DEAD Box Protein DhR1 Is a Global Regulator Involved in the Bacterial Fitness and Virulence of Riemerella anatipestifer.

DEAD box proteins perform diverse cellular functions in bacteria. Our group previously reported that the transposon Tn4531 insertion in Riean_0395 (designated dhR1), which encodes a putative DEAD box helicase, attenuated the virulence of R. anatipestifer strain YZb1. Here, we show that, compared to the wild-type (WT) R. anatipestifer strain Yb2, the growth or survival of the ΔdhR1 mutant in tryptic soy broth (TSB) was significantly decreased in response to cold, pH, osmotic stress, ethanol, Triton X-100, and oxidative stress, and the dhR1 deletion significantly reduced biofilm formation and the adhesion capacity to Vero cells, whereas the growth of ΔdhR1 was less impaired in iron-limited TSB. Moreover, the virulence of ΔdhR1 in ducklings was attenuated by about 80-fold, compared to the WT. In addition, a transcriptome analysis showed that the dhR1 deletion in the strain Yb2 affected the expression of 58 upregulated genes and 98 downregulated genes that are responsible for various functions. Overall, our work reveals that the deletion of DhR1 results in a broad effect on the bacterial fitness, biofilm formation, iron utilization, and virulence of R. anatipestifer, which makes it a global regulator. IMPORTANCE R. anatipestifer infection has been a continued and serious problem in many duck farms, but little is known about the mechanism underlying the pathogenesis of R. anatipestifer and how R. anatipestifer adapts to the external environment and thereby persists in duck farms. The results of this study demonstrate that the DEAD box protein DhR1 is required for the tolerance of R. anatipestifer to cold, pH, and other stresses, and it is also necessary for biofilm formation, iron utilization, and virulence in ducklings, demonstrating multiple functions of DhR1.

Extracellular vesicles derived from PM2.5-exposed alveolar epithelial cells mediate endothelial adhesion and atherosclerosis in ApoE-/- mice.

Epidemiological studies suggested that PM2.5 (particle matters with an aerodynamic diameter ≤2.5 µm) exposure is associated with atherosclerosis. Extracellular vesicles (EVs) are messengers between intracellular communications which are important in diseases procession. At present, whether EVs derived from PM2.5-exposed alveolar epithelial cells (P-EVs) involve in atherosclerosis has not been clearly understood. This study is performed to investigate the effects of P-EVs on the development of endothelium adhesion and atherosclerosis. Here, ApoE-/- mice were randomized into different groups receiving one of the following treatments, filtered air (FA), PM2.5, PBS, PBS-treated alveolar epithelial cells-derived EVs (EVs), or P-EVs. Then the atherosclerosis level in aortas or aorta sections was evaluated by oil red O staining. The results indicated that ApoE-/- mice treated with P-EVs or PM2.5 showed more obvious atherosclerosis plaques in aortas and aortic arches than those treated with EVs or PBS. Endothelial cells (ECs) were treated with PBS, EVs, P-EVs, or PM2.5. The adhesion property, miRNAs level and expressions of IκBα, phosphorylated IκBα, NF-κB p65, phosphorylated NF-κB p65, and VCAM1 in ECs were determined. It was found that P-EVs activated IκBα-NF-κB-VCAM1 signaling and increased adhesion of ECs, and such effects could be reversed by adalimumab (the TNF-α inhibitor) or miR-326-3p inhibitor. Further study suggested that P-EVs induced upregulation of TNF-α and miR-326-3p in recipient ECs and contributed to the phosphorylation of NF-κB p65. Collectively, EVs derived from PM2.5-exposed alveolar epithelial cells played an important role in the development of atherosclerosis via activating IκBα-NF-κB-VCAM1 signaling.

Extracellular Vesicles Derived from Adipose Mesenchymal Stem Cells Alleviate PM2.5-Induced Lung Injury and Pulmonary Fibrosis.

BACKGROUND Exposure to PM2.5 (fine particulate matter ≤2.5 µm in aerodynamic diameter) in air increases the risk of lung injury and pulmonary fibrosis (PF). Extracellular vesicles (EVs) derived from adipose mesenchymal stem cells (ADSCs) have been identified as a potential treatment based on the proteins or RNAs delivery and immunomodulatory properties. Here, we assessed the protective effects and mechanisms of ADSCs-EVs on PM2.5-induced lung injury or PF. MATERIAL AND METHODS Rats (male, 6 weeks old) were exposed to PBS or PM2.5 (1.5 mg/kg/day) for 3 days a week for 4 weeks. ADSCs-EVs were extracted by ultracentrifugation. PBS and ADSCs-EVs were administrated through intratracheal instillation. After the end of exposure, the rats were anesthetized and killed. Lung tissues with different treatments were collected for Western blot analysis and HE, IHC, and IF staining analysis. Cells exposed to PM2.5 or "PM2.5+ADSCs-EVs" in vitro were also collected for further Western blotting, qRT-PCR, and IF staining evaluation. RESULTS The results indicated that the initial response of lungs exposed to PM2.5 was lung injury with oxidative stress and inflammation. Long-term PM2.5 exposure resulted in obvious PF in rats. Treatment with ADSCs-EVs decreased PM2.5-induced apoptosis and necrosis in type II alveolar epithelial cells and alleviated lung injury and PF in rats. ADSCs-EVs suppressed reactive oxygen species (ROS) levels and inflammation induced by PM2.5. Furthermore, ADSCs-EVs inhibited TGF-ßRI by transferring let-7d-5p and further mitigated PF. CONCLUSIONS Our results suggest that EVs derived from ADSCs can alleviate PM2.5-induced lung injury and PF.

ACE-2/ANG1-7 ameliorates ER stress-induced apoptosis in seawater aspiration-induced acute lung injury.

Previous studies have shown that apoptosis of alveolar cells can be regulated by autocrine of angiotensin (ANG)II and its counter regulatory ACE-2/ANG1-7 axis. Our earlier study has shown that endoplasmic reticulum (ER) stress in response to seawater aspiration eventually led to apoptosis in lung tissue. In this study, we examined the hypothesis that ER stress-induced apoptosis in seawater aspiration-induced acute lung injury (ALI) might also be regulated by the ANGII/ANG1-7 system. ER stress was induced by seawater stimulation and proteasome inhibitor MG132 (an ER stress inductor). Moreover, ER stress in seawater-stimulated lung tissues and rat pulmonary microvascular endothelial cells (RPMVECs) promoted ANGII expression and decreased ACE-2/ANG1-7 expression. ER stress induced by seawater stimulation also led to apoptosis. Apoptosis induced by seawater stimulation and MG132 were inhibited by ANGII receptor blocker and abrogated by the addition of ANG1-7. These results suggest that apoptosis induced by ER stress in seawater aspiration-induced ALI is regulated by ANG II/ANG1-7 in lung tissues and RPMVECs. In addition, the active form of X-box binding protein 1 (XBP1), spliced XBP1 (XBP1s), a transcription factor that regulates ER-associated degradation genes during ER stress was significantly activated in seawater stimulated cells. Based on this phenomenon we designed a tandem gene, Wfs1 promoter (a target gene promoter of XBP1s)- ACE2 and ANG1-7 and transfected this tandem gene into seawater-stimulated cells. ACE-2/ANG1-7 expression were significantly promoted and apoptosis was inhibited in cells transfected with the tandem gene. These results suggest that stimulation of ACE-2/ANG1-7 may be a therapeutic target of ER stress-induced apoptosis in seawater aspiration-induced ALI.

PET Imaging of VCAM-1 Expression and Monitoring Therapy Response in Tumor with a 68Ga-Labeled Single Chain Variable Fragment.

Vascular cell adhesion molecule-1 (VCAM-1) is a transmembrane glycoprotein closely related to tumorigenicity as well as tumor metastasis. It is also a well-known candidate for detecting tumors. LY2409881, an IKKß inhibitor, could induce apoptosis of VCAM-1 positive cells. Our purpose is to prepare a novel tracer to evaluate its feasibility of detecting VCAM-1 expression and monitoring LY2409881 tumor curative effect. The tracer was prepared by conjugating the single chain variable fragment (scFv) of VCAM-1 and NOTA-NHS-ester and then labeled with 68Ga. 68Ga-NOTA-VCAM-1scFv was successfully prepared with high radiochemical yield. VCAM-1 overexpression and underexpression melanoma cell lines, B16F10 and A375m, were used in this study. The results of microPET/CT imaging in small animals indicated that the uptake of 68Ga-NOTA-VCAM-1scFv in B16F10 tumor was much higher than that of A375m, which was also confirmed by the biodistribution and autoradiography results. LY2409881 inhibits the growth of B16F10 melanoma in vivo by inducing dose- and time-dependent growth inhibition and apoptosis of the cells. The LY2409881 treated group and DMSO control group were established and imaged by microPET/CT. In the LY2409881 group, uptake of the tracer in tumor was decreased at the first week, and then gradually recovered to the initial level. In DMSO control, the uptake of the tracer remained at the same level during the whole time. The results suggested that LY2409881 inhibits the expression of VCAM-1 and suppresses tumor growth. 68Ga-NOTA-VCAM-1scFv, an easily synthesized probe, has a potential clinical application in the visual monitoring of IKKß inhibitor intervention on VCAM-1 positive tumors.

The impacts of climate change and human activities on biogeochemical cycles on the Qinghai-Tibetan Plateau.

With a pace of about twice the observed rate of global warming, the temperature on the Qinghai-Tibetan Plateau (Earth's 'third pole') has increased by 0.2 °C per decade over the past 50 years, which results in significant permafrost thawing and glacier retreat. Our review suggested that warming enhanced net primary production and soil respiration, decreased methane (CH(4)) emissions from wetlands and increased CH(4) consumption of meadows, but might increase CH(4) emissions from lakes. Warming-induced permafrost thawing and glaciers melting would also result in substantial emission of old carbon dioxide (CO(2)) and CH(4). Nitrous oxide (N(2)O) emission was not stimulated by warming itself, but might be slightly enhanced by wetting. However, there are many uncertainties in such biogeochemical cycles under climate change. Human activities (e.g. grazing, land cover changes) further modified the biogeochemical cycles and amplified such uncertainties on the plateau. If the projected warming and wetting continues, the future biogeochemical cycles will be more complicated. So facing research in this field is an ongoing challenge of integrating field observations with process-based ecosystem models to predict the impacts of future climate change and human activities at various temporal and spatial scales. To reduce the uncertainties and to improve the precision of the predictions of the impacts of climate change and human activities on biogeochemical cycles, efforts should focus on conducting more field observation studies, integrating data within improved models, and developing new knowledge about coupling among carbon, nitrogen, and phosphorus biogeochemical cycles as well as about the role of microbes in these cycles.

Isolation of Enterococcus faecium and determination of its mechanism for promoting the growth and development of Drosophila.

Intestinal symbiotic microorganisms have a strong capacity to regulate the physiological functions of their host, and Drosophila serves as a useful model. Enterococcus faecium (E. faecium) is a member of the normal intestinal flora of animals. Lactic acid bacteria (LAB) such as E. faecium can promote the growth and development of Drosophila, but the mechanism of regulation of Drosophila is poorly understood. In this study, we found that E. faecium used a carbon source to produce probiotic acids. E. faecium is a symbiotic bacterium for Drosophila, and adult flies passed on parental flora to offspring. E. faecium promoted the growth and development of Drosophila, especially under poor nutritional conditions. E. faecium shortened the developmental process for Drosophila and accelerated the transformation from larva to pupa. Finally, E. faecium promoted the growth and development of Drosophila through TOR and insulin signalling pathways.

Plasma exosomal miR-1290 and miR-29c-3p as diagnostic biomarkers for lung cancer.

Background: Enhancing the diagnostic efficacy of early-stage lung cancer is crucial for improving prognosis. The objective of this study was to ascertain dependable exosomal miRNAs as biomarkers for the diagnosis of lung cancer. Methods: Exosomal miRNA candidates were identified through miRNA sequencing and subsequently validated in various case-control sets using real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The correlation between the expression of exosomal miRNAs and the clinicopathological features of lung cancer was investigated. To assess the diagnostic efficacy of exosomal miRNAs for lung cancer, the receiver operating characteristic (ROC) curve analysis was conducted. The optimal cutoff value of exosomal miRNAs was determined in the testing cohort and subsequently confirmed in the validation cohort. Results: The results showed that the expression of exosomal miR-1290 was significantly elevated, while that of miR-29c-3p was significantly decreased in the plasma of lung cancer patients, especially in those with early-stage lung cancer, compared to individuals with benign lung conditions (P < 0.01). Exosomal miR-1290 and miR-29c-3p demonstrated superior diagnostic efficacy compared to conventional tumor biomarkers in distinguishing between lung cancer and benign lung diseases, as evidenced by their respective area under the curve (AUC) values of 0.934 and 0.868. Furthermore, exosomal miR-1290 and miR-29c-3p exhibited higher diagnostic efficiency in early-stage lung cancer than traditional tumor markers, with AUC values of 0.947 and 0.895, respectively. Notably, both exosomal miR-1290 and miR-29c-3p displayed substantial discriminatory capacity in distinguishing between non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), as indicated by their respective AUC values of 0.810 and 0.842. Conclusions: The findings of this study provided evidence that exosomal miR-1290 and miR-29c-3p hold significant potential as biomarkers for the early detection of lung cancer, as well as for differentiating between NSCLC and SCLC.

PaR1 secreted by the type IX secretion system is a protective antigen of Riemerella anatipestifer.

Riemerella anatipestifer mainly infects domestic ducks, geese, turkeys, and other birds, and causes considerable economic losses to the global duck industry. Previous studies have shown that concentrated cell-free culture filtrates of R. anatipestifer induce highly significant protection against homologous challenge. In this study, 12 immunogenic proteins were identified in the culture supernatant of R. anatipestifer strain Yb2 with immunoproteomic analysis. Of these, three immunogenic proteins, AS87_RS06600 (designated "PaR1" in this study), AS87_RS09020, and AS87_RS09965, which appeared in more than three spots on the western-blotted membrane, were expressed in Escherichia coli and purified. Animal experiments showed that the recombinant PaR1 (rPaR1) protein protected 41.67% of immunized ducklings against challenge with virulent Yb2, whereas rAS87_RS09020 or rAS87_RS09965 did not, and that ducklings immunized once with rPaR1 were 20, 40, and 0% protected from challenge with R. anatipestifer strains WJ4 (serotype 1), Yb2 (serotype 2), and HXb2 (serotype 10), respectively. In addition, rPaR1 immunized rabbit serum showed bactericidal activity against strain Yb2 at a titer of 1:8. These results indicate that rPaR1 of strain Yb2 protects against homologous challenge. Amino acid homology analysis show that PaR1 is a non-serotype-specific protein among different R. anatipestifer serotypes. Furthermore, PaR1 is mainly secreted outside the cell through the T9SS. Overall, our results demonstrate that R. anatipestifer PaR1 is a non-serotype-specific protective protein secreted by the T9SS.

Importance of local non-fossil sources to carbonaceous aerosols at the eastern fringe of the Tibetan Plateau, China: Δ14C and δ13C evidences.

Carbonaceous particles are an important radiative forcing agent in the atmosphere, with large temporal and spatial variations in their concentrations and compositions, especially in remote regions. This study reported the Δ14C and δ13C of total carbon (TC) and water-insoluble particulate carbon (IPC) of the total suspended particles (TSP) and PM2.5 at a remote site of the eastern Tibetan Plateau (TP), a region that is influenced by heavy air pollution from Southwest China. The average organic carbon and elemental carbon concentrations of TSP samples in this study were 3.20 ± 2.38 µg m-3 and 0.68 ± 0.67 µg m-3, respectively, with low and high values in summer and winter, respectively. The fossil fuel contributions of TC in TSP and PM2.5 samples were 18.91 ± 7.22% and 23.13 ± 12.52%, respectively, both of which were far lower than that in Southwest China, indicating the importance of non-fossil contributions from local sources. The δ13C of TC in TSP samples of the study site was -27.06 ± 0.96‰, which is between the values of long-range transported sources (e.g., Southwest China) and local biomass combustion emissions. Therefore, despite the contribution from the long-range transport of particles, aerosols emitted from local biomass combustion also have an important influence on carbonaceous particles at the study site. The findings of this work can be applied to other remote sites on the eastern TP and should be considered in related research in the future.

Photodynamic therapy for primary tracheobronchial malignancy in Northwestern China.

BACKGROUND: Photodynamic therapy (PDT) has been routinely performed to treat tracheobronchial malignancy. However, the experience in tracheobronchial adenoid cystic carcinoma (ACC) and peripheral lung cancer is still insufficient. This study aimed to share the experience of PDT for patients with primary tracheobronchial malignancy, especially the adenoid cystic carcinoma and peripheral lung cancer, and evaluated the efficacy and safety of PDT in Northwestern Chinese patients. METHODS: This study retrospectively analyzed the clinical data of 23 patients with primary tracheobronchial malignancy receiving PDT in our center. The short-term effect was evaluated by the objective tumor response and the clinical response. The long-term effect was estimated by recurrence-free survival (RFS). RESULTS: Of 23 patients, SR was achieved in 18 patients and MR in 3 patients. The clinical symptoms and the quality of life were significantly improved after PDT (P<0.05). And the mean RFS was 8.9 ± 1.9 months. SR for 6 cases of ACC were achieved with significant improvement of clinical symptoms and quality of life. No procedure-related complications appeared. And PDT was successfully performed for the peripheral lung cancer with the guidance of electromagnetic navigation bronchoscopy (ENB). CONCLUSIONS: This study demonstrated that PDT achieved satisfactory efficacy and safety for Northwestern Chinese patients with primary tracheobronchial malignancy. Patients with ACC can benefit from PDT. And ENB-guided PDT is a novel and available option for the peripheral lung cancer. In short, this study accumulated valuable experience for the application of PDT in Chinese patients with primary tracheobronchial malignancy.

Nitrous oxide fluxes from the littoral zone of a lake on the Qinghai-Tibetan Plateau.

Nitrous oxide (N(2)O) fluxes were measured in six littoral mirco-zones of Lake Huahu on Qinghai-Tibetan Plateau in the peak growing season of years of 2006 and 2007. The weighted mean N(2)O flux rate was 0.08 mg N m(-2) h(-1) (ranged from -0.07 to 0.35 mg N m(-2) h(-1)). The result was relatively high in the scope of N(2)O fluxes from boreal and temperate lakes. Emergent plant zones (Hippuris vulgaris and Glyceria maxima stands) recorded the highest N(2)O flux rate (0.11 ± 0.24 and 0.08 ± 0.17 mg N m(-2) h(-1), respectively). Non-vegetated lakeshore recorded the lowest N(2)O flux (0.03 ± 0.11 mg N m(-2) h(-1)), lower than that from the floating mat zone of Carex muliensis (0.05 ± 0.18 mg N m(-2) h(-1)), the floating-leaved plant zone of Polygonum amphibium (0.07 ± 0.11 mg N m(-2) h(-1)), and the wet meadow (0.07 ± 0.15 mg N m(-2) h(-1)). Standing water depths were important factors to explain such spatial variations in N(2)O fluxes. Significant temporal variations in N(2)O fluxes were also found. Such temporal variation in N(2)O flux in the littoral zone may be dependent on the interaction of water regime and thermal conditions, instead of the latter solely. These results showed the importance of the littoral zone of lake, especially the emergent plant zone, as a hotspot of N(2)O fluxes in such grazing meadows.

Nitrogen addition, rather than altered precipitation, stimulates nitrous oxide emissions in an alpine steppe.

Anthropogenic-driven global change, including changes in atmospheric nitrogen (N) deposition and precipitation patterns, is dramatically altering N cycling in soil. How long-term N deposition, precipitation changes, and their interaction influence nitrous oxide (N2O) emissions remains unknown, especially in the alpine steppes of the Qinghai-Tibetan Plateau (QTP). To fill this knowledge gap, a platform of N addition (10 g m-2 year-1) and altered precipitation (±50% precipitation) experiments was established in an alpine steppe of the QTP in 2013. Long-term N addition significantly increased N2O emissions. However, neither long-term alterations in precipitation nor the co-occurrence of N addition and altered precipitation significantly affected N2O emissions. These unexpected findings indicate that N2O emissions are particularly susceptible to N deposition in the alpine steppes. Our results further indicated that both biotic and abiotic properties had significant effects on N2O emissions. N2O emissions occurred mainly due to nitrification, which was dominated by ammonia-oxidizing bacteria, rather than ammonia-oxidizing archaea. Furthermore, the alterations in belowground biomass and soil temperature induced by N addition modulated N2O emissions. Overall, this study provides pivotal insights to aid the prediction of future responses of N2O emissions to long-term N deposition and precipitation changes in alpine ecosystems. The underlying microbial pathway and key predictors of N2O emissions identified in this study may also be used for future global-scale model studies.

Adipose mesenchymal stem cell-derived antioxidative extracellular vesicles exhibit anti-oxidative stress and immunomodulatory effects under PM2.5 exposure.

PM2.5 exposure elevates the level of reactive oxygen species (ROS) in the lungs and leads to lung injury or other pulmonary conditions. Nrf2 is a key antioxidative regulator that suppresses ROS production. Extracellular vesicles (EVs) secreted by adipose mesenchymal stem cells (ADSCs) have been identified as therapeutic as well as potential drug/gene/protein carriers. In this study, we established rat (PM2.5, 100 µL, 5 mg/mL) or cell (PM2.5, 50 µg/mL) models to conduct in vivo and in vitro studies on the adverse pulmonary effects of PM2.5. Our findings indicated that the initial responses to PM2.5 exposure were robust oxidative stress and inflammation. EVs and antioxidative EVs (Antioxi-EVs, derived from ADSCs that overexpress Nrf2) had been tested as interventions in PM2.5-treated rat or cell models through tracheal instillation or co-incubation. Treatment with EVs or Antioxi-EVs (3 × 1010 particles in vivo and 1 × 109in vitro) was found to have a suppressive effect on the levels of ROS and inflammatory cytokines, with Antioxi-EVs having a superior effect on anti-oxidative stress. In particular, the occurrence of lung injury or cell apoptosis correlated positively with the ROS level, and inhibition of ROS by upregulating Nrf2 alleviated lung injury and cell apoptosis. Furthermore, treatment with EVs or Antioxi-EVs increased the level of M2-like macrophages as compared to treatment with PBS and further reduced IL-6 and TNF-α levels. Our results suggest that Antioxi-EVs can reduce the severity of oxidative stress, inflammation, and lung injury induced by PM2.5via anti-oxidative stress and immunomodulation pathways.

Diurnal variation of methane emissions from an alpine wetland on the eastern edge of Qinghai-Tibetan Plateau.

Alpine wetland is a source for CH(4), but little is known about methane emission from such wetland, especially about its diurnal pattern. In this study we tried to probe the diurnal variation in methane emission from alpine wetland vegetation. The average methane emission rate was 9.6 +/- 3.4 mg CH(4) m(-2) h(-1). There was an apparent diurnal variation pattern in methane emission with one minor peak at 06:00 and a major one at 15:00. The sunrise peak was consistent with a two-way transport mechanism for plants (convective at daytime and diffusive at night-time). CH(4) emission was found significantly correlated with redox potentials. The afternoon peak could not be explained by diurnal variation in soil temperature, but could be attributable to changes in CH(4) oxidation and production driven by plant gas transport mechanism. The results have important implications for sampling and scaling strategies for estimating methane emission from alpine wetlands.

Evaluation of bronchoalveolar lavage fluid combined with the loop-mediated isothermal amplification assay in lower respiratory tract infections.

The clinical application of the loop-mediated isothermal amplification (LAMP) assay has been problematic because of conflicting results obtained from the LAMP assay and bacterial culture. In order to eliminate the interference of oral microorganisms and more accurately evaluate the diagnostic performance of the LAMP assay, we utilized bronchoalveolar lavage fluid (BALF) as a sample to test whether the LAMP assay and bacteria culture yielded similar results. A total of 1092 BALF samples from patients with suspected lower respiratory tract infections were collected. For each sample, parallel studies using both bacterial culture and the LAMP assay were carried out. We were the first to utilize BALF as a sample to study the consistency between the LAMP assay and bacterial culture results. The present study demonstrated that the positive rate from the LAMP assay was higher than that from bacterial culture, and the two methods had a better consistency than previously reported.

VHL mutation-mediated SALL4 overexpression promotes tumorigenesis and vascularization of clear cell renal cell carcinoma via Akt/GSK-3ß signaling.

BACKGROUND: Although ongoing development of therapeutic strategies contributes to the improvements in clinical management, clear cell renal cell carcinoma (ccRCC) deaths originate mainly from radiochemoresistant and metastatic disease. Transcription factor SALL4 has been implicated in tumorigenesis and metastasis of multiple cancers. However, it is not known whether SALL4 is involved in the pathogenesis of ccRCC. METHODS: Analyses of clinical specimen and publicly available datasets were performed to determine the expression level and clinical significance of SALL4 in ccRCC. The influence of SALL4 expression on ccRCC tumor growth, metastasis and vascularity was evaluated through a series of in vitro and in vivo experiments. Western blotting, immunofluorescence staining and integrative database analysis were carried out to investigate the underlying mechanism for SALL4-mediated oncogenic activities in ccRCC. RESULTS: SALL4 expression was increased in ccRCC and positively correlated with tumor progression and poor prognosis. SALL4 could promote ccRCC cell proliferation, colony formation, cell cycle progression, migration, invasion and tumorigenicity and inhibit cell senescence. Further investigation revealed a widespread association of SALL4 with individual gene transcription and the involvement of SALL4 in endothelium development and vasculogenesis. In the context of ccRCC, SALL4 promoted tumor vascularization by recruiting endothelial cells. In addition, we found that SALL4 could exert its tumor-promoting effect via modulating Akt/GSK-3ß axis and VEGFA expression. VHL mutation and DNA hypomethylation may be involved in the upregulation of SALL4 in ccRCC. CONCLUSIONS: Overall, our results provide evidence that upregulated SALL4 can function as a crucial regulator of tumor pathogenesis and progression in ccRCC, thus offering potential therapeutic strategies for future treatment.

Extracellular vesicle-mediated delivery of miR-101 inhibits lung metastasis in osteosarcoma.

Rationale: Extracellular vesicles (EVs) have emerged as novel mediators of cell-to-cell communication that are capable of the stable transfer of therapeutic microRNAs (miRNAs), and thus, EVs hold immense promise as a miRNA delivery system for cancer therapy. Additionally, as miRNA-containing EVs are secreted into circulation, miRNAs contained within plasma EVs may represent ideal biomarkers for diseases. The objective of this study was to characterize a potential tumor suppressor miRNA, miR-101, and explore the potential of miR-101 delivery via EVs for in vivo therapy of metastatic osteosarcoma as well as the potential value of plasma EV-packaged miR-101 (EV-miR-101) level for predicting osteosarcoma metastasis. Methods: The relationship of miR-101 expression and osteosarcoma progression was investigated in osteosarcoma specimens by in situ hybridization (ISH), and the potential inhibitory effect of miR-101 was further investigated using in vivo models. Using prediction software analysis, the mechanism of action of miR-101 in osteosarcoma was explored using quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blotting and dual-luciferase assay. Adipose tissue-derived mesenchymal stromal cells (AD-MSCs) were transduced with lentiviral particles to obtain miR-101-enriched EVs. A Transwell assay and lung metastasis models of osteosarcoma were used to observe the effect of miR-101-enriched EVs on osteosarcoma invasiveness and metastasis. Detection of plasma EV-miR-101 levels was carried out in osteosarcoma patients and healthy controls by qRT-PCR. Results: miR-101 expression was markedly lower in metastatic osteosarcoma specimens compared to non-metastatic specimens. Significantly fewer metastatic lung nodules were formed by Saos-2 cells overexpressing miR-101 and SOSP-9607 cells overexpressing miR-101 injected into mice. With increased miR-101 expression, B cell lymphoma 6 (BCL6) mRNA and protein expression levels were reduced, and miR-101 was found to exert its effects by directly targeting BCL6. AD-MSCs were successfully engineered to secrete miR-101-enriched EVs. Once taken up by osteosarcoma cells, these EVs showed suppressive effects on cell invasion and migration in vitro, and systemic administration of these EVs effectively suppressed metastasis in vivo with no significant side effects. Finally, the EV-miR-101 level was lower in osteosarcoma patients than in healthy controls and even lower in osteosarcoma patients with metastasis than in those without metastasis. Conclusion: Our data support the function of miR-101 as a tumor suppressor in osteosarcoma via downregulation of BCL6. AD-MSC derived miR-101-enriched EVs represent a potential innovative therapy for metastatic osteosarcoma. EV-miR-101 also represents a promising circulating biomarker of osteosarcoma metastasis.

Spatial variations on methane emissions from Zoige alpine wetlands of Southwest China.

This study was aimed to understand the spatial variation of CH(4) emissions from alpine wetlands in Southwest China on a field-scale in two phenological seasons, namely the peak growing season and the spring thaw. Methane emission rates were measured at 30 plots, which included three kinds of environmental types: dry hummock, Carex muliensis and Eleocharis valleculosa sites. There were highly spatial variations of methane emissions among and within different environmental types in both phenological seasons. Mean methane emission rates ranged from 1.1 to 37.0 mg CH(4) m(-2) h(-1) in the peak growing season and from 0.004 to 0.691 mg CH(4) m(-)(2) h(-1) in the spring thaw. In the peak growing season, coefficients of variation (CV) averaged 38% among environmental types and 64% within environmental types; while in the spring thaw, CV were on the average 61% among environmental types and 96% within environmental types. The key influencing factors were the standing water table and the plant community height in the peak growing season, while in the spring thaw, no significant correlations between factors and methane emissions were found.