Therapeutic In Vivo Gene Editing Achieved by a Hypercompact CRISPR-Cas12f1 System Delivered with All-in-One Adeno-Associated Virus.

CRISPR-based gene therapies are making remarkable strides toward the clinic. But the large size of most widely used Cas endonucleases including Cas9 and Cas12a restricts their efficient delivery by the adeno-associated virus (AAV) for in vivo gene editing. Being exceptionally small, the recently engineered type V-F CRISPR-Cas12f1 systems can overcome the cargo packaging bottleneck and present as strong candidates for therapeutic applications. In this study, the pairwise editing efficiencies of different engineered Cas12f1/sgRNA scaffold combinations are systemically screened and optimized, and the CasMINI_v3.1/ge4.1 system is identified as being able to significantly boost the gene editing activity. Moreover, packaged into single AAV vectors and delivered via subretinal injection, CasMINI_v3.1/ge4.1 achieves remarkably high in vivo editing efficiencies, over 70% in transduced retinal cells. Further, the efficacy of this Cas12f1 system-based gene therapy to treat retinitis pigmentosa in RhoP23H mice is demonstrated by the therapeutic benefits achieved including rescued visual function and structural preservation. And minimal bystander editing activity is detected. This work advances and expands the therapeutic potential of the miniature Cas12f1 system to support efficient and accurate in vivo gene therapy.

Potential interactions between triazole antifungal agents and lorlatinib based on ultra-performance liquid chromatography-tandem mass spectrometry in rat plasma.

AIM: Our study is to investigate the effects of triazole antifungal drugs on the pharmacokinetics of lorlatinib in rats. METHODS: The samples were precipitated with methanol. Chromatographic separation was performed on a ultra-performance liquid chromatography (UPLC) system using a BEH C18 column. The mobile phase consisted of 0.1% formic acid water and methanol. Lorlatinib and crizotinib (internal standard) were detected in multiple reaction monitoring mode. The fragment ions were 407.3-228.07 for lorlatinib and m/z 450.3-260.0 for crizotinib. Lorlatinib and different triazole antifungal drugs were given to Sprague Dawley rats by gavage, and blood was collected from the tail vein at a certain time point. The validated UPLC-MS/MS method was applied to a drug interaction study of ketoconazole, voriconazole, itraconazole, and posaconazole with lorlatinib in rats. RESULTS: Ketoconazole and voriconazole significantly inhibited lorlatinib metabolism. When administration with ketoconazole and voriconazole, the area under the curve from time zero to infinity of lorlatinib increased by 49.0% and 104.3%, respectively; the clearance decreased by 40.0% and 40.0%, respectively. While itraconazole and posaconazole did not affect lorlatinib pharmacokinetics. CONCLUSION: The UPLC-MS/MS-based assay is helpful to further understand the pharmacokinetics of lorlatinib in rats, and confirmed the findings that the combination of lorlatinib with CYP3A inhibitors should be avoided as predicted by our pre-clinical studies.

A circadian clock gene-related signature for predicting prognosis and its association with sorafenib response in hepatocellular carcinoma.

Background: Hepatocellular carcinoma (HCC), one of the highest causes of cancer-associated death, has effective treatments, especially for patients with advanced HCC. Circadian rhythm participates in several important physiological functions, and its chronic disruption results in many disordered diseases, including cancer. However, the role of circadian rhythm in the overall survival (OS) of patients with HCC remains unclear. Methods: We investigated the expression, copy number variation (CNV), and mutation profiles of core circadian clock genes in normal and tumor tissues. We developed and validated a messenger RNA signature (mRNASig) based on prognostic circadian clock genes. A set of bioinformatic tools were applied for functional annotation and tumor-associated microenvironment (TME) analysis. Results: Core circadian clock genes were disrupted in terms of the transcription and CNV of HCC samples. The mRNASig, including NPAS2, NR1D1, PER1, RORC, and TIMELESS, was constructed. We divided patients with HCC into high-risk group and low-risk group based on the median value of the risk score. The high-risk group had a poorer prognosis than the low-risk group. The high-risk group was associated with malignant processes (e.g., proliferation, oncogenic pathway, DNA repair), metabolism, and tumor mutational burden (TMB). Surprisingly, the low-risk group was associated with enriched angiogenesis and was linked to enhanced response to sorafenib. Moreover, the high-risk group showed poor infiltration of CD8 T cells and natural killer cells accompanied by higher expression of CTLA4, PDCD1, TIGIT, and TIM3. Additionally, the mRNASig was associated with TMB. Conclusions: The mRNASig based on core circadian clock genes is a potential prognostic signature and therapeutic strategy and is significantly associated with the malignant biology of HCC.

A Markov model-based cost-effectiveness analysis comparing zanubrutinib to ibrutinib for treating relapsed and refractory chronic lymphocytic leukemia.

OBJECTIVE: This article examined the cost-effectiveness of zanubrutinib and ibrutinib for managing relapsed and refractory chronic lymphocytic leukemia from the viewpoint of payers in China and the US. METHODS: Markov models were employed to conduct comparisons. Baseline characteristics and clinical data were extracted from the ALPINE study. The cost-effectiveness outcome indicators encompassed cost, quality-adjusted life years, and the incremental cost-effectiveness ratio. RESULTS: The Markov model analysis revealed that the zanubrutinib group incurred an incremental cost per patient of $-24,586.53 compared to the ibrutinib group. The zanubrutinib group exhibited an incremental utility per capita of 0.28 quality-adjusted life years, resulting in an incremental cost-effectiveness ratio of $-88,068.16 per quality-adjusted life year, which is lower than the payment threshold in China. The willingness-to-pay value in China for 2022 was three times the country's gross domestic product per capita. In the US, patients in the zanubrutinib group experienced per capita incremental costs of $-79,421.56, per capita incremental utility of 0.28 quality-adjusted life years, and an incremental cost-effectiveness ratio of $-284,485.45 per quality-adjusted life year. CONCLUSION: For Chinese payers, zanubrutinib exhibited superior cost-effectiveness compared to ibrutinib. Zanubrutinib proved to be a more affordable option for US payers when considering the payment threshold.

Efficacy of Baricitinib in Patients with Refractory Adult-Onset Still's Disease.

BACKGROUND AND OBJECTIVE: Adult-onset Still's disease (AOSD) is an idiopathic systemic inflammatory disease of unknown aetiology. Some patients exhibit resistance to conventional treatment during long-term therapy. Janus kinase inhibitors (JAKinibs) may contribute to the improvement in AOSD symptoms via the JAK-signal transducer and activator of transcription (STAT) pathway. We aimed to explore the efficacy and safety of baricitinib in patients with refractory AOSD. METHODS: Patients were enrolled if they fulfilled the Yamaguchi AOSD classification criteria in China between 2020 and 2022. All patients were recognized as having refractory AOSD and were treated with oral baricitinib at a dosage of 4 mg once daily. A systemic score and prednisone dosage were used to evaluate the efficacy of baricitinib at months 1, 3, and 6 and at the last follow-up visit. The safety profiles were recorded and analysed at every assessment. RESULTS: Seven female patients with refractory AOSD received baricitinib. The median age was 31 (IQR 10) years. Treatment was terminated in one patient due to progressive macrophage activation syndrome (MAS). Others continued baricitinib treatment until the last assessment. The systemic score decreased significantly at 3 months (p = 0.0216), 6 months (p = 0.0007), and the last follow-up visit (p = 0.0007) compared with baseline. One month after the initiation of baricitinib, the rates of improvement in fever, rash, sore throat, and myalgia symptoms were 71.4% (5/7), 40% (2/5), 80% (4/5), and 66.7% (2/3), respectively. Five patients remained symptom-free at the last follow-up visit. In most patients, their laboratory values had returned to normal by the last follow-up visit. A significant reduction in the levels of C-reactive protein (CRP) (p = 0.0165) and ferritin (p = 0.0047) was observed at the last visit compared with baseline. The daily prednisolone dosage significantly decreased from 35.7 ± 15.1 mg/day at baseline to 8.8 ± 4.4 mg/day by month 6 (p = 0.0256), and it was 5.8 ± 4.7 mg/day at the last assessment (p = 0.0030). Leukopenia due to MAS was noted in one patient. Except for mild abnormalities in lipid parameters, no other severe adverse events occurred during follow-up. CONCLUSIONS: Our findings suggest that baricitinib therapy could provide rapid and durable clinical and laboratory improvement in patients with refractory AOSD. Treatment seemed to be well tolerated by these patients. The long-term efficacy and safety of baricitinib therapy for AOSD should be assessed further in prospective controlled clinical trials in the future. TRIAL REGISTRATION: Trial registration number (TRN): ChiCTR2200061599. Date of registration: 29 June 2022 (retrospectively registered).

Distinct immune and inflammatory response patterns contribute to the identification of poor prognosis and advanced clinical characters in bladder cancer patients.

Due to the molecular heterogeneity, most bladder cancer (BLCA) patients show no pathological responses to immunotherapy and chemotherapy yet suffer from their toxicity. This study identified and validated three distinct and stable molecular clusters of BLCA in cross-platform databases based on personalized immune and inflammatory characteristics. H&E-stained histopathology images confirmed the distinct infiltration of immune and inflammatory cells among clusters. Cluster-A was characterized by a favorable prognosis and low immune and inflammatory infiltration but showed the highest abundance of prognosis-related favorable immune cell and inflammatory activity. Cluster-B featured the worst prognosis and high immune infiltration, but numerous unfavorable immune cells exist. Cluster-C had a favorable prognosis and the highest immune and inflammatory infiltration. Based on machine learning, a highly precise predictive model (immune and inflammatory responses signature, IIRS), including FN1, IL10, MYC, CD247, and TLR2, was developed and validated to identify the high IIRS-score group that had a poor prognosis and advanced clinical characteristics. Compared to other published models, IIRS showed the highest AUC in 5 years of overall survival (OS) and a favorable predictive value in predicting 1- and 3- year OS. Moreover, IIRS showed an excellent performance in predicting immunotherapy and chemotherapy's response. According to immunohistochemistry and qRT-PCR, IIRS genes were differentially expressed between tumor tissues with corresponding normal or adjacent tissues. Finally, immunohistochemical and H&E-stained analyses were performed on the bladder tissues of 13 BLCA patients to further demonstrate that the IIRS score is a valid substitute for IIR patterns and can contribute to identifying patients with poor clinical and histopathology characteristics. In conclusion, we established a novel IIRS depicting an IIR pattern that could independently predict OS and acts as a highly precise predictive biomarker for advanced clinical characters and the responses to immunotherapy and chemotherapy.

Comparison of dual mTORC1/2 inhibitor AZD8055 and mTORC1 inhibitor rapamycin on the metabolism of breast cancer cells using proton nuclear magnetic resonance spectroscopy metabolomics.

Dual mTORC1/2 inhibitors may be more effective than mTORC1 inhibitor rapamycin. Nevertheless, their metabolic effects on breast cancer cells have not been reported. We compared the anti-proliferative capacity of rapamycin and a novel mTORC1/2 dual inhibitor (AZD8055) in two breast cancer cell lines (MDA-MB-231 and MDA-MB-453) and analyzed their metabolic effects using proton nuclear magnetic resonance (1H NMR) spectroscopy-based metabolomics. We found that AZD8055 more strongly inhibited breast cancer cell proliferation than rapamycin. The half-inhibitory concentration of AZD8055 in breast cancer cells was almost one-tenth that of rapamycin. We identified 22 and 23 metabolites from the 1H NMR spectra of MDA-MB-231 and MDA-MB-453 cells. The patterns of AZD8055- and rapamycin-treated breast cancer cells differed significantly; we then selected the metabolites that contributed to these differences. For inhibiting glycolysis and reducing glucose consumption, AZD8055 was likely to be more potent than rapamycin. For amino acids metabolism, although AZD8055 has a broad effect as rapamycin, their effects in degrees were not exactly the same. AZD8055 and rapamycin displayed cell-specific metabolic effects on breast cancer cells, a finding that deserves further study. These findings help fill the knowledge gap concerning dual mTORC1/2 inhibitors and provide a theoretical basis for their development.

Prognostic Value of Platelet-Albumin-Bilirubin Grade in Child-Pugh A and B Patients With Hepatocellular Carcinoma: A Meta-Analysis.

Background: Numerous studies showed that preoperative platelet-albumin-bilirubin (PALBI) grade was closely related to the prognostic outcome of patients with hepatocellular carcinoma (HCC). However, the conclusions were inconsistent. Therefore, we implemented the study to comprehensively evaluate the association between PALBI grade and prognosis in patients with HCC. Methods: Relevant articles were collected from the specified databases until February 10, 2022. We included all studies exploring the relationship between PALBI grade and prognosis in HCC patients. We used the hazard ratio (HR) and 95% confidence interval (CI) to calculate the comprehensive analysis. All data analyses were performed using STATA 12.0. Results: Thirteen retrospective articles containing 15534 patients were included in the meta-analysis. The pooled results displayed that the high PALBI grade was obviously correlated with poor overall survival (OS) (HR: 1.71, 95% CI: 1.46-2.02) and disease-free survival/relapse-free survival (DFS/RFS) (HR:1.31; 95% CI: 1.11-1.54). Subgroup analyses further confirmed the reliability of the comprehensive results. Conclusions: PALBI may be a valid prognostic indicator in HCC patients. More investigations were needed to test our findings.

MgIG exerts therapeutic effects on crizotinib-induced hepatotoxicity by limiting ROS-mediated autophagy and pyroptosis.

Crizotinib (CRIZO) has been widely employed to treat non-small-cell lung cancer. However, hepatic inflammatory injury is the major toxicity of CRIZO, which limits its clinical application, and the underlying mechanism of CRIZO-induced hepatotoxicity has not been fully explored. Herein, we used cell counting kit-8 assay and flow cytometry to detect CRIZO-induced cytotoxicity on human hepatocytes (HL-7702). CRIZO significantly reduced the survival rate of hepatocytes in a dose-dependent manner. Furthermore, the reactive oxygen species (ROS) assay kit showed that CRIZO treatment strongly increased the level of ROS. In addition, CRIZO treatment caused the appearance of balloon-like bubbles and autophagosomes in HL-7702 cells. Subsequently, Western blotting, quantitative real-time PCR and ELISA assays revealed that ROS-mediated pyroptosis and autophagy contributed to CRIZO-induced hepatic injury. Based on the role of ROS in CRIZO-induced hepatotoxicity, magnesium isoglycyrrhizinate (MgIG) was used as an intervention drug. MgIG activated the Nrf2/HO-1 signalling pathway and reduced ROS level. Additionally, MgIG suppressed hepatic inflammation by inhibiting NF-κB activity, thereby reducing CRIZO-induced hepatotoxicity. In conclusion, CRIZO promoted autophagy activation and pyroptosis via the accumulation of ROS in HL-7702 cells. MgIG exerts therapeutic effects on CRIZO-induced hepatotoxicity by decreasing the level of ROS.

Neuropsychiatric lupus erythematosus with neurogenic pulmonary edema and anti-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor limbic encephalitis: a case report.

BACKGROUND: Systemic lupus erythematosus (SLE) is a chronic multisystem autoimmune inflammatory disease predominantly found in women of child-bearing age. Neurogenic pulmonary edema (NPE) is a recalcitrant complication that occurs after injury to the central nervous system and has an acute onset and rapid progression. Limbic encephalitis is an inflammatory encephalopathy caused by viruses, immune responses, or other factors involving the limbic system. NPE caused by SLE is rare. CASE PRESENTATION: Here, we report a case of a 21-year-old woman with SLE who experienced five episodes of generalized tonic-clonic seizure after headache and dyspnea. Anti-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) 2 antibody was tested positive in the serum and cerebrospinal fluid. Electrocardiography (EEG) indicated paroxysmal or sporadic medium amplitude theta activity. In addition, chest computed tomography (CT) showed multiple diffuse consolidations and ground-glass opacities. We finally considered a diagnosis of NPE and AMPAR limbic encephalitis. The patient's symptoms improved obviously after methylprednisolone pulse therapy and antiepileptic treatment. CONCLUSIONS: NPE can be a complication of neuropsychiatric lupus erythematosus (NPSLE). AMPAR2 antibodies may be produced in NPSLE patients, especially in those with high polyclonal IgG antibody titers. More basic and clinical studies are required to confirm these observations and elucidate the pathogenicity of encephalitis-related autoantibodies in SLE patients.

Prognostic value of STC1 in solid tumors: a meta-analysis.

Aim: The prognostic value of STC1 has been evaluated in solid tumors. However, the results remain controversial. Materials & methods: Relevant studies published up to 27 February 2021 were identified by a comprehensive search of the PubMed, EMBASE and Web of Science databases. Hazard ratios (HRs) and odds ratios with 95% CIs were applied to explore the association between STC1 and survival outcome and clinical characteristics. Results: Sixteen articles involving 2942 participants were included in this meta-analysis. The pooled analysis showed that high STC1 expression was significantly associated with worse overall survival (HR: 1.91; 95% CI: 1.63-2.24) and disease-free survival/progression-free survival/relapse-free survival (HR: 2.01; 95% CI: 1.34-3.02). Conclusion: STC1 may be an effective prognostic marker in solid tumors.

Comprehensive Analysis of Prognostic Value and Immune Infiltration of the NT5DC Family in Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world, and its incidence is obviously increasing. The NT5DC family has been shown to be involved in the progression of many tumors. However, the biological function of NT5DC family members in HCC is still not well understood. METHODS: Oncomine, Gene Expression Profiling Interactive Analysis (GEPIA), UALCAN, Kaplan-Meier plotter, cBioPortal, GeneMANIA, Metascape, and TIMER were applied to assess the biological function of NT5DC family members in HCC. RESULTS: Most of the NT5DC family members were highly expressed in HCC. High expression of NT5C2, NT5DC2, and NT5DC3 was closely associated with higher tumor stage and poor overall survival (OS). In addition, high NT5DC2 and NT5DC3 expression also predicted poor disease-free survival (DFS). Enrichment analysis revealed that the NT5DC family in HCC mainly involved the IMP metabolic process, purine ribonucleoside monophosphate metabolic process, and purine nucleoside monophosphate metabolic process. The expression of NT5DC family members was closely related to the infiltration of some immune cells, such as B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells. CONCLUSION: Our findings provided new insights into the biological function and prognostic value of NT5DC family members in HCC.

NTF3 Correlates With Prognosis and Immune Infiltration in Hepatocellular Carcinoma.

Background: The potential role of Neurotrophic factor-3(NTF3) in liver cancer is unknown. Therefore, we aimed to explore the clinical value of NTF3 in hepatocellular carcinoma (HCC). Methods: We used a variety of databases to analyze the expression, relationship with prognosis and immune significance of NTF3 in liver cancer through bioinformatics. Results: NTF3 was low expressed in HCC and was an independent prognostic factor in patients with HCC. CIBERSORT analysis indicated that NTF3 expression was positively correlated with CD4+ cells, mast cells, NK cells, macrophages and B cells in the tumor microenvironment. Furthermore, we found that NTF3 expression was negatively correlated with the immune checkpoints PD-L1, TIGIT and TIM-3. Functional network analysis revealed that NTF3 regulates HCC progression through a variety of cancer-related kinases, transcription factors and signaling pathways. Conclusions: We demonstrate that NTF3 correlates with prognosis and immune infiltration in HCC.

Defending the homeland: microbiome molecules provide protection to their vertebrate hosts.

Aim: The resident bacterial microbiome may shape and protect the health of vertebrate host. An array of molecules secreted by microbiome may contribute to the ecological stability of the microbiome itself. Material & methods: ELISA, radioactivity, immunofluorescence and cytokines measurements were used to observe the bioactivity and stability of colicin Ia level in oviparous and viviparous animal circulation. Results: Colicin Ia, a protein antimicrobial produced by Escherichia coli, is not present in animals at birth, but increases in concentration with the establishment of a stable gut microbiome and drops when the microbiome is experimentally disrupted. Colicin introduced in vivo is transported to tissues at concentrations able to prevent or eliminate bacterial infection. Conclusion: Our findings suggest an unexpected benefit provided by the presence of a resident microbiome in the form of active, circulating, bacterially-synthesized antimicrobial molecules.

Piezoelectric Performance of a Symmetrical Ring-Shaped Piezoelectric Energy Harvester Using PZT-5H under a Temperature Gradient.

With the rapid development of microelectronics technology, low-power electronic sensors have been widely applied in many fields, such as Internet of Things, aerospace, and so on. In this paper, a symmetrical ring-shaped piezoelectric energy harvester (SR-PEH) is designed to provide energy for the sensor to detect the ambient temperature. The finite element method is used by utilizing software COMSOL 5.4, and the electromechanical coupling model of the piezoelectric cantilever is established. The output performance equations are proposed; the microelectromechanical system (MEMS) integration process of the SR-PEH, circuit, and sensor is stated; and the changing trend of the output power density is explained from an energy perspective. In the logarithmic coordinate system, the results indicate that the output voltage and output power are approximately linear with the temperature when the resistance is constant. In addition, the growth rate of the output voltage and output power decreases with an increase of resistance under the condition of constant temperature. In addition, with an increase of temperature, the growth rate of the output power is faster than that of the output voltage. Furthermore, resistance has a more dramatic effect on the output voltage, whereas temperature has a more significant effect on the output power. More importantly, the comparison with the conventional cantilever-shaped piezoelectric energy harvester (CC-PEH) shows that the SR-PEH can improve the output performance and broaden the frequency band.

A Novel Cold-Adapted Leucine Dehydrogenase from Antarctic Sea-Ice Bacterium Pseudoalteromonas sp. ANT178.

l-tert-leucine and its derivatives are useful as pharmaceutical active ingredients, in which leucine dehydrogenase (LeuDH) is the key enzyme in their enzymatic conversions. In the present study, a novel cold-adapted LeuDH, psleudh, was cloned from psychrotrophic bacteria Pseudoalteromonas sp. ANT178, which was isolated from Antarctic sea-ice. Bioinformatics analysis of the gene psleudh showed that the gene was 1209 bp in length and coded for a 42.6 kDa protein containing 402 amino acids. PsLeuDH had conserved Phe binding site and NAD⁺ binding site, and belonged to a member of the Glu/Leu/Phe/Val dehydrogenase family. Homology modeling analysis results suggested that PsLeuDH exhibited more glycine residues, reduced proline residues, and arginine residues, which might be responsible for its catalytic efficiency at low temperature. The recombinant PsLeuDH (rPsLeuDH) was purified a major band with the high specific activity of 275.13 U/mg using a Ni-NTA affinity chromatography. The optimum temperature and pH for rPsLeuDH activity were 30 °C and pH 9.0, respectively. Importantly, rPsLeuDH retained at least 40% of its maximum activity even at 0 °C. Moreover, the activity of rPsLeuDH was the highest in the presence of 2.0 M NaCl. Substrate specificity and kinetic studies of rPsLeuDH demonstrated that l-leucine was the most suitable substrate, and the catalytic activity at low temperatures was ensured by maintaining a high kcat value. The results of the current study would provide insight into Antarctic sea-ice bacterium LeuDH, and the unique properties of rPsLeuDH make it a promising candidate as a biocatalyst in medical and pharmaceutical industries.

An engineered multidomain fungicidal peptide against plant fungal pathogens.

Fungal pathogens represent major problems for human health and agriculture. As eukaryotic organisms, fungi share some important features with mammalian cells. Therefore, current anti-fungal antibiotics often can not distinguish between fungi and mammalian cells, resulting in serious side effects in mammalian cells. Accordingly, there is strong impetus to develop antifungal alternatives that are both safe and effective. The E1 family of colicin are channel-forming bacteriocins produced by Escherichia coli, which are bactericidal only to E. coli and related species. To target the channel-forming domain of colicin to fungal cell membrane, we engineered a sexual mating pheromone of Candida albicans, α-factor pheromone to colicin Ia. A peptide was constructed consisting of an α mating pheromone of C. albicans fused to the channel-forming domain of colicin Ia to create a new fusion protein, pheromonicin-CA (PMC-CA). Indirect immunolabeling showed that the PMC-CA bound to fungal cells and inhibited growth in the laboratory and field. In the field, the protective activity of pheromonicin against rice blast disease was significantly greater, on a molar basis, than that of triazoles, tricyclazole or isoprothiolane. These results suggest that fusion peptides may be of value as fungicidal agents under agricultural conditions.

[Experimental study on the induced differentiation of human amnion mesenchymal cells into osteoblasts].

OBJECTIVE: To investigate the feasibility of inducing differentiation of the human amniotic mesenchymal cells (hAMCs) into osteoblasts in vitro, so as to provide the seed cells for bone tissue engineering. METHODS: The hAMCs were isolated from abandoned human amnion and cultured in osteogenic media to induce the osteogenic differentiation in vitro. After hAMCs were induced by osteogenic media for 15 days, morphological observation, immunocytochemistry and western blot were used to study the cellular morphology and expression of alkaline phosphatase (ALP), type I collagen, osteopontin and osteocalcin. RESULTS: The primary cultured hAMCs had long spindle shape or irregular shape, which were distributed evenly. The cells were usually suheultured in 5 or 7 days. After subculture, the cells became larger. After cultured by osteogenic media for 15 days, the hAMCs were detected to express ALP, osteocalcin and osteopontin, and secrete type I collagen. CONCLUSIONS: The hAMCs are isolated, cultured and amplified easily in vitro. The induced differentiated cells by osteogenic media have typical osteoblast morphological and functional characteristics, which can be used as seed cells for bone tissue engineering.

[Changes of bacterial community structure on reusing domestic sewage of Daoxianghujing Hotel to landscape water].

A 16S rDNA library was used to evaluate the bacterial diversity and identify dominant groups of bacteria in different treatment pools in the domestic sewage system of the Beijing Daoxianghujing Hotel. The results revealed that there were many types of bacteria in the hotel domestic sewage, and the bacterial Shannon-Weaver diversity index was 3.12. In addition, epsilon Proteobacteria was found to be the dominant group with the ratio of 32%. In addition, both the CFB phylum, Fusobacteria, gamma Proteobacteria and Firmicutes were also reached to 9%-15%. After treated with the reclaimed water station, the bacterial Shannon-Weaver diversity index was reduced to 2. 41 and beta Proteobacteria became the dominant group and occupied 73% of the total clones. However, following artificial wetland training, the bacterial Shannon-Weaver diversity index in the sample increased to 3.38, Actinobacteria arrived to 33% and became the most dominant group; Cyanobacteria reached to 26%, and was the second dominant group. But, the control sample comprised 38% Cyanobacteria, and mainly involved in Cyanobium, Synechoccus and Microcystis, with ratios of 47.1%, 17.6% and 8.8%, respectively. Some bacteria of Microcystis aenruginosa were also detected, which probably resulted in the light bloom finally. Therefore, the bacterial diversity and community structures changed in response to treatment of the hotel domestic sewage; there was no cyanobacteria bloom explosion in the treated water. This study will aid in investigation the changes of microbial ecology in different types of water and providing the useful information for enhancing the cyanobacteria blooms control from ecological angle.

[Effect of free surface flow wetland and subsurface flow wetland on bacterial diversity in Beijing Cuihu Wetland Park].

To achieve the effects of artificial wetland on the bacterial diversity, the culturable bacteria and total cell counts of three wetland cells, including sewage pond (SP), free surface wetland (SF) and subsurface flow wetland (SSF), were investigated using the traditional culture-dependent approach and flow cytometry method, based on the detecting the water quality. The bacterial diversity and dominant groups were also compared by PCR-DGGE profiles and 16S rDNA library technique based on its V3 region. Results show that SF and SSF cells can remove the nutrients effectively, the highest removal ratio of COD, total nitrogen, and total phosphorus reach to 42.33%, 52.92% and 41.4%, respectively; The total microbes are increased continuously with the treatment by SF and SSF, and the culturable bacteria clones are decreased after treatment by SF, and increased after further train by SSF. The Shannon-Weaver index is increased to 3.2850 from 3.0819 while the water flowing through SF, but decreased to 3.0181 after flowing through SSF; The dominant groups in SP include Actinobacteria, Cyanobacteria and alpha-Proteobacteria, reach to 38%, 18% and 18%, respectively; but the most dominant bacteria is changed to beta-Proteobacteria with the ratio of 32% and 44%, after treatment by SF and SSF, respectively. Cytophagal Flexibacter/Bacteroides (CFB) phylum is also increased to 24% finally. Therefore, while the Cuihu Wetland removing the nutrients,the bacterial counts, diversity and dominant groups are also changed,some beneficial bacteria in beta-Proteobacteria and CFB phylum increased, and part of those deleterious bacteria in Actinobacteria and Cyanobacteria decreased.