate the sequence-derived hypothesis and to disclose the microbial functions in cave ecosystems. In this study, the microbiomes of two karst caves in Guizhou Province in southwest China were examined. A total of 3,562 bacterial strains were cultivated from rock, water, and sediment samples, and 329 species (including 14 newly described species) of 102 genera were found. We created a cave bacterial genome collection of 218 bacterial genomes from a karst cave microbiome through the extraction of 204 database-derived genomes and de novo sequencing of 14 new bacterial genomes. The cultivated genome collection obtained in this study and the metagenome data from previous studies were used to investigate the bacterial metabolism and potential involvement in the carbon, nitrogen, and sulfur biogeochemical cycles in the cave ecosystem. New N2-fixing Azospirillum and alkane-oxidizing Oleomonas species were documented in the karst cave microbiome. Two pcaIJ clusters of the ß-ketoadipate pathway that were abundant in both the cultivated microbiomes and the metagenomic data were identified, and their representatives from the cultivated bacterial genomes were functionally demonstrated. This large-scale cultivation of a cave microbiome represents the most intensive collection of cave bacterial resources to date and provides valuable information and diverse microbial resources for future cave biogeochemical research.IMPORTANCE Karst caves are oligotrophic environments that are dark and humid and have a relatively stable annual temperature. The diversity of bacteria and their metabolisms are crucial for understanding the biogeochemical cycling in cave ecosystems. We integrated large-scale bacterial cultivation with metagenomic data mining to explore the compositions and metabolisms of the microbiomes in two karst cave systems. Our results reveal the presence of a highly diversified cave bacterial community, and 14 new bacterial species were described and their genomes sequenced. In this study, we obtained the most intensive collection of cultivated microbial resources from karst caves to date and predicted the various important routes for the biogeochemical cycling of elements in cave ecosystems.

Candidatus Kaistella beijingensis sp. nov., Isolated from a Municipal Wastewater Treatment Plant, Is Involved in Sludge Foaming.

Biological foaming (or biofoaming) is a frequently occurring problem in wastewater treatment plants (WWTPs) and is attributed to the overwhelming growth of filamentous bulking and foaming bacteria (BFB). Biological foaming has been intensively investigated, with BFB like Microthrix and Skermania having been identified from WWTPs and implicated in foaming. Nevertheless, studies are still needed to improve our understanding of the microbial diversity of WWTP biofoams and how microbial activities contribute to foaming. In this study, sludge foaming at the Qinghe WWTP of China was monitored, and sludge foams were investigated using culture-dependent and culture-independent microbiological methods. The foam microbiomes exhibited high abundances of Skermania, Mycobacterium, Flavobacteriales, and Kaistella. A previously unknown bacterium, Candidatus Kaistella beijingensis, was cultivated from foams, its genome was sequenced, and it was phenotypically characterized. Ca. K. beijingensis exhibits hydrophobic cell surfaces, produces extracellular polymeric substances (EPS), and metabolizes lipids. Ca. K. beijingensis abundances were proportional to EPS levels in foams. Several proteins encoded by the Ca. K. beijingensis genome were identified from EPS that was extracted from sludge foams. Ca. K. beijingensis populations accounted for 4 to 6% of the total bacterial populations in sludge foam samples within the Qinghe WWTP, although their abundances were higher in spring than in other seasons. Cooccurrence analysis indicated that Ca. K. beijingensis was not a core node among the WWTP community network, but its abundances were negatively correlated with those of the well-studied BFB Skermania piniformis among cross-season Qinghe WWTP communities. IMPORTANCE Biological foaming, also known as scumming, is a sludge separation problem that has become the subject of major concern for long-term stable activated sludge operation in decades. Biological foaming was considered induced by foaming bacteria. However, the occurrence and deterioration of foaming in many WWTPs are still not completely understood. Cultivation and characterization of the enriched bacteria in foaming are critical to understand their genetic, physiological, phylogenetic, and ecological traits, as well as to improve the understanding of their relationships with foaming and performance of WWTPs.

Submerged macrophytes recruit unique microbial communities and drive functional zonation in an aquatic system.

Aquatic and wetland systems are widely used for landscapes and water regeneration. Microbiomes and submerged macrophytes (hydrophytes) play essential roles in conversions of organic and inorganic compounds in those ecosystems. The systems were extensively investigated for microbial diversities and compositions. However, little is known about how hydrophytes recruited diverse microbiota and affected functional zonation in aquatic systems. To address this issue, epiphytic leaf and root, sediment, and surrounding water samples were collected from the dragon-shape aquatic system in Beijing Olympic Park. Metagenomic DNAs were extracted and subjected to sequencing. Results showed that epiphytic leaf and root microbiomes and metabolic marker genes were remarkably different from that of surrounding environment. Twenty indicator bacterial genera for epiphytic microbiomes were identified and 50 metabolic marker genes were applied to evaluate the function of epiphytic leaf and root, water, and sediment microbiomes. Co-occurrence analysis revealed highly modularized pattern of metabolic marker genes and indicator bacterial genera related to metabolic functions. These results suggested that hydrophytes shaped microbiomes and drove functional zonation in aquatic systems. KEY POINTS: • Microbiomes of hydrophytes and their surrounding environments were investigated. • Twenty indicator bacterial genera highly specific to epiphytic biofilms were identified. • Epiphytes recruited unique microbiomes and drove functional zonation in aquatic systems.

Casimicrobium huifangae gen. nov., sp. nov., a Ubiquitous "Most-Wanted" Core Bacterial Taxon from Municipal Wastewater Treatment Plants.

Microorganisms in wastewater treatment plants (WWTPs) play a key role in the removal of pollutants from municipal and industrial wastewaters. A recent study estimated that activated sludge from global municipal WWTPs harbors 1 × 109 to 2 × 109 microbial species, the majority of which have not yet been cultivated, and 28 core taxa were identified as "most-wanted" ones (L. Wu, D. Ning, B. Zhang, Y. Li, et al., Nat Microbiol 4:1183-1195, 2019, https://doi.org/10.1038/s41564-019-0426-5). Cultivation and characterization of the "most-wanted" core bacteria are critical to understand their genetic, physiological, phylogenetic, and ecological traits, as well as to improve the performance of WWTPs. In this study, we isolated a bacterial strain, designated SJ-1, that represents a novel cluster within Betaproteobacteria and corresponds to OTU_16 within the 28 core taxa in the "most-wanted" list. Strain SJ-1 was identified and nominated as Casimicrobium huifangae gen. nov., sp. nov., of a novel family, Casimicrobiaceae. C. huifangae is ubiquitously distributed and is metabolically versatile. In addition to mineralizing various carbon sources (including carbohydrates, aromatic compounds, and short-chain fatty acids), C. huifangae is capable of nitrate reduction and phosphorus accumulation. The population of C. huifangae accounted for more than 1% of the bacterial population of the activated sludge microbiome from the Qinghe WWTP, which showed seasonal dynamic changes. Cooccurrence analysis suggested that C. huifangae was an important module hub in the bacterial network of Qinghe WWTP.IMPORTANCE The activated sludge process is the most widely applied biotechnology and is one of the best ecosystems to address microbial ecological principles. Yet, the cultivation of core bacteria and the exploration of their physiology and ecology are limited. In this study, the core and novel bacterial taxon C. huifangae was cultivated and characterized. This study revealed that C. huifangae functioned as an important module hub in the activated sludge microbiome, and it potentially plays an important role in municipal wastewater treatment plants.

Crenobacter cavernae sp. nov., isolated from a karst cave, and emended description of the genus Crenobacter.

A Gram-stain-negative, rod-shaped, motile and strictly aerobic novel bacterial isolate, designated strain K1W11S-77T, was obtained from a water sample that was collected from a karst cave in Guizhou province, PR China. The results of a phylogenetic analysis based on 16S rRNA gene sequences indicated that K1W11S-77T represented a member of the genus Crenobacter within the family Neisseriaceae of the phylum Proteobacteria. K1W11S-77T was phylogenetically closely related to Crenobacter luteusYIM 78141T (Their 16S rRNA gene sequence similarity is 95.02 %). Growth of K1W11S-77T occurred at 10-30 °C, at pH 7-9, and in the presence of 0-1 % (w/v) NaCl. The major cellular fatty acids were C12 : 0, C16 : 0, C18:1ω7c and summed feature 3. The major isoprenoid quinone was Q-8. The major polar lipids were phosphatidylethanolamine, diphosphatidylglycerol and one unidentified phospholipid. The genome of K1W11S-77T was 3.27 Mb long and encoded 3167 annotated genes. The DNA G+C content of the genomic DNA was 65.3 mol%. On the basis of phylogenetic, phenotypic and chemotaxonomic characteristics, K1W11S-77T is considered to represent a novel species of the genus Crenobacter, for which the name Crenobactercavernae sp. nov. is proposed. The type strain is K1W11S-77T (=CGMCC 1.13527T=NBRC 113452T).

Cohnella faecalis sp. nov., isolated from animal faeces in a karst cave.

A Gram-stain-positive, rod-shaped, endospore-forming, motile and aerobic bacterial isolate, designated strain K2E09-144T, was obtained from animal faeces that were collected from a karst cave in Guizhou province, China. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain K2E09-144T represents a novel member of the genus Cohnella within the family Paenibacillaceae of the phylum Firmicutes. Strain K2E09-144T was phylogenetically closely related to Cohnella nanjingensis D45T (16S rRNA gene sequence similarity 97.0 %). The major cellular fatty acids were anteiso-C15:0, iso-C16:0 and C16 : 0. The major isoprenoid quinone was menaqinone 7 (MK-7). The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, one unidentified phospholipid, four unidentified aminophospholipids, one glycolipid and one unidentified lipid. The isomer type of diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. The genome of strain K2E09-144T comprised 6.43 Mb, and encoded 6029 genes in total. The DNA G+C content of the genomic DNA was 53.3 mol%. Based on its phylogenetic, phenotypic and chemotaxonomic characteristics, strain K2E09-144T is considered to represent a novel species of the genus Cohnella, for which the name Cohnella faecalis sp. nov. is proposed. The type strain is K2E09-144T (=CGMCC 1.13587T=NBRC 113454T).

Hymenobacter cavernae sp. nov., isolated from a karst cave.

A novel Gram-stain-negative, rod-shaped, non-motile and red-pigmented bacterial strain, designated K1E01-27T, was isolated from an animal excrement sample which was found in a karst cave located in Guizhou province, China. The phylogenetic analysis based on 16S rRNA gene sequences indicated that strain K1E01-27T represented a member of the genus Hymenobacter within the family Cytophagaceae of the phylum Bacteroidetes. Strain K1E01-27T was most closely related to Hymenobacter algoricola VUG-A23aT, with 95.1 % 16S rRNA gene sequence similarity. Growth of strain K1E01-27T occurred at 4-35 °C, at pH 5-9 and in the presence of 0-0.5 % (w/v) NaCl. The major fatty acids were iso-C15 : 0, anteiso-C15 : 0, C16 : 1ω5c, summed feature 3 (comprising C16 : 1ω6c and/or C16 : 1ω7c) and summed feature 4 (comprising iso-C17 : 1 I/anteiso-C17 : 1 B). The major isoprenoid quinone was menaquinone 7 (MK-7). The major polar lipids were phosphatidylethanolamine, five unidentified aminophospholipids, three unidentified glycolipids, two unidentified phospholipids and one unidentified polar lipid. The DNA G+C content of the genomic DNA was 54.9 mol%. On the basis of phylogenetic, phenotypic and chemotaxonomic characteristics, strain K1E01-27T is considered to represent a novel species of the genus Hymenobacter, for which the name Hymenobacter cavernae sp. nov. is proposed. The type strain is K1E01-27T (=CGMCC 1.15197T=NBRC 112610T).

Parapedobacter defluvii sp. nov., isolated from the sewage treatment packing of a coking chemical plant.

Strain WY-1T, a Gram-stain-negative, non-spore-forming, rod-shaped, non-motile bacterium, was isolated from the sewage treatment packing of a coking chemical plant. Strain WY-1T grew over a temperature range of 15-45 °C (optimum, 30-37 °C), a pH range of 5.5-11.0 (optimum, pH 6.5-7.0) and an NaCl concentration range of 0-3 % (w/v; optimum, 0 %). 16S rRNA gene sequence analysis showed that strain WY-1T was closely related to Parapedobacter indicus RK1T with the highest sequence similarity of 96.0 %. The predominant cellular fatty acids of the novel strain were iso-C15 : 0, summed feature 3(C16 : 1ω6c and/or C16 : 1ω7c), iso-C17 : 0 3-OH, iso-C17 : 1ω9c, iso-C15 : 0 3-OH and C16 : 0. The respiratory quinone of the cells was menaquinone 7 (MK-7). The main polar lipid was phosphatidylethanolamine, an unidentified phospholipid, two unidentified aminolipids and two unknown lipids. The G+C content of the DNA was 47.1 mol%. Chemotaxonomic characteristics and phylogenetic analyses revealed that strain WY-1T belonged to the genus Parapedobacter. Strain WY-1T showed a range of phenotypic characteristics that differentiated it from species of the genus Parapedobacter with validly published names, including its assimilation from carbon sources, enzyme activities and having a wider pH range for growth. Based on these results, it is concluded that strain WY-1T represents a novel species of the genus Parapedobacter, for which the name Parapedobacter defluvii sp. nov. is proposed. The type strain is WY-1T (=NBRC 112611T=CGMCC 1.15342T).

Chitosan combined with molecular beacon for mir-155 detection and imaging in lung cancer.

Lung cancer is the major cause of cancer-related deaths worldwide, thus developing effective methods for its early diagnosis is urgently needed. In recent years, microRNAs (miRNAs, miR) have been reported to play important roles in carcinogenesis and have become potential biomarkers for cancer diagnosis and treatment. Molecular beacon (MB) technology is a universal technology to detect DNA/RNA expression in living cells. As a natural polymers, chitosan (CS) nanoparticles could be used as a carrier for safe delivery of nucleic acid. In this study, we developed a probe using nanoparticles of miR-155 MB self assembled with CS (CS-miR-155 MB) to image the expression of miR-155 in cancer cells. Hybridization assay showed that the locked nucleic acid (LAN) modified miR-155 MB could target miR-155 effectively and sensitively. The miR-155 MB self-assembly with CS nanoparticles formed stable complexes at the proper weight ratio. The CS nanoparticles showed higher fluorescence intensity and transfection efficiency than the lipid-based formulation transfection agent by confocal microscopy and flow cytometry analysis. The CS-MB complexes were found to be easily synthesized and exhibited strong enzymatic stability, efficient cellular uptake, high target selectivity and biocompatibility. The CS-MB complexes can also be applied in other cancers just by simply changing for a targeted miRNA highly expressed in those cancer cells. Therefore, it is a promising vehicle used for detecting miRNA expression in living cells.

[Expression of mRNA and protein of p38, Osx, PI3K and Akt1 in rat bone with chronic fluorosis].

OBJECTIVE: To investigate the expressions of mRNA and protein of p38, Osx, PI3K, Akt1 in the rats bone with chronic fluorosis. METHODS: Dental fluorosis were observed and the fluoride contents in the urine and bone were detected by fluorin-ion selective electrode. The morphologic changes and ultrastructure of rats' bone were observed by light and electronic microscopy. The expressions of protein and mRNA of p38, Osx, PI3K and Akt1 were detected by immunohistochemistry and real-time PCR, respectively. The contents of BALP and BGP in serum were detected by ELISA. RESULTS: The rates of dental fluorosis in the fluorosis rats were increased, and the fluoride contents in bone and urine of the fluorosis rats were increased compared to the control group, the difference was statistically significant (P < 0.05). The bone trabeculae thickness and density and the thickness of bone cortex in fluorosis rats were remarkably increased, the space of bone trabeculae was reduced, and in accordance with the matching morphometrical indices, the difference was statistically significant (P < 0.05) as compared with the control rats. The contents of BALP [(54.61 ± 2.27) U/L] and BGP [(2.38 ± 0.16) µg/L]in the fluoride groups were higher than those in the control group, the difference was statistically significant (P < 0.05). Ultrastructurally, the broadening of the osseouslacuna was observed. The reduced protuberances of the osteocytes, the unclear organelle structure, pyknosis, karyotheca increasation and edged chromatin were also observed. Compared to the control group, the expressions of protein and its mRNA of p38, Osx, PI3K and Akt1 were higher in the fluorosis rats than those in the control rats, and the difference was statistically significant (P < 0.05). There is no any expression of p38, Osx, PI3K and Akt1 in the osteocytes in fluorosis rats. CONCLUSIONS: The over-expression of p38, Osx, PI3K and Akt1 in bone tissue of fluorosis rats may relate to the accumulation of fluorine in the body. The bone injury mainly occur in the stage of the differentiation and proliferation. The upregulation of P38MARK signal path and PI3K/Akt1 signal path may be involved in the pathogenesis of bone injury caused by fluoride.

Microbial roles in cave biogeochemical cycling.

Among fundamental research questions in subterranean biology, the role of subterranean microbiomes playing in key elements cycling is a top-priority one. Karst caves are widely distributed subsurface ecosystems, and cave microbes get more and more attention as they could drive cave evolution and biogeochemical cycling. Research have demonstrated the existence of diverse microbes and their participance in biogeochemical cycling of elements in cave environments. However, there are still gaps in how these microbes sustain in caves with limited nutrients and interact with cave environment. Cultivation of novel cave bacteria with certain functions is still a challenging assignment. This review summarized the role of microbes in cave evolution and mineral deposition, and intended to inspire further exploration of microbial performances on C/N/S biogeocycles.

Droplet microfluidics-based high-throughput bacterial cultivation for validation of taxon pairs in microbial co-occurrence networks.

Co-occurrence networks inferred from the abundance data of microbial communities are widely applied to predict microbial interactions. However, the high workloads of bacterial isolation and the complexity of the networks themselves constrained experimental demonstrations of the predicted microbial associations and interactions. Here, we integrate droplet microfluidics and bar-coding logistics for high-throughput bacterial isolation and cultivation from environmental samples, and experimentally investigate the relationships between taxon pairs inferred from microbial co-occurrence networks. We collected Potamogeton perfoliatus plants (including roots) and associated sediments from Beijing Olympic Park wetland. Droplets of series diluted homogenates of wetland samples were inoculated into 126 96-well plates containing R2A and TSB media. After 10 days of cultivation, 65 plates with > 30% wells showed microbial growth were selected for the inference of microbial co-occurrence networks. We cultivated 129 bacterial isolates belonging to 15 species that could represent the zero-level OTUs (Zotus) in the inferred co-occurrence networks. The co-cultivations of bacterial isolates corresponding to the prevalent Zotus pairs in networks were performed on agar plates and in broth. Results suggested that positively associated Zotu pairs in the co-occurrence network implied complicated relations including neutralism, competition, and mutualism, depending on bacterial isolate combination and cultivation time.

Detection of miR-155-5p and imaging lung cancer for early diagnosis: in vitro and in vivo study.

PURPOSE: Currently, the routine screening program has insufficient capacity for the early diagnosis of lung cancer. Therefore, a type of chitosan-molecular beacon (CS-MB) probe was developed to recognize the miR-155-5p and image the lung cancer cells for the early diagnosis. METHODS: Based on the molecular beacon (MB) technology and nanotechnology, the CS-MB probe was synthesized self-assembly. There are four types of cells-three kinds of animal models and one type of histopathological sections of human lung cancer were utilized as models, including A549, SPC-A1, H446 lung cancer cells, tumor-initiating cells (TICs), subcutaneous and lung xenografts mice, and lox-stop-lox(LSL) K-ras G12D transgenic mice. The transgenic mice dynamically displayed the process from normal lung tissues to atypical hyperplasia, adenoma, carcinoma in situ, and adenocarcinoma. The different miR-155-5p expression levels in these cells and models were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The CS-MB probe was used to recognize the miR-155-5p and image the lung cancer cells by confocal microscopy in vitro and by living imaging system in vivo. RESULTS: The CS-MB probe could be used to recognize the miR-155-5p and image the lung cancer cells significantly in these cells and models. The fluorescence intensity trends detected by the CS-MB probe were similar to the expression levels trends of miR-155 tested by qRT-PCR. Moreover, the fluorescence intensity showed an increasing trend with the tumor progression in the transgenic mice model, and the occurrence and development of lung cancer were dynamically monitored by the differen fluorescence intensity. In addition, the miR-155-5p in human lung cancer tissues could be detected by the miR-155-5p MB. CONCLUSION: Both in vivo and in vitro experiments demonstrated that the CS-MB probe could be utilized to recognize the miR-155-5p and image the lung cancer cells. It provided a novel experimental and theoretical basis for the early diagnosis of the disease. Also, the histopathological sections of human lung cancer research laid the foundation for subsequent preclinical studies. In addition, different MBs could be designed to detect other miRNAs for the early diagnosis of other tumors.