Search

The deposition of CeO2 nanoparticles on supported lipid bilayers was governed by DLVO interactions at various pH values and electrolyte concentrations.

Microbial electrosynthesis (MES) of acetate is a process using electrical energy to reduce CO2to acetic acid in an integrated bioelectrochemical system. MES powered by excess renewable electricity produces carbon-neutral acetate while benefitting from inexpensive but intermittent energy sources. Interruptions in electricity supply also cause energy limitation and starvation of the microbial cells performing MES. Here, we studied the effect of intermittent electricity supply on the performance of hydrogen-mediated MES of acetate. Thermoanaerobacter kivui produced acetic acid for more than 4 months from intermittent electricity supplied in 12 h on-off cycles in a semicontinuously-fed MES system. After current interruptions, hydrogen utilization and acetate synthesis rates were severely diminished. They did not recover to the steady-state rates of continuous MES within the 12 h current-on period under most conditions. Accumulating high product (acetate) concentration exacerbated this effect and prolonged recovery. However, supply of a low background current of 1-5% of the maximum current during "off-times" reduced the impact of current interruptions on subsequent MES performance. This study presents sustained MES at a rate of up to 2 mM h-1acetate at an average concentration of 60-90 mM by a pure thermophilic microbial culture powered by intermittent electricity. We identified product inhibition of accumulating acetic acid as a key challenge to improving the efficiency of intermittently powered MES.

Methanotrophs require copper for their activity as it plays a critical role in the oxidation of methane to methanol. To sequester copper, some methanotrophs secrete a copper-binding compound termed methanobactin (MB). MB, after binding copper, is reinternalized via a specific outer membrane TonB-dependent transporter (TBDT). Methylosinus trichosporium OB3b has two such TBDTs (MbnT1 and MbnT2) that enable M. trichosporium OB3b to take up not only its own MB (MB-OB3b) but also heterologous MB produced from other methanotrophs, e.g., MB of Methylocystis sp. strain SB2 (MB-SB2). Here, we show that uptake of copper in the presence of heterologous MB-SB2 can either be achieved by initiating transcription of

Increasing evidence suggests that long noncoding RNAs (lncRNAs) are pivotal regulators in oncogenesis. However, the role of numerous lncRNAs has never been unmasked in clear cell renal cell carcinoma (ccRCC). Presently, we investigated the function of long intergenic nonprotein coding RNA 1426 (LINC01426) in ccRCC, as The Cancer Genome Atlas data indicated that LINC01426 was highly expressed in ccRCC tissues and its overexpression was correlated with disappointing prognosis. First, we verified that LINC01426 was indeed upregulated in ccRCC cell lines and its depletion restrained ccRCC cell proliferation and migration. Besides, we proved that LINC01426 facilitated ccRCC tumorigenesis via forkhead box M1 (FOXM1). Moreover, it was revealed that miR-423-5p was downregulated and directly targeted FOXM1 in ccRCC, and that LINC01426 positively regulated FOXM1 via its inhibition on miR-423-5p. Notably, we also uncovered that miR-423-5p was transcriptionally silenced by CTBP1 and HDAC2. Of importance, LINC01426 was certified to distribute both in the cytoplasm and the nucleus of ccRCC cells, and it increased CTBP1 expression through recruiting insulin like growth factor 2 mRNA binding protein 1 (IGF2BP1) in cytoplasm whereas interacted with CTBP1 protein to improve the transcriptional repression on miR-423-5p in nucleus. Jointly, our observations unveiled that LINC01426 aggravates ccRCC progression via IGF2BP1/CTBP1/HDAC2/miR-423-5p/FOXM1 axis, highlighting LINC01426 as a novel promising target for ccRCC treatment.

As the only enzyme currently known to reduce dinitrogen (N2) to ammonia (NH3), nitrogenase is of significant interest for bio-inspired catalyst design and for new biotechnologies aiming to produce NH3 from N2. In order to reduce N2, nitrogenase must also hydrolyze at least 16 equivalents of adenosine triphosphate (MgATP), representing the consumption of a significant quantity of energy available to biological systems. Here, we review natural and engineered electron transfer pathways to nitrogenase, including strategies to redirect or redistribute electron flow in vivo towards NH3 production. Further, we also review strategies to artificially reduce nitrogenase in vitro, where MgATP hydrolysis is necessary for turnover, in addition to strategies that are capable of bypassing the requirement of MgATP hydrolysis to achieve MgATP-independent N2 reduction.

BackgroundThe transient receptor potential vanilloid (TRPV) channels family, TRPV1-6, has been identified to profoundly affect a wide spectrum of pathological processes in various cancers. However, the biological function and prognostic value of TRPVs in clear cell renal cell carcinoma (ccRCC) are still largely unknown.MethodsWe obtained the gene expression data and clinical information of 539 ccRCC patients from The Cancer Genome Atlas (TCGA) database. A series of databases were used for data processing and visualization, including GEPIA, GeneMANIA, MethSurv, GSCA, TIMER, and starBase databases.ResultsThe mRNA expression of TRPV2/3 was upregulated while the expression of TRPV5/6 was downregulated in ccRCC tumor tissues. TRPV family members in ccRCC were rarely mutated (nearly 7 frequencies). The ROC curve showed that TRPV2/5/6 had a high diagnostic ability in discriminating ccRCC from the control samples (AUC>0.9). Higher levels of TRPV3 expression were associated with poor prognosis of ccRCC patients, while higher expression of TRPV4 was associated with favorable prognosis. The expression of TRPV3 in normal and ccRCC tissues was validated by Immunohistochemistry, and its expression was remarkably related to high histologic grade and advanced stage. Besides, TRPV3 exhibit a reduction of DNA methylation level with tumor progression, and 12 CpGs of TRPV3 were associated with a significant prognosis. In addition, TRPV3 expression was significantly associated with the accumulation of several tumor-infiltrating immune cells, especially regulatory T cells. Furthermore, high levels of TRPV3 induced the expression of immune checkpoints such as LAG3, CTLA4, PDCD1, and TIGIT. Finally, we predicted a key SNHG3/AL513497.1-miR-10b-5p-TRPV3 axis linking to carcinogenesis and progression of ccRCC.ConclusionOur study may uncover TRPV channels–associated molecular mechanisms involved in the tumorigenesis and progression of ccRCC. TRPV family members might be diagnosed and prognostic markers and potential therapeutic targets for ccRCC patients.

Background Non-muscle invasive bladder cancer (NMIBC) represents the majority of bladder neoplasms. It is unusual for NMIBC metastasizing distantly without regional progression, namely metastatic NMIBC (mNMIBC), which is still poorly understood and easily omitted based on current management policies. So far, description of mNMIBC is limited to a few case reports. Methods We reported a 70-year-old man with NMIBC who suffered from cervical metastasis without pelvic recurrence at 41 months after initial diagnosis. Then we performed a collective analysis of this case together with published mNMIBC cases searched from PubMed, Embase, and Web of Science, aiming to illustrate baseline clinicopathologic parameters, metastatic patterns, and treatment outcomes of these patients and analyze associated influencing factors. Results After scrupulous review, 45 cases previous reported and the one from our center were incorporated into the aggregated cohort of mNMIBC, including 34 males and 12 females. Primary tumors from 46.7% of patients were high-grade (HG) or grade 3 (G3) and 65.1% had T1 lesions. Aberrant biomarker expression was found in tumors of some cases. Most (40/46) metastases of mNMIBC occurred at a single site, mainly in lung, bone and lymph nodes. Apart from three cases of de novo mNMIBC, the mean metastasis-free survival (MFS) interval of metachronous mNMIBC was 42.5 months, which was obviously longer than conventional metastatic bladder cancer. Shortened MFS interval was associated with old age, T1 or HG/G3 primary tumors, and non-lung metastases. Systemic chemotherapy and metastasectomy or radiotherapy for oligometastatic lesion were main therapeutic approaches of mNMIBC, and immunotherapy was adopted for the case from our center. Lung and bone metastases correlated with relatively favorable and unfavorable survival outcomes, respectively. Compared with monotherapy, chemotherapy, or immunotherapy combined with local cytoreduction got more favorable outcomes. Conclusion Although rare, mNMIBC occurs more in tumors with high-risk features. Usually, mNMIBC metastasizes later than conventional metastatic bladder cancer and manifests as solitary lesion. Outcomes of mNMIBC would be influenced by metastatic site and post-metastatic treatment. Systemic treatment combined with local cytoreduction may render survival benefit in selected patients.

Catabolic and anabolic processes are finely coordinated in microorganisms to provide optimized fitness under varying environmental conditions. Understanding this coordination and the resulting physiological traits reveals fundamental strategies of microbial acclimation. Here, we characterized the system-level physiology of Methanococcus maripaludis, a niche-specialized methanogenic archaeon, at different dilution rates ranging from 0.09 to 0.003 h

Methanobactins (MBs) are ribosomally synthesized and post-translationally modified peptides (RiPPs) produced by methanotrophs for copper uptake. The post-translational modification that define MBs is the formation of two heterocyclic groups with associated thioamines from X-Cys dipeptide sequences. Both heterocyclic groups in the MB from Methylosinus trichosporium OB3b (MB-OB3b) are oxazolone groups. The precursor gene for MB-OB3b, mbnA, which is part of a gene cluster that contains both annotated and unannotated genes. One of those unannotated genes, mbnC, is found in all MB operons, and in conjunction with mbnB, is reported to be involved in the formation of both heterocyclic groups in all MBs. To determine the function of mbnC, a deletion mutation was constructed in M. trichosporium OB3b, and the MB produced from the ΔmbnC mutant was purified and structurally characterized by UV-visible absorption spectroscopy, mass spectrometry and solution NMR spectroscopy. MB-OB3b from ΔmbnC was missing the C-terminal Met and also found to contain a Pro and a Cys in place of the pyrrolidiny-oxazolone-thioamide group. These results demonstrate MbnC is required for the formation of the C-terminal pyrrolidinyl-oxazolone-thioamide group from the Pro-Cys dipeptide, but not for the formation of the N-terminal 3-methylbutanol-oxazolone-thioamide group from the N-terminal dipeptide Leu-Cys. IMPORTANCE A number of environmental and medical applications have been proposed for MBs, including bioremediation of toxic metals, nanoparticle formation, as well as for the treatment of copper- and iron-related diseases. However, before MBs can be modified and optimized for any specific application, the biosynthetic pathway for MB production must be defined. The discovery that mbnC is involved in the formation of the C-terminal oxazolone group with associated thioamide but not for the formation of the N-terminal oxazolone group with associated thioamide in M. trichosporium OB3b suggests the enzymes responsible for post-translational modification(s) of the two oxazolone groups are not identical.

Optical density (OD) measurement is the gold standard to estimate microbial cell density in aqueous systems. Recording microbial growth curves is essential to assess substrate utilization, gauge sensitivity to inhibitors or toxins, or determine the perfect sampling point. Manual sampling for cuvette-photometer-based measurements can cause disturbances and impact growth, especially for strictly anaerobic or thermophilic microbes. For slow growing microbes, manual sampling can cause data gaps that complicate analysis. Online OD measurement systems provide a solution, but are often expensive and ill-suited for applications such as monitoring microbial growth in custom or larger anaerobic vessels. Furthermore, growth measurements of thermophilic cultures are limited by the heat sensitivity of complex electronics. Here, we present two simple, low-cost, self-assembled photometers—a “TubeOD” for online measurement of anaerobic and thermophilic cultures in Hungate tubes and a “ClampOD” that can be attached to virtually any transparent growth vessel. Both OD-meters can be calibrated in minutes. We detail the manufacturing and calibration procedure and demonstrate continuous acquisition of high quality cell density data of a variety of microbes, including strict anaerobes, a thermophile, and gas-utilizing strains in various glassware. When calibrated and operated within their detection limits (ca. 0.3–90% of the photosensor voltage range), these self-build OD-meters can be used for continuous measurement of microbial growth in a variety of applications, thereby, simplifying and enhancing everyday lab operations.

Copper is an important component of methanotrophic physiology as it controls the expression and activity of alternative forms of methane monooxygenase (MMO). To collect copper, some methanotrophs secrete a chalkophore or copper-binding compound called methanobactin (MB). MB is a ribosomally synthesized post-translationally modified polypeptide (RiPP) that, after binding copper, is collected by MbnT, a TonB-dependent transporter (TBDT). Structurally different forms of MB have been characterized, and here we show that different forms of MB are collected by specific TBDTs. Further, we report that in the model methanotroph, Methylosinus trichosporium OB3b, expression of the TBDT required for uptake of a different MB made by Methylocystis sp. strain SB2 (MB-SB2), is induced in the presence of MB-SB2, suggesting that methanotrophs have developed specific machinery and regulatory systems to actively take up MB from other methanotrophs for copper collection. Moreover, the canonical "copper-switch" in Ms. trichosporium OB3b that controls expression of alternative MMOs is apparent if one of the two TBDTs required for MB-OB3b and MB-SB2 uptake is knocked out, but is disrupted if both TBDTs are knocked out. These data indicate that MB uptake, including the uptake of exogenous MB, plays an important role in the copper switch in M. trichosporium OB3b and thus overall activity. Based on these data, we propose a revised model for the "copper-switch" in this methanotroph that involves MB uptake. IMPORTANCE In this study, we demonstrate that different TonB-dependent transporters (TBDTs) in the model methanotroph Methylosinus trichosporium OB3b are responsible for uptake of either endogenous MB or exogenous MB. Interestingly, the presence of exogenous MB induces expression of its specific TBDT in M. trichosporium OB3b, suggesting that this methanotroph is able to actively take up MB produced by others. This work contributes to our understanding of how microbes collect and compete for copper, and also helps inform how such uptake coordinates the expression of different forms of methane monooxygenase. Such studies are likely to be very important to develop a better understanding of methanotrophic interactions via synthesis and secretion of secondary metabolites such as methanobactin and thus provide additional means whereby these microbes can be manipulated for a variety of environmental and industrial purposes.

Bioelectrochemical power-to-gas presents a promising technology for long-term storage of excess renewable energy in the form of methane. The transition of the technology from laboratory to applied scale is currently challenged by low volumetric production rates, energy losses at the cathode, as well as the unknown physiology of the microbes in an electrochemical reactor. Here, we introduce a stable electromethanogenesis system based on efficient in situ hydrogen production by non-precious-metal catalysts and effective hydrogen uptake by the methanogenic microorganisms. Using NiMo-cathodes and pure cultures of hydrogenotrophic Methanococcus maripaludis, our system achieved an unprecedented volumetric methane production rate from CO2 of 1.4 L methane per L per day. The system performed stably for over 4 weeks with columbic efficiencies steadily above 90%. A physiological analysis of cells in the electromethanogenic reactor revealed robustly-growing cells with nearly identical protein expression patterns to gas-fed controls. Local pH fluctuations at the surface of cathode and cation exchange membrane resulted in a small but noticeable fraction of cell lysis. Our data collectively indicate that physiologically uncompromised cells of a pure methanogenic culture can perform methanogenesis robustly at high specific rate in a biocompatible electromethanogenic reactor using inexpensive, earth-abundant cathode materials.

Multimodal neuroimaging plays an important role in neuroscience research. Integrated noninvasive neuroimaging modalities, such as magnetoencephalography (MEG), electroencephalography (EEG) and functional near-infrared spectroscopy (fNIRS), allow neural activity and related physiological processes in the brain to be precisely and comprehensively depicted, providing an effective and advanced platform to study brain function. Noncryogenic optically pumped magnetometer (OPM) MEG has high signal power due to its on-scalp sensor layout and enables more flexible configurations than traditional commercial superconducting MEG. Here, we integrate OPM-MEG with EEG and fNIRS to develop a multimodal neuroimaging system that can simultaneously measure brain electrophysiology and hemodynamics. We conducted a series of experiments to demonstrate the feasibility and robustness of our MEG-EEG-fNIRS acquisition system. The complementary neural and physiological signals simultaneously collected by our multimodal imaging system provide opportunities for a wide range of potential applications in neurovascular coupling, wearable neuroimaging, hyperscanning and brain-computer interfaces.

Hypoxic tumor-associated macrophages (TAMs) are related to poor prognosis of patients with clear cell renal cell carcinoma (ccRCC). Exosomes are small lipid-bilayer vesicles that implicated in tumor progression and metastasis. However, whether hypoxic TAM-derived exosomes affect RCC progression within the hypoxic tumor microenvironment has not been elucidated. GSE analysis identified miR-155-5p was upregulated in RCC. Moreover, we quantified levels of miR-155-5p using RT-qPCR, performed immunohistochemical staining in 79 pairs of primary RCC specimens and related them to clinicopathological parameters. Higher miR-155-5p levels were related to more CD163 + TAM infiltration and elevated HIF-1a expression in our cohort. In the in vitro studies, we initially purified and characterized the exosomes from the supernatant of TAMs subjected to normoxia or hypoxia, and then transfected antagomiR-155-5p or control into these TAMs to produce corresponding exosomes. Gain and loss-of-function studies further investigated the effect of transferred hypoxic exosomal miR-155-5p on the cross-talk between TAMs and RCC cells in xenograft model and in vitro co-culture experiments. The results of RNA immunoprecipitation analyses elucidated that miR-155-5p could directly interact with human antigen R (HuR), thus increasing IGF1R mRNA stability. Mechanistically, hypoxic TAM-Exo transferred miR-155-5p promoted RCC progression partially through activating IGF1R/PI3K/AKT cascades. Taken together, transfer of miR-155-5p from hypoxic TAMs by exosomes to renal cancer cells explains the oncogenic manner, in which M2 macrophages confer the malignant phenotype to RCC cells by enhancing HuR-mediated mRNA stability of IGF1R.

Most microorganisms in nature spend the majority of time in a state of slow or zero growth and slow metabolism under limited energy or nutrient flux rather than growing at maximum rates. Yet, most of our knowledge has been derived from studies on fast-growing bacteria. Here, we systematically characterized the physiology of the methanogenic archaeon Methanococcus maripaludis during slow growth. M. maripaludis was grown in continuous culture under energy (formate)-limiting conditions at different dilution rates ranging from 0.09 to 0.002 h(−1), the latter corresponding to 1% of its maximum growth rate under laboratory conditions (0.23 h(−1)). While the specific rate of methanogenesis correlated with growth rate as expected, the fraction of cellular energy used for maintenance increased and the maintenance energy per biomass decreased at slower growth. Notably, proteome allocation between catabolic and anabolic pathways was invariant with growth rate. Unexpectedly, cells maintained their maximum methanogenesis capacity over a wide range of growth rates, except for the lowest rates tested. Cell size, cellular DNA, RNA, and protein content as well as ribosome numbers also were largely invariant with growth rate. A reduced protein synthesis rate during slow growth was achieved by a reduction in ribosome activity rather than via the number of cellular ribosomes. Our data revealed a resource allocation strategy of a methanogenic archaeon during energy limitation that is fundamentally different from commonly studied versatile chemoheterotrophic bacteria such as E. coli.

Most microorganisms in nature spend the majority of time in a state of slow or zero growth and slow metabolism under limited energy or nutrient flux rather than growing at maximum rates. Yet, most of our knowledge has been derived from studies on fast-growing bacteria. Here, we systematically characterized the physiology of the methanogenic archaeon Methanococcus maripaludis during slow growth. M. maripaludis was grown in continuous culture under energy (formate)-limiting conditions at different dilution rates ranging from 0.09 to 0.002 h-1, the latter corresponding to 1% of its maximum growth rate under laboratory conditions (0.23 h-1). While the specific rate of methanogenesis correlated with growth rate as expected, the fraction of cellular energy used for maintenance increased and the maintenance energy per biomass decreased at slower growth. Notably, proteome allocation between catabolic and anabolic pathways was invariant with growth rate. Unexpectedly, cells maintained their maximum methanogenesis capacity over a wide range of growth rates, except for the lowest rates tested. Cell size, cellular DNA, RNA, and protein content as well as ribosome numbers also were largely invariant with growth rate. A reduced protein synthesis rate during slow growth was achieved by a reduction in ribosome activity rather than via the number of cellular ribosomes. Our data revealed a resource allocation strategy of a methanogenic archaeon during energy limitation that is fundamentally different from commonly studied versatile chemoheterotrophic bacteria such as E. coli.

Objective We aimed to evaluate the role of tumor size in predicting tumor risk for localized prostate cancer (PCa) patients undergoing radical prostatectomy (RP). Methods Twenty‐five thousand, one hundred twenty‐seven men with PCa receiving RP from 2010 to 2015 were extracted from the Surveillance, Epidemiology, and End Results database. Kaplan–Meier plots and multivariable Cox regression analyses were used to illustrate overall survival (OS) according to the tumor size. The tumor size was confirmed by postoperative pathology after RP. Results Among overall localized PCa, 84.6% were high‐risk PCa, 9.2% were intermediate‐risk PCa, and 6.2% were low‐risk PCa. Multivariate analyses demonstrated that tumor size ≥21 mm was an independent risk predict factor of low‐risk PCa (odds ratio [OR]: 11.940; 95% CI, 9.404–15.161; p, We quantified the relationship between tumor size and localized prostate cancer grade, and hoped to play a positive role in early diagnosis and treatment of clinical work.

Unique means of copper scavenging have been identified in proteobacterial methanotrophs, particularly the use of methanobactin, a novel ribosomally synthesized, post-translationally modified polypeptide that binds copper with very high affinity. The possibility that copper sequestration strategies of methanotrophs may interfere with copper uptake of denitrifiers in situ and thereby enhance N(2)O emissions was examined using a suite of laboratory experiments performed with rice paddy microbial consortia. Addition of purified methanobactin from Methylosinus trichosporium OB3b to denitrifying rice paddy soil microbial consortia resulted in substantially increased N(2)O production, with more pronounced responses observed for soils with lower copper content. The N(2)O emission-enhancing effect of the soil’s native mbnA-expressing Methylocystaceae methanotrophs on the native denitrifiers was then experimentally verified with a Methylocystaceae-dominant chemostat culture prepared from a rice paddy microbial consortium as the inoculum. Finally, with microcosms amended with various cell numbers of methanobactin-producing Methylosinus trichosporium OB3b before CH(4) enrichment, microbiomes with different ratios of methanobactin-producing Methylocystaceae to gammaproteobacterial methanotrophs incapable of methanobactin production were simulated. Significant enhancement of N(2)O production from denitrification was evident in both Methylocystaceae-dominant and Methylococcaceae-dominant enrichments, albeit to a greater extent in the former, signifying the comparative potency of methanobactin-mediated copper sequestration, while implying the presence of alternative copper abstraction mechanisms for Methylococcaceae. These observations support that copper-mediated methanotrophic enhancement of N(2)O production from denitrification is plausible where methanotrophs and denitrifiers cohabit. IMPORTANCE Proteobacterial methanotrophs—groups of microorganisms that utilize methane as a source of energy and carbon—have been known to employ unique mechanisms to scavenge copper, namely, utilization of methanobactin, a polypeptide that binds copper with high affinity and specificity. Previously the possibility that copper sequestration by methanotrophs may lead to alteration of cuproenzyme-mediated reactions in denitrifiers and consequently increase emission of potent greenhouse gas N(2)O has been suggested in axenic and coculture experiments. Here, a suite of experiments with rice paddy soil slurry cultures with complex microbial compositions were performed to corroborate that such copper-mediated interplay may actually take place in environments cohabited by diverse methanotrophs and denitrifiers. As spatial and temporal heterogeneity allows for spatial coexistence of methanotrophy (aerobic) and denitrification (anaerobic) in soils, the results from this study suggest that this previously unidentified mechanism of N(2)O production may account for a significant proportion of N(2)O efflux from agricultural soils.

Background Recent progress in optically pumped magnetometers (OPMs) and high-temperature superconducting quantum interference devices (SQUIDs) has facilitated the development of an on-scalp magnetoencephalography (MEG) system that offers high signal intensity and flexibility at a lower cost. While the on-scalp sensor array has high flexibility, it brings new challenges to accurate sensor-to-brain coregistration, which is essential for MEG source localization. New method A novel automatic filtering algorithm based on plane segmentation was proposed to locate on-scalp MEG sensors in 3D images reconstructed from optical scanning. Global image registration was employed for the automatic alignment of anatomical images and sensor positions. Results Seventy-one sensor dummies on the scalp were located and registered to brain anatomical images. The deviations of the sensor location and orientation from the averaged result of 10 measurements were less than 1 mm and 0.6°, respectively. The entire process could be completed in less than 4 min. Comparison with existing methods Compared with existing methods that involve various manual procedures, such as moving digitizers to fiducials and repeatedly pulling out sensors, our proposed coregistration method is more efficient and accurate. Conclusion An automatic method for the coregistration of anatomical structure and on-scalp sensors that will have a large impact on the practical use of on-scalp MEG is developed.

Unique means of copper scavenging have been identified in proteobacterial methanotrophs, particularly the use of methanobactin, a novel ribosomally synthesized post-translationally modified polypeptide that binds copper with very high affinity. The possibility that copper sequestration strategies of methanotrophs may interfere with copper uptake of denitrifiers in situ and thereby enhance N2O emissions was examined using a suite of laboratory experiments performed with rice paddy microbial consortia. Addition of purified methanobactin from Methylosinus trichosporium OB3b to denitrifying rice paddy soil microbial consortia resulted in substantially increased N2O production, with more pronounced responses observed for soils with lower copper content. The N2O emission-enhancing effect of the soil’s native mbnA-expressing Methylocystaceae methanotrophs on the native denitrifiers was then experimentally verified with a Methylocystaceae-dominant chemostat culture prepared from a rice paddy microbial consortium as the inoculum. Lastly, with microcosms amended with varying cell numbers of methanobactin-producing Methylosinus trichosporium OB3b before CH4 enrichment, microbiomes with different ratios of methanobactin-producing Methylocystaceae to gammaproteobacterial methanotrophs incapable of methanobactin production were simulated. Significant enhancement of N2O production from denitrification was evident in both Methylocystaceae-dominant and Methylococcaceae-dominant enrichments, albeit to a greater extent in the former, signifying the comparative potency of methanobactin-mediated copper sequestration while implying the presence of alternative copper abstraction mechanisms for Methylococcaceae. These observations support that copper-mediated methanotrophic enhancement of N2O production from denitrification is plausible where methanotrophs and denitrifiers cohabit.ImportanceProteobacterial methanotrophs, groups of microorganisms that utilize methane as source of energy and carbon, have been known to utilize unique mechanisms to scavenge copper, namely utilization of methanobactin, a polypeptide that binds copper with high affinity and specificity. Previously the possibility that copper sequestration by methanotrophs may lead to alteration of cuproenzyme-mediated reactions in denitrifiers and consequently increase emission of potent greenhouse gas N2O has been suggested in axenic and co-culture experiments. Here, a suite of experiments with rice paddy soil slurry cultures with complex microbial compositions were performed to corroborate that such copper-mediated interplay may actually take place in environments co-habited by diverse methanotrophs and denitrifiers. As spatial and temporal heterogeneity allow for spatial coexistence of methanotrophy (aerobic) and denitrification (anaerobic) in soils, the results from this study suggest that this previously unidentified mechanism of N2O production may account for significant proportion of N2O efflux from agricultural soils.

Background Currently, the integrated curriculum of urology is still on the road of complete integration. Co-teaching, an existing concept in education, however, may be used to promote the integration of urology. Therefore, the present study was designed to explore the effect of co-teaching method compare with the traditional teaching method.Methods Co-teaching and traditional teaching in the topics hematuria and acute kidney injure (AKI) were prepared and validated. 98 undergraduate medical students of Tongji University were randomly assigned to groups A and B. Group A was taught hematuria by Co-teaching (CT) and AKI by traditional teaching (TT) and Group B was taught hematuria by TT and AKI by CT. A knowledge assessment tool of 20 multiple choice items was administered to assess the pre, post and retention knowledge scores. Change between knowledge scores was analyzed using inferential statistics. One students’ questionnaire and one tutors’ questionnaire were designed and used to investigate the perception on teaching-learning experience.Results Both traditional teaching and co-teaching were significantly effective in increasing the knowledge scores with no significant difference in learning outcomes between these two methods. However, co-teaching showed better knowledge retention compared to conventional teaching. At the same time, the feedback from students’ questionnaire showed co-teaching method better than traditional teaching; otherwise, the feedback from tutors’ questionnaire showed it needs more time and cooperation for the co-teaching. ConclusionsCo-teaching can promote the curriculum to be deeply integrated, achieve comparatively better retention of knowledge and improve the teaching effect as revealed by the findings.

Melatonin is a well-known anti-inflammatory and antioxidant molecule, which plays a crucial role in various physiological functions. In this study, mice received a single dose of 15 Gy radiation delivered to the lungs and daily intraperitoneal administration of melatonin. After 7 days, mice were processed to harvest either bronchoalveolar lavage fluid for cytokine assays or lungs for flow cytometry and histopathological studies. Herein, we showed that melatonin markedly alleviated the oxidative stress and injury, especially suppressing the infiltration of macrophages (CD11b+CD11c−) and neutrophils (CD11b+Ly6G+) to the irradiated lungs. Moreover, in the irradiated RAW 264.7 cells, melatonin blocked the NLRP3 inflammasome activation accompanied with the inhibition of the IL-1β release and caspase-1 activity. However, melatonin restored the downregulated miR-30e levels. Quantitative PCR analysis of miR-30e and NLRP3 indicated the negative correlation between them. Notably, immunofluorescence staining showed that overexpression of miR-30e dramatically diminished the increased NLRP3 expression. Luciferase reporter assay confirmed that NLRP3 was a target gene of miR-30e. Western blotting revealed that transfection with miR-30e mimics markedly reduced the expressions of NLRP3 and cleaved caspase-1, whereas this phenomenon was reversed by the miR-30e inhibitor. Consistent with this, the beneficial effect of melatonin under irradiated exposure was blunted in cells transfected with anti-miR-30e. Collectively, our results demonstrate that the NLRP3 inflammasome contributed to the pathogenesis of radiation-induced lung injury. Meanwhile, melatonin exerted its protective effect through negatively regulating the NLRP3 inflammasome in macrophages. The melatonin-mediated miR-30e/NLRP3 signaling may provide novel therapeutic targets for radiation-induced injury.

Molecular hydrogen is a major high-energy carrier for future energy technologies, if produced from renewable electrical energy. Hydrogenase enzymes offer a pathway for bioelectrochemically producing hydrogen that is advantageous over traditional platforms for hydrogen production because of low overpotentials and ambient operating temperature and pressure. However, electron delivery from the electrode surface to the enzyme's active site is often rate-limiting. Here, it is shown that three different hydrogenases from Clostridium pasteurianum and Methanococcus maripaludis, when immobilized at a cathode in a cobaltocene-functionalized polyallylamine (Cc-PAA) redox polymer, mediate rapid and efficient hydrogen evolution. Furthermore, it is shown that Cc-PAA-mediated hydrogenases can operate at high faradaic efficiency (80-100 %) and low apparent overpotential (-0.578 to -0.593 V vs. SHE). Specific activities of these hydrogenases in the electrosynthetic Cc-PAA assay were comparable to their respective activities in traditional methyl viologen assays, indicating that Cc-PAA mediates electron transfer at high rates, to most of the embedded enzymes.

Endothelial progenitor cell (EPC) has potential to attenuate pulmonary inflammation and injury. As a pivotal paracrine entity of stem cells, whether EPC-derived exosomes (EPC-Exos) contribute to acute lung injury (ALI) remains unknown. Exosomes were purified from conditional medium of EPCs, and then characterized by electron micrograph and immunoblotting. A model of ALI was induced by lipopolysaccharide (LPS) and then rats were transplanted with EPC-Exos. The underlying mechanisms of action of EPC-Exos were examined in vitro endothelial functional assays including the TEER, proliferation (CKK-8), angiogenesis and migration. A possible underlying mechanism was examined by western blotting and further animal studies. Administration of EPC-Exos ameliorated LPS-induced ALI and restored the in vivo pulmonary integrity. EPC-Exos enhanced the proliferation, migration and tube formation of the endothelial cells (ECs). Furthermore, we found that miR-126 was enriched in EPC-Exos and can be delivered onto ECs. Modification of EPCs through miR-126 knockdown can diminish their exosomes function in vitro, indicative of the abilities of EPC-Exos to protect against LPS were inherited by the horizontal shuttled miR-126. Luciferase reporter assays confirmed that miR-126 could target SPRED1. Additionally, the miR-126 transferred to target endothelial cells resulted in subsequent downregulation of SPRED1 and promoted RAF/ERK signaling pathways and subsequent improvement in endothelial cell function. Our study revealed a novel role of exosomal miRNAs in EPC-mediated therapy, suggesting that the clinical application of EPC-Exos might represent a strategy in ALI/ARDS.

Methanotrophs synthesize methanobactin, a secondary metabolite that binds copper with an unprecedentedly high affinity. Such a strategy may provide methanotrophs a "copper monopoly" that can inhibit the activity of copper-containing enzymes of other microbes, e.g., copper-dependent N2O reductases. Here, we show that methanobactin from Methylosinus trichosporium OB3b inhibited N2O reduction in denitrifiers. When Pseudomonas stutzeri DCP-Ps1 was incubated in cocultures with M. trichosporium OB3b or with purified methanobactin from M. trichosporium OB3b, stoichiometric N2O production was observed from NO3- reduction, whereas no significant N2O accumulation was observed in cocultures with a mutant defective in methanobactin production. Copper uptake by P. stutzeri DCP-Ps1 was inhibited by the presence of purified methanobactin, leading to a significant downregulation of nosZ transcription. Similar findings were observed with three other denitrifier strains. These results suggest that in situ stimulation of methanotrophs can inadvertently increase N2O emissions, with the potential for increasing net greenhouse gas emissions.

Correction for ‘In situ electrochemical H2 production for efficient and stable power-to-gas electromethanogenesis’ by Frauke Kracke et al., Green Chem., 2020, 22, 6194–6203, DOI: 10.1039/D0GC01894E.

Zinc finger and BTB domain containing 7A (ZBTB7) is a ZBTB protein family member of transcriptional repressors that serves a critical role in cell transformation and malignancy. However, the association between ZBTB7 expression in bladder cancer tissues and the prognosis of patients remains unclear. The aim of the current study was to detect the expression of ZBTB7 in transitional cell carcinoma (TCC) of the bladder and normal bladder mucous tissues to evaluate the diagnostic and prognostic value of ZBTB7 in TCC of the bladder. A total of 100 TCC specimens were analyzed and the expression of ZBTB7 mRNA was examined via reverse transcription-quantitative polymerase chain reaction. The expression of ZBTB7 protein was examined by western blotting and immunohistochemistry. The association between ZBTB7 expression and the clinical prognosis of patients from the TCGA database was analyzed. High expression of ZBTB7 mRNA and protein in TCC tissue was detected and TCC expression was significantly higher in TCC tissue than in normal bladder mucous tissues (P

Clear cell renal cell carcinoma (ccRCC) is regarded as the most aggressive subtype of RCC, with high rates of metastasis and recurrence. An extensive body of studies had proved long noncoding RNAs (lncRNAs) play pivotal parts in the development and evolution of diverse malignant tumors. However, the potential of LINC01094 in ccRCC tumorigenesis is still unexplored. In the present research, with the aid of the TCGA database, we found that LINC01094 was highly expressed in ccRCC tissues. Upregulation of LINC01094 was also confirmed in ccRCC cell lines, and functional experiments delineated that LINC01094 knockdown led to inhibition on ccRCC cell growth and metastasis. Moreover, LINC01094 was activated by FOXM1 at the transcriptional level. Further assay demonstrated that LINC01094 worked as a sponge of microRNA 224-5p (miR-224-5p) and CHSY1 was a miR-224-5p-targeted mRNA. Further, we verified that LINC01094 acted as a competing endogenous RNA in ccRCC to regulate CHSY1 expression via competitively bind to miR-224-5p. Lastly, our results expounded that LINC01094 exerted its tumor-promoting performance in ccRCC development through miR-224-5p/CHSY1 regulatory axis, which shed light on the molecular mechanism underlying LINC01094 in ccRCC and opened a new prospective for the treatment of ccRCC.

Objective We aimed to investigate the roles of the lncRNA MALAT1 in renal cell carcinoma (RCC) progression. Methods qRT‐PCR was used for the assessment of BIRC5, miRNA‐203 and MALAT1 expression. Furthermore, the targeted relationships between miR‐203 and BIRC5, as well as MALAT1 and miR‐203, were predicted by the miRanda/starBase database and verified by dual‐luciferase reporter gene assay. The effects of MALAT1, miRNA‐203 and BIRC5 on cell proliferation, cell cycle, cell apoptosis, cell invasion and cell migration were studied by using CCK‐8, flow cytometry, transwell and wound healing assays, respectively. In addition, the effects of MALAT1 on RCC tumorigenesis were evaluated in vivo by nude mouse tumorigenesis. Results The expression levels of BIRC5 and MALAT1 were higher in RCC tissues and cell lines than in adjacent normal tissues and a normal renal cortex proximal tubule epithelial cell line. In contrast, the expression of miRNA‐203 in RCC tissues and cell lines was higher than that in adjacent normal tissues and a normal renal cortex proximal tubule epithelial cell line. BIRC5 and MALAT1 promoted cell proliferation yet decreased the percentage of RCC cells at G0/G1 phase. Conclusions Our study demonstrated that MALAT1 functions as a miR‐203 decoy to increase BIRC5 expression in RCC.

Radiation-induced pulmonary fibrosis (RIPF) is a life-threatening complication of thoracic radiotherapy, which contributes to continued deterioration in pulmonary function. Sphingosine-1 phosphate receptor 3 (S1PR3) has been identified as a crucial molecule in fibrosis. Accumulating evidence indicated that the inhibition of the S1PRs ameliorates fibrogenesis. Thus, this study aims to explore whether S1PR3 participates in RIPF and elucidates the molecular mechanisms underlying S1PR3-modulated epithelial-mesenchymal transition (EMT) in transforming growth factor-β1-induced pulmonary epithelia. A recombinant adeno-associated viral-mediated S1PR3 (AAV-S1PR3) gene therapy analyzed the effect of S1PR3 gene deficiency on the altered histology structure and molecular mechanisms in the lung of mice with whole-lung irradiation. Compared with the AAV-negative control mice, AAV-mediated S1PR3 knockdown in the lung of mice attenuated pulmonary fibrosis induced by the radiation, as indicated by the alleviation of collagen accumulation, lessened histopathological alterations, and the suppression of inflammatory cells infiltration. S1PR3 deficiency reversed the RIPF concomitantly with abrogated EMT-related protein (α-smooth muscle actin). Consistently, S1PR3-deficient pulmonary epithelia inhibited the EMT process changes and fibrosis formation. Furthermore, S1PR3 was designated as one of the target genes for microRNA-495-3p (miR-495-3p). The inhibition of miR-495-3p promoted the expression of S1PR3 in pulmonary epithelia, whereas the overexpression of miR-495-3p inhibited the S1PR3/SMAD2/3 pathway and suppressed the EMT process. Collectively, miR-495-3p might be a negative regulator of the EMT process in fibrosis formation by inhibiting the targeted S1PR3 gene. These results established a link between the S1PR3 gene, the EMT process, and the fibrosis, suggesting the pharmacological blockage of S1PR3 as a potential therapeutic strategy for RIPF.

Methanotrophs have remarkable redundancy in multiple steps of the central pathway of methane oxidation to carbon dioxide. For example, it has been known for over 30 years that two forms of methane monooxygenase, responsible for oxidizing methane to methanol, exist in methanotrophs, i.e., soluble methane monooxygenase (sMMO) and particulate methane monooxygenase (pMMO), and that expression of these two forms is controlled by the availability of copper. Specifically, sMMO expression occurs in the absence of copper, while pMMO expression increases with increasing copper concentrations. More recently, it was discovered that multiple forms of methanol dehydrogenase (MeDH), Mxa MeDH and Xox MeDH, also exist in methanotrophs and that the expression of these alternative forms is regulated by the availability of cerium. That is, expression of Xox MeDH increases in the presence of cerium, while Mxa MeDH expression decreases in the presence of cerium. As it had been earlier concluded that pMMO and Mxa MeDH form a supercomplex in which electrons from Mxa MeDH are back donated to pMMO to drive the initial oxidation of methane, we speculated that Mxa MeDH could be rendered inactive through marker-exchange mutagenesis but growth on methane could still be possible if cerium was added to increase the expression of Xox MeDH under sMMO-expressing conditions. Here we report that mxaF , encoding the large subunit of Mxa MeDH, could indeed be knocked out in Methylosinus trichosporium OB3b, yet growth on methane was still possible, so long as cerium was added. Interestingly, growth of this mutant occurred in both the presence and the absence of copper, suggesting that Xox MeDH can replace Mxa MeDH regardless of the form of MMO expressed.

The purpose of the present meta-analysis was to evaluate the efficacy and safety of testosterone replacement therapy in men with hypogonadism. A search was conducted for appropriate randomized controlled trials and the data from 16 trials were pooled. The intended primary outcome of the present study was to determine the efficacy and safety of testosterone replacement therapy. The current data demonstrated that scores for Aging Male Symptoms (AMS) were significantly reduced following testosterone replacement therapy, with a mean decrease in AMS score of 1.52 [95% confidence interval (CI), 0.72 to 2.32; P=0.0002]. Testosterone replacement therapy increased lean body mass [mean difference (MD), 1.22; 95% CI, 0.33 to 2.11; P=0.007], reduced fat mass in a non-significantly manner (MD, −0.85; 95% CI, −1.74 to 0.04; P=0.06) and significantly reduced total cholesterol (MD, −0.16; 95% CI, −0.29 to −0.03; P=0.01). No significant differences were identified in body weight (MD, 0.09; 95% CI, −1.13 to 1.31; P=0.89), body mass index (MD, 0.10; 95% CI, −0.62 to 0.82; P=0.78) or bone mineral density (MD, −0.01; 95% CI, −0.03 to 0.02; P=0.60). Average prostate volume increased (MD, 1.58; 95% CI, 0.6 to 2.56; P=0.002) following testosterone replacement therapy, but the levels of prostate-specific antigen (PSA) (MD, 0.10; 95% CI, −0.03 to 0.22; P=0.14) and the International Prostate Symptom Scores (MD, 0.01; 95% CI, −0.37 to 0.39; P=0.96) did not change. In conclusion, testosterone replacement therapy improves quality of life, increases lean body mass, significantly decreases total cholesterol, and is well-tolerated and safe for men with hypogonadism who are exhibiting PSA levels of

Although emerging evidence demonstrates increased circulating endothelial progenitor cells in patients with solid tumors, to our knowledge it is still unknown whether such cells can be cultured from patients with highly angiogenic renal cell carcinoma. We cultured and characterized circulating endothelial progenitor cells from patients with renal cell carcinoma.The circulating endothelial progenitor cell level (percent of CD45(-)CD34(+) VEGF-R2(+) cells in total peripheral blood mononuclear cells) was quantified in 47 patients with renal cell carcinoma and 40 healthy controls. Peripheral blood mononuclear cells were then isolated from 33 patients with renal cell carcinoma and 30 healthy controls to culture and characterize circulating endothelial progenitor cells.The circulating endothelial progenitor cell level was significantly higher in patients with renal cell carcinoma than in healthy controls (0.276% vs 0.086%, p0.001). A colony of circulating endothelial progenitor cells first emerged significantly earlier in patient than in control preparations (6.72 vs 14.67 days, p0.001). The culture success rate (87.8% vs 40.0% of participants) and the number of colonies (10.06 vs 1.83) were significantly greater for patients than for controls (each p0.001). The circulating endothelial progenitor cell level correlated positively with the number of patient colonies (r = 0.762, p0.001). Cells cultured from patients and controls showed a similar growth pattern, immunophenotype, ability to uptake Ac-LDL and bind lectin, and form capillary tubes in vitro. However, significantly more VEGF-R2(+) circulating endothelial progenitor cells were found in preparations from patients with renal cell carcinoma than from healthy controls (21.1% vs 13.4%, p0.001).Earlier emergence of circulating endothelial progenitor cell colonies, a higher cell culture success rate and more colonies were found for patients with renal cell carcinoma than for healthy controls. Results indicate the important significance of VEGF-R2(+) circulating endothelial progenitors in patients with renal cell carcinoma.

Chronic intermittent hypoxia (CIH), as the foremost pathophysiological change of obstructive sleep apnea (OSA), contributes to continued deterioration in renal function. Nucleotide-binding domain like receptor protein 3 (NLRP3) inflammasome is a multiprotein complex that triggers innate immune responses to infection and cell stress through activation of caspase-1 and maturation of inflammatory pro-interleukin-1β cytokine. Emerging evidence indicates that inhibition of the NLRP3 inflammasome ameliorates renal injury. Nevertheless, it is uncertain whether NLRP3 inflammasome participates in CIH-induced renal injury. The molecular mechanisms modulating NLRP3 inflammasome activation remain to be elucidated. Compared with wild-type mice, NLRP3 knockout mice dramatically protected them from kidney injury, as indicated by the restoration of creatinine levels, lessened histopathological alterations, and the suppression of macrophages infiltration stained with F4/80. NLRP3 deficiency notably reversed CIH-induced oxidative stress (malondialdehyde and superoxide dismutase), concomitantly with the abrogated apoptosis-related proteins and proinflammatory signaling pathway. Consistently, NLRP3-deficient tubular cells remarkably inhibited reactive oxygen species generation and NLRP3 inflammasome activation. Furthermore, our study revealed that microRNA-155 (miR-155) was augmented in the renal tissue and HK-2 cells exposed to CIH. In addition, we investigated the role of miR-155 in the regulation of NLRP3 inflammasome. Inhibition of miR-155 suppressed the CIH-induced NLRP3 inflammasome activation in renal tubular cells, whereas overexpression of miR-155 promoted oxidation and enhanced NLRP3 pathway. Collectively, we demonstrated that miR-155 might be a positive-regulator of NLRP3 pathway by inhibiting the targeted FOXO3a gene. These results established a link between the miR-155/FOXO3a pathway and the NLRP3 inflammasome, suggesting pharmacological blockage of NLRP3 as a potential therapeutic strategy for OSA-associated chronic kidney disease.

Methanotrophs synthesize methanobactin, a secondary metabolite that binds copper with an unprecedentedly high affinity. Such a strategy may provide methanotrophs a “copper monopoly” that can inhibit the activity of copper-containing enzymes of other microbes, e.g., copper-dependent N(2)O reductases. Here, we show that methanobactin from Methylosinus trichosporium OB3b inhibited N(2)O reduction in denitrifiers. When Pseudomonas stutzeri DCP-Ps1 was incubated in cocultures with M. trichosporium OB3b or with purified methanobactin from M. trichosporium OB3b, stoichiometric N(2)O production was observed from NO(3)(−) reduction, whereas no significant N(2)O accumulation was observed in cocultures with a mutant defective in methanobactin production. Copper uptake by P. stutzeri DCP-Ps1 was inhibited by the presence of purified methanobactin, leading to a significant downregulation of nosZ transcription. Similar findings were observed with three other denitrifier strains. These results suggest that in situ stimulation of methanotrophs can inadvertently increase N(2)O emissions, with the potential for increasing net greenhouse gas emissions.

In aerobic methanotrophs, copper and cerium control the expression and activity of different forms of methane monooxygenase and methanol dehydrogenase, respectively. To exploit methanotrophy for the valorization of methane, it is crucial to determine if these metals exert more global control on gene expression in methanotrophs. Using RNA-Seq analysis we compared the transcriptome of Methylosinus trichosporium OB3b grown in the presence of varying amounts of copper and cerium. When copper was added in the absence of cerium, expression of genes encoding for both soluble and particulate methane monooxygenases varied as expected. Genes encoding for copper uptake, storage, and efflux also increased, indicating that methanotrophs must carefully control copper homeostasis. When cerium was added in the absence of copper, expression of genes encoding for alternative methanol dehydrogenases varied as expected, but few other genes were found to have differential expression. When cerium concentrations were varied in the presence of copper, few genes were found to be either up- or downregulated, indicating that copper over rules any regulation by cerium. When copper was increased in the presence of cerium, however, many genes were upregulated, most notably multiple steps of the central methane oxidation pathway, the serine cycle, and the ethylmalonyl-CoA pathway. Many genes were also downregulated, including those encoding for nitrogenase and hydrogenase. Collectively, these data suggest that copper plays a larger role in regulating gene expression in methanotrophs, but that significant changes occur when both copper and cerium are present.

Certain methanotrophs can take up and degrade methylmercury, signifying a potentially important demethylation pathway in the environment., Methylmercury (CH3Hg+) is a potent neurotoxin produced by certain anaerobic microorganisms in natural environments. Although numerous studies have characterized the basis of mercury (Hg) methylation, no studies have examined CH3Hg+ degradation by methanotrophs, despite their ubiquitous presence in the environment. We report that some methanotrophs, such as Methylosinus trichosporium OB3b, can take up and degrade CH3Hg+ rapidly, whereas others, such as Methylococcus capsulatus Bath, can take up but not degrade CH3Hg+. Demethylation by M. trichosporium OB3b increases with increasing CH3Hg+ concentrations but was abolished in mutants deficient in the synthesis of methanobactin, a metal-binding compound used by some methanotrophs, such as M. trichosporium OB3b. Furthermore, addition of methanol (>5 mM) as a competing one-carbon (C1) substrate inhibits demethylation, suggesting that CH3Hg+ degradation by methanotrophs may involve an initial bonding of CH3Hg+ by methanobactin followed by cleavage of the C–Hg bond in CH3Hg+ by the methanol dehydrogenase. This new demethylation pathway by methanotrophs indicates possible broader involvement of C1-metabolizing aerobes in the degradation and cycling of toxic CH3Hg+ in the environment.

Methanotrophs or methane-oxidizing bacteria exhibit a unique 'copper-switch' where expression of two forms of methane monooxygenase (MMO) is controlled by the availability of copper. In the absence of copper, a cytoplasmic or soluble methane monooxygenase (sMMO) is expressed. In the presence of copper, a membrane-bound or particulate methane monooxygenase (pMMO) is expressed. These two forms of MMO have very different properties, and elucidation of the basis of the copper-switch is of significant interest as methanotrophs are becoming increasingly popular for the valorization of methane. Recently, it was suggested via characterization of a mutant of Methylosinus trichosporium OB3b that expresses sMMO in the presence of copper (smmoC mutant) that the copper-switch may be based on copCD. These genes encode for a periplasmic copper-binding protein and an inner membrane protein, respectively, and are used by other bacteria for copper uptake. Specific knockouts of copCD in M. trichosporium OB3b wild type, however, show that these genes are not part of the copper-switch in methanotrophs, nor do they appear to be critical for copper uptake. Rather, it appears that the constitutive expression of sMMO in the smmoC mutant of M. trichosporium OB3b may be due to multiple lesions as smmoC was generated via random chemical mutagenesis.

In an effort to contribute in further reducing pollution and fuel consumption, the effects of various combustion modes on the combustion process and emissions were investigated on a modified single cylinder from a six-cylinder engine using diesel, gasoline, and diesel from direct coal liquefaction (DDCL). The engine was run at 1000 r/min with an indicated mean effective pressure (IMEP) of 0.7 MPa. Additionally, we experimentally evaluated the impact of exhaust gas recirculation (EGR) and did comparisons among the tested fuels. Gasoline was injected into the intake port to form a homogeneous mixture and compression ignition, which led to homogeneous charge compression ignition (HCCI). Diesel was injected near top dead center (TDC), which was dominated by diffused combustion depending on the mixing rate of diesel and air. DDCL was also injected near TDC. In comparison with diesel, DDCL has a longer ignition delay (ID) than diesel due to low cetane number and high volatility. It also had a heat release rate between that of gasoline and diesel. Among the tested fuels, gasoline displayed the highest gross indicated thermal efficiency, followed by DDCL, and then diesel. Moreover, emission test results showed that gasoline had the lowest NOx and soot emissions, but had the highest CO and HC emissions. Different particle modes were present under different EGR conditions. For instance, at an EGR ratio lower than 20%, the particles of the tested fuels were composed mainly of nucleation-mode particles. When the EGR ratio was higher than 20%, the particles of DDCL and diesel were of accumulation-mode, whereas those of gasoline were of small-diameter nucleation-mode.

Objective This study aimed at detecting Pokemon expression in bladder cancer cell and investigating the relationship between Pokemon and epithelial–mesenchymal transition. Furthermore, we investigated the functions of Pokemon in the carcinogenesis and development of bladder cancer. This study was also designed to observe the inhibitory effects of siRNA expression vector on Pokemon in bladder cancer cell. Methods The siRNA expression vectors which were constructed to express a short hairpin RNA against Pokemon were transfected to the bladder cancer cells T24 with a liposome. Levels of Pokemon, E-cadherin and β-catenin mRNA and protein were examined by real-time quantitative-fluorescent PCR and Western blot analysis, respectively. The effects of Pokemon silencing on epithelial–mesenchymal transition of T24 cells were evaluated with wound-healing assay. Results Pokemon was strongly inhibited by siRNA treatment, especially siRNA3 treatment group, as it was reflected by Western blot and real-time PCR. The gene and protein of E-cadherin expression level showed increased markedly after Pokemon was inhibited by RNA interference. While there were no differences in the levels of gene and protein of β-catenin among five groups. The bladder cancer cell after Pokemon siRNA interference showed a significantly reduced wound-closing efficiency at 6, 12 and 24 h. Conclusions Our findings suggest Pokemon may inhibit the expression of E-cadherin. The low expression of E-cadherin lead to increasing the phenotype and apical-base polarity of epithelial cells. These changes of cells may result in the recurrence and progression of bladder cancer at last.

The bacterial anti-adhesive properties of polysulfone (PSU) microfiltration membranes modified with poly(allylamine hydrochloride) (PAH)/poly(acrylic acid) (PAA) polyelectrolyte multilayers (PEMs) were investigated in this study. Using a direct microscopic observation membrane filtration system, the deposition kinetics of Escherichia coli cells on the membrane surfaces, as well as the reversibility of bacterial deposition, were examined in the absence and presence of calcium. The PEM-modified membranes exhibited significantly improved bacterial anti-adhesive properties compared to the PSU base membranes in both the tested solution chemistries. Specifically, the bacterial deposition kinetics on the modified membranes were slower than the deposition kinetics on the base membranes. Furthermore, the bacterial removal efficiency was significantly enhanced from o10% to as high as 99% after PEM modification. Interaction force measurements conducted through atomic force microscopy revealed strong, long-ranged repulsive interactions between a carboxylate modified latex colloid probe and PEMmodified membranes, while attractive interactions were detected between the colloid probe and PSU base membranes. The bacterial anti-adhesive properties exhibited by the PEM-modified membranes were attributed to the highly swollen and hydrated PEMs that inhibit the direct contact or close approach of bacteria to the underlying PSU membranes.

Gene expression in methanotrophs has been shown to be affected by the availability of a variety of metals, most notably copper-regulating expression of alternative forms of methane monooxygenase. A copper-binding compound, or chalkophore, called methanobactin plays a key role in copper uptake in methanotrophs. Methanobactin is a ribosomally synthesized and posttranslationally modified peptide (RiPP) with two heterocyclic rings with an associated thioamide for each ring, formed from X-Cys dipeptide sequences that bind copper. The gene coding for the precursor polypeptide of methanobactin, mbnA , is part of a gene cluster, but the role of other genes in methanobactin biosynthesis is unclear. To begin to elucidate the function of these genes, we constructed an unmarked deletion of mbnABCMN in Methylosinus trichosporium OB3b and then homologously expressed mbnABCM using a broad-host-range cloning vector to determine the function of mbnN , annotated as coding for an aminotransferase. Methanobactin produced by this strain was found to be substantially different from wild-type methanobactin in that the C-terminal methionine was missing and only one of the two oxazolone rings was formed. Rather, in place of the N-terminal 3-methylbutanoyl-oxazolone-thioamide group, a leucine and a thioamide-containing glycine (Gly-Ψ) were found, indicating that MbnN is used for deamination of the N-terminal leucine of methanobactin and that this posttranslational modification is critical for closure of the N-terminal oxazolone ring in M. trichosporium OB3b. These studies provide new insights into methanobactin biosynthesis and also provide a platform for understanding the function of other genes in the methanobactin gene cluster. IMPORTANCE Methanotrophs, microbes that play a critical role in the carbon cycle, are influenced by copper, with gene expression and enzyme activity changing as copper levels change. Methanotrophs produce a copper-binding compound, or chalkophore, called methanobactin for copper uptake, and methanobactin plays a key role in controlling methanotrophic activity. Methanobactin has also been shown to be effective in the treatment of Wilson disease, an autosomal recessive disorder where the human body cannot correctly assimilate copper. It is important to characterize the methanobactin biosynthesis pathway to understand how methanotrophs respond to their environment as well as to optimize the use of methanobactin for the treatment of copper-related diseases such as Wilson disease. Here we show that mbnN , encoding an aminotransferase, is involved in the deamination of the N-terminal leucine and necessary for the formation of one but not both of the heterocyclic rings in methanobactin that are responsible for copper binding.

In this study, we investigate the expression and role of special AT-rich sequence-binding protein-2 (SATB2) in clear cell renal cell carcinoma (ccRCC) tissue, and to evaluate the clinical and prognostic significance of SATB2 protein in patients with ccRCC. The expression of SATB2 and SATB1 was examined in ccRCC tissue by Western blotting, real-time PCR and immunohistochemical staining. The association between clinicopathological features and SATB2 level was investigated. The correlation of SATB2 expression with overall survival was also analyzed. The expression of SATB2 protein in tumor tissues was much lower than that in paired normal tissues. The overall survival of the patients with high SATB2 expression was significantly higher than that of the low SATB2 expression group. Low or negative SATB2 expression was significantly correlated with AJCC staging and Furman grade in ccRCC. In contrast, the expression of SATB1 was significantly higher in adjacent tumor tissue than that in normal and tumor tissues. This study provides the first evidence of the expression and clinical significance of SATB2 in ccRCC. Our data suggest that SATB2 functions as a tumor suppressor in the development and progression of ccRCC, and is thereby implicated as a valuable prognostic marker for ccRCC patients.

It is well known that Methylosinus trichosporium OB3b has two forms of methane monooxygenase (MMO) responsible for the initial conversion of methane to methanol, a cytoplasmic (soluble) methane monooxygenase and a membrane-associated (particulate) methane monooxygenase, and that copper strongly regulates expression of these alternative forms of MMO. More recently, it has been discovered that M. trichosporium OB3b has multiple types of the methanol dehydrogenase (MeDH), i.e. the Mxa-type MeDH (Mxa-MeDH) and Xox-type MeDH (Xox-MeDH), and the expression of these two forms is regulated by the availability of the rare earth element (REE), cerium. Here, we extend these studies and show that lanthanum, praseodymium, neodymium and samarium also regulate expression of alternative forms of MeDH. The effect of these REEs on MeDH expression, however, was only observed in the absence of copper. Further, a mutant of M. trichosporium OB3b, where the Mxa-MeDH was knocked out, was able to grow in the presence of lanthanum, praseodymium and neodymium, but was not able to grow in the presence of samarium. Collectively, these data suggest that multiple levels of gene regulation by metals exist in M. trichosporium OB3b, but that copper overrides the effect of other metals by an as yet unknown mechanism.

The substrate-reducing proteins of all nitrogenases (MoFe, VFe, and FeFe) are organized as alpha 2ss2(gamma 2) multimers with two functional halves. While their dimeric organization could afford improved structural stability of nitrogenases in vivo, previous research has proposed both negative and positive cooperativity contributions with respect to enzymatic activity. Here, a 1.4 kDa peptide was covalently introduced in the proximity of the P cluster, corresponding to the Fe protein docking position. The Strep-tag carried by the added peptide simultaneously sterically inhibits electron delivery to the MoFe protein and allows the isolation of partially inhibited MoFe proteins (where the half-inhibited MoFe protein was targeted). We confirm that the partially functional MoFe protein retains its ability to reduce N2 to NH3, with no significant difference in selectivity over obligatory/parasitic H2 formation. Our experiment concludes that wild-type nitrogenase exhibits negative cooperativity during the steady state regarding H2 and NH3 formation (under Ar or N2), with one-half of the MoFe protein inhibiting turnover in the second half. This emphasizes the presence and importance of long-range (>95 angstrom) protein-protein communication in biological N2 fixation in Azotobacter vinelandii.