Partial nitritation and nitrogen removal of vacuum toilet wastewater from high-speed trains in a sequential batch reactor.

Owing to the high contents of organics and nitrogen in vacuum toilet wastewater (VTW) generated from high-speed trains, onsite pretreatment is usually required before VTW can be discharged into municipal sewers. In this study, a partial nitritation process was stably established in a sequential batch reactor to efficiently utilize the organics in synthetic and real VTWs for nitrogen removal and to produce an effluent suitable for anaerobic ammonia oxidation. In spite of the high fluctuations of COD and nitrogen in VTW, the organics used for nitrogen removal stabilized at 1.97 ± 0.18 mg COD mg N-1 removed, and the effluent NO2--N/NH4+-N ratios were maintained at 1.26 ± 0.13. The removal efficiencies of nitrogen and COD were 31.8 ± 3.5% and 65.2 ± 5.3% under the volumetric loading rates of 1.14 ± 0.15 kg N m-3 d-1 and 1.03 ± 0.26 kg COD m-3 d-1 for real VTW, respectively. Microbial community analysis revealed that Nitrosomonas (0.95%-1.71%) was the dominant autotrophic ammonium-oxidizing bacterial genus, but nitrite-oxidizing bacteria, Nitrolancea, was severely inhibited, with a relative abundance less than 0.05%. The relative abundance of denitrifying bacteria increased by 7.34% when the influent was switched to real VTW. Functional profile predictions of the biomass showed that the decrease in the COD/N ratio and the switch of reactor influent from synthetic to real VTW increased the relative abundance of enzymes and modules involved in carbon and nitrogen metabolisms.

One-stage freehand minimally invasive pedicle screw fixation combined with mini-access surgery through OLIF approach for the treatment of lumbar tuberculosis.

OBJECTIVE: To compare one-stage freehand minimally invasive pedicle screw fixation (freehand MIPS) combined with mini-access surgery through OLIF approach with posterior approach for treatment of lumbar tuberculosis (TB), and evaluate its feasibility, efficacy and safety in debridement, bone graft fusion and internal fixation. METHODS: 48 patients with single segment lumbar TB from June 2014 to June 2017 were included. Among them, 22 patients underwent one-stage freehand MIPS combined with mini-access surgery through OLIF approach (group 1), 26 patients were treated with posterior open surgery (group 2). Duration of operation, blood loss, and stay time in hospital were compared. Pre- and postoperative visual analog scale (VAS) pain scores, Oswestry disability index (ODI), erythrocyte sedimentation rate, complications and images were also recorded. RESULTS: Patients in group 1 showed significantly less blood loss (165 ± 73 ml vs 873 ± 318 ml, P < 0.001), shorter stay time in hospital (6/4-8 days vs 12/8-15 days, P < 0.001), while longer duration of operation (185 ± 14 min vs 171 ± 12 min, P < 0.001) than group 2 did. VAS scores significantly decreased after surgery in both groups, however, VAS scores of group 1 were significantly lower than that of group 2 immediately after surgery and during follow-ups (P < 0.001). ODI of group 1 was also significantly lower than that of group 2 at 12-month after surgery (P < 0.001). CONCLUSION: One-stage freehand MIPS combined with mini-access surgery through OLIF approach is a feasible, efficient and safe method in treating single segment lumbar TB. It shows advantages of less surgical trauma and faster postoperative recovery.

Efficacy and safety of anterior cervical discectomy and fusion (ACDF) through mini-incision and posterior laminoplasty (LAMP) for treatment of long-level cervical spondylosis: a retrospective cohort study.

BACKGROUND: The efficacy and safety of anterior cervical discectomy and fusion (ACDF) through mini-incision and posterior laminoplasty for long-level cervical spondylosis were investigated. METHOD: From January 2018 to September 2019, clinical patients data with 3-4 segments (C3-7) cervical spondylotic radiculopathy, cervical spondylotic myelopathy, or mixed cervical spondylosis who received ACDF (42 cases) throughwith mini-incision or LAMP (36 cases) treatment were retrospectively collected and analyzed. The operative time, bleeding volume, incisive length, and hospital stay were recorded. Moreover, the intervertebral height, functional segment height, cervical lordosis, cervical hyperextension and hyperflexion range-of-motion (ROM) and ROM in all directions of the cervical spine before and after the operation were measured. Additionally, all relevant postoperative complications were also recorded. Then, the therapeutic effects of both surgical methods were investigated. RESULTS: Patients in the ACDF group had less bleeding, shorter incision, and fewer hospitalization days than the LAMP group. There was no significant difference in JOA, VAS score of the upper limb, NDI score after surgery between two groups. Postoperative intervertebral height and functional segment height in the ACDF group were significantly higher than those before the operation, and postoperative functional segment height of the ACDF group was significantly higher than that of the LAMP group. Moreover, the postoperative cervical lordosis angle in the ACDF group was significantly larger than the LAMP group. There was no significant difference between preoperative and postoperative ROM in all directions of the cervical spine for the two groups. CONCLUSIONS: Both ACDF through mini-incision and LAMP are effective treatments for long-level cervical spondylosis. However, ACDF through mini-incision shows minor trauma, less bleeding, fast recovery, and it is beneficial for cervical lordosis reconstruction.

Knockdown of lncRNA LINC00662 suppresses malignant behaviour of osteosarcoma cells via competition with miR-30b-3p to regulate ELK1 expression.

PURPOSE: Osteosarcoma is a type of bone malignancy that mainly occurred in teenagers. This investigation is aimed to clarify the effect of long non-coding RNA (lncRNA) LINC00662 on the proliferation, migration, and invasion in osteosarcoma and explore the underlying action mechanisms. METHODS: The mRNA expression of LINC00662 was determined by real-time quantitative polymerase chain reaction. Cell proliferation, migration, and invasion were evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, wound healing, and transwell assays, respectively. A dual-luciferase reporter assay was used to validate the target relationships Between microRNA (miR)-30b-3p and LINC00662/ ETS domain-containing protein 1 (ELK1). Western blotting was performed to determine the protein expression of ELK1. Xenograft model was established to evaluate the effects of LINC00662 silencing on tumor growth in vivo. RESULTS: LncRNA LINC00662 and ELK1 were significantly increased, while miR-30b-3p was reduced in osteosarcoma tissues. The results of functional experiments indicated that transfection of small hairpin (sh)-LINC00662 and miR-30b-3p mimics repressed the migration, invasion, and proliferation of osteosarcoma cells. LncRNA LINC00662 also appeared to sponge miR-30b-3p in order to affect the expression of ELK1. Simultaneously, there were weak negative correlations between the expression of miR-30b-3p and LINC00662/ELK1 in osteosarcoma tissues. Rescue experiments suggested that ELK1 overexpression and downregulation of miR-30b-3p reversed the suppressive effects of sh-LINC00662 on the cell migration, invasion, and proliferation in osteosarcoma. CONCLUSIONS: The current study indicated that knockdown of LINC00662 repressed cell migration, invasion, and proliferation through sponging miR-30b-3p to regulate the expression of ELK1 in osteosarcoma. These results may uncover a promising target for the treatment of osteosarcoma.

Transcriptionally inactive hepatitis B virus episome DNA preferentially resides in the vicinity of chromosome 19 in 3D host genome upon infection.

The hepatitis B virus (HBV) infects 257 million people worldwide. HBV infection requires establishment and persistence of covalently closed circular (ccc) DNA, a viral episome, in nucleus. Here, we study cccDNA spatial localization in the 3D host genome by using chromosome conformation capture-based sequencing analysis and fluorescence in situ hybridization (FISH). We show that transcriptionally inactive cccDNA is not randomly distributed in host nucleus. Rather, it is preferentially accumulated at specialized areas, including regions close to chromosome 19 (chr.19). Activation of the cccDNA is apparently associated with its re-localization, from a pre-established heterochromatin hub formed by 5 regions of chr.19 to transcriptionally active regions formed by chr.19 and nearby chromosomes including chr.16, 17, 20, and 22. This active versus inactive positioning at discrete regions of the host genome is primarily controlled by the viral HBx protein and by host factors including the structural maintenance of chromosomes protein 5/6 (SMC5/6) complex.

Mesenchymal stem cell-derived exosomes carrying microRNA-150 suppresses the proliferation and migration of osteosarcoma cells via targeting IGF2BP1.

BACKGROUND: MicroRNA-150 (miR-150) plays a critical role in varied types of human cancers. In this study, we explored the effect and mechanism of mesenchymal stem cell (MSC)-derived exosomes (exo) carrying miR-150 (MSC-Exo-150) on the proliferation, migration, invasion, and apoptosis of osteosarcoma (OS) cells. METHODS: MiR-150 expression in OS cell lines was assessed by quantitative reverse-transcription PCR (qRT-PCR). MSCs were transfected with cell-miR-67 or has-miR-150, and grouped as MSC-67 or MSC-150. Exosomes were isolated from each group, and separately named MSC-Exo-67, MSC-Exo-150 and MSC-Exo. MTT or flow cytometry assay was used to analyze the proliferation or apoptosis of U2SO and HOS cells, respectively. Wound healing or transwell assay was utilized to examine the migration or invasion of U2SO and HOS cells, respectively. The target relationship of miR-150 and insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) was established using StarBase2.0 and verified by dual-luciferase reporter gene analysis. Xenografted tumor model was established in rats to confirm the inhibitory effect of MSC-Exo-150 on the growth of xenografted tumor in vivo. RESULTS: The expression of miR-150 was downregulated in OS cell lines, and significantly higher in MSC-150 cells than that in MSCs. MiR-150 was overexpressed in MSC-Exo-150 group compared with MSC-Exo group. After transfection of MSC-Exo-150 into U2SO and HOS cells, cell viability, mobility and invasion rate were decreased, and the cell apoptosis was increased. MiR-150 targeted IGF2BP1 and IGF2BP1 expression was negatively modulated by miR-150. Overexpression of IGF2BP1 reversed the anti-tumor effect of MSC-Exo-150 on HOS cells. CONCLUSIONS: MSC-Exo-150 inhibited proliferation, migration, invasion, and induced apoptosis of OS cells by targeting IGF2BP1.

A quantitative method for hepatitis B virus covalently closed circular DNA enables distinguishing direct acting antivirals from cytotoxic agents.

Studying the biogenesis of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) and developing anti-HBV agents require analytical methods to quantify viral DNA levels inside host cells. The well-accepted Southern blotting method is only semi-quantitative, while the other widely used methods (based on qPCR) have been questioned as to their fidelity for cccDNA quantification. In addition, Southern blotting and qPCR results barely reflect the number of host cells present in an analytical sample. We here developed new techniques that substantially improve cccDNA detection and quantification, including a sample pretreatment method that i) exploits high temperature and exonuclease V (an ATP-dependent, bidirectional exonuclease) digestion to effectively increase the amplification efficiency of HBV cccDNA by removing rcDNA and denaturing the cccDNA template, and ii) a method that splits cell samples and uses separate extraction technologies to facilitate "host normalization" based on host genomic DNA signals. Our study introduces new analytical techniques that should be useful for the basic biology and translational studies of HBV.

The p.Ser267Phe variant of sodium taurocholate cotransporting polypeptide (NTCP) supports HBV infection with a low efficiency.

Sodium taurocholate cotransporting polypeptide (NTCP) is a functional receptor for human hepatitis B virus (HBV) and its satellite virus Hepatitis D virus (HDV). Physiologically, NTCP is responsible for the majority of sodium-dependent bile acids uptake by hepatocytes. The p.Ser267Phe (S267F) variant of NTCP is a single nucleotide polymorphism (SNP) previously found to cause substantial loss of ability to support HBV and HDV infection and its taurocholic acid uptake function in vitro. Intriguingly, ten individuals were identified as S267F homozygotes in population studies of chronic hepatitis B (CHB) patients. In this study, we identified new HBV isolates from one homozygous S267F mutation carrier and confirmed new isolates also use wildtype-NTCP as a cellular receptor. Furthermore, we demonstrated S267F variant of NTCP, though inefficient, is still a functional receptor for HBV entry. This study advances our understanding of NTCP-mediated HBV infection.

DNA Polymerase κ Is a Key Cellular Factor for the Formation of Covalently Closed Circular DNA of Hepatitis B Virus.

Hepatitis B virus (HBV) infection of hepatocytes begins by binding to its cellular receptor sodium taurocholate cotransporting polypeptide (NTCP), followed by the internalization of viral nucleocapsid into the cytoplasm. The viral relaxed circular (rc) DNA genome in nucleocapsid is transported into the nucleus and converted into covalently closed circular (ccc) DNA to serve as a viral persistence reservoir that is refractory to current antiviral therapies. Host DNA repair enzymes have been speculated to catalyze the conversion of rcDNA to cccDNA, however, the DNA polymerase(s) that fills the gap in the plus strand of rcDNA remains to be determined. Here we conducted targeted genetic screening in combination with chemical inhibition to identify the cellular DNA polymerase(s) responsible for cccDNA formation, and exploited recombinant HBV with capsid coding deficiency which infects HepG2-NTCP cells with similar efficiency of wild-type HBV to assure cccDNA synthesis is exclusively from de novo HBV infection. We found that DNA polymerase κ (POLK), a Y-family DNA polymerase with maximum activity in non-dividing cells, substantially contributes to cccDNA formation during de novo HBV infection. Depleting gene expression of POLK in HepG2-NTCP cells by either siRNA knockdown or CRISPR/Cas9 knockout inhibited the conversion of rcDNA into cccDNA, while the diminished cccDNA formation in, and hence the viral infection of, the knockout cells could be effectively rescued by ectopic expression of POLK. These studies revealed that POLK is a crucial host factor required for cccDNA formation during a de novo HBV infection and suggest that POLK may be a potential target for developing antivirals against HBV.

microRNA expression in hepatitis B virus infected primary treeshrew hepatocytes and the independence of intracellular miR-122 level for de novo HBV infection in culture.

Infection of Hepatitis B virus (HBV) in hepatocytes has been known to be controlled by multiple cellular factors, while the relationship of the infection and liver microRNAs remains obscure. In this study, a miRNA database, containing 168 unique mature miRNA members from primary hepatocytes of a primate-like animal, northern treeshrew (Tupaia belangeri) that is the only species susceptible for HBV infection other than human and chimpanzee, was established. The relative level of a liver predominant microRNA, miR-122, was markedly increased upon HBV infection of the primary tupaia hepatocyte (PTH). However, introducing neither miR-122 nor its antagonist anti-miR-122 into PTHs, or, HepG2-NTCP that is HepG2 cells with the newly identified receptor sodium taurocholate cotransporting polypeptide (NTCP) did not alter the viral infection on these cells. These data suggest that de novo HBV infection of cultured hepatocytes does not depend on the expression level of intracellular miR-122 of the target cells.

Molecular determinants of hepatitis B and D virus entry restriction in mouse sodium taurocholate cotransporting polypeptide.

Human hepatitis B virus (HBV) and its satellite virus, hepatitis D virus (HDV), primarily infect humans, chimpanzees, or tree shrews (Tupaia belangeri). Viral infections in other species are known to be mainly restricted at the entry level since viral replication can be achieved in the cells by transfection of the viral genome. Sodium taurocholate cotransporting polypeptide (NTCP) is a functional receptor for HBV and HDV, and amino acids 157 to 165 of NTCP are critical for viral entry and likely limit viral infection of macaques. However, the molecular determinants for viral entry restriction in mouse NTCP (mNTCP) remain unclear. In this study, mNTCP was found to be unable to support either HBV or HDV infection, although it can bind to pre-S1 of HBV L protein and is functional in transporting substrate taurocholate; comprehensive swapping and point mutations of human NTCP (hNTCP) and mNTCP revealed molecular determinants restricting mNTCP for viral entry of HBV and HDV. Remarkably, when mNTCP residues 84 to 87 were substituted by human counterparts, mNTCP can effectively support viral infections. In addition, a number of cell lines, regardless of their species or tissue origin, supported HDV infection when transfected with hNTCP or mNTCP with residues 84 to 87 replaced by human counterparts, highlighting the central role of NTCP for viral infections mediated by HBV envelope proteins. These studies advance our understanding of NTCP-mediated viral entry of HBV and HDV and have important implications for developing the mouse model for their infections.

Sodium taurocholate cotransporting polypeptide mediates woolly monkey hepatitis B virus infection of Tupaia hepatocytes.

Primary Tupaia hepatocytes (PTHs) are susceptible to woolly monkey hepatitis B virus (WMHBV) infection, but the identity of the cellular receptor(s) mediating WMHBV infection of PTHs remains unclear. Recently, sodium taurocholate cotransporting polypeptide (NTCP) was identified as a functional receptor for human hepatitis B virus (HBV) infection of primary human and Tupaia hepatocytes. In this study, a synthetic pre-S1 peptide from WMHBV was found to bind specifically to cells expressing Tupaia NTCP (tsNTCP) and it efficiently blocked WMHBV entry into PTHs; silencing of tsNTCP in PTHs significantly inhibited WMHBV infection. Ectopic expression of tsNTCP rendered HepG2 cells susceptible to WMHBV infection. These data demonstrate that tsNTCP is a functional receptor for WMHBV infection of PTHs. The result also indicates that NTCP's orthologs likely act as a common cellular receptor for all known primate hepadnaviruses.

Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.

Human hepatitis B virus (HBV) infection and HBV-related diseases remain a major public health problem. Individuals coinfected with its satellite hepatitis D virus (HDV) have more severe disease. Cellular entry of both viruses is mediated by HBV envelope proteins. The pre-S1 domain of the large envelope protein is a key determinant for receptor(s) binding. However, the identity of the receptor(s) is unknown. Here, by using near zero distance photo-cross-linking and tandem affinity purification, we revealed that the receptor-binding region of pre-S1 specifically interacts with sodium taurocholate cotransporting polypeptide (NTCP), a multiple transmembrane transporter predominantly expressed in the liver. Silencing NTCP inhibited HBV and HDV infection, while exogenous NTCP expression rendered nonsusceptible hepatocarcinoma cells susceptible to these viral infections. Moreover, replacing amino acids 157-165 of nonfunctional monkey NTCP with the human counterpart conferred its ability in supporting both viral infections. Our results demonstrate that NTCP is a functional receptor for HBV and HDV.DOI:http://dx.doi.org/10.7554/eLife.00049.001.

Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.

Human hepatitis B virus (HBV) infection and HBV-related diseases remain a major public health problem. Individuals coinfected with its satellite hepatitis D virus (HDV) have more severe disease. Cellular entry of both viruses is mediated by HBV envelope proteins. The pre-S1 domain of the large envelope protein is a key determinant for receptor(s) binding. However, the identity of the receptor(s) is unknown. Here, by using near zero distance photo-cross-linking and tandem affinity purification, we revealed that the receptor-binding region of pre-S1 specifically interacts with sodium taurocholate cotransporting polypeptide (NTCP), a multiple transmembrane transporter predominantly expressed in the liver. Silencing NTCP inhibited HBV and HDV infection, while exogenous NTCP expression rendered nonsusceptible hepatocarcinoma cells susceptible to these viral infections. Moreover, replacing amino acids 157-165 of nonfunctional monkey NTCP with the human counterpart conferred its ability in supporting both viral infections. Our results demonstrate that NTCP is a functional receptor for HBV and HDV.