Metals and Methanotrophs: 1. Genetic and Biochemical Characterization of the Uptake and Synthesis of Methanobactin; 2. Bioinformatic Analyses of the Effect of Rare Earth Elements on Gene Expression.

Methanotrophs are a group of bacteria that use methane as their sole carbon and energy source. These microbes have various applications including methane removal, biodegradation of halogenated hydrocarbons, and valorization of methane to various products including biofuels, bioplastics, and single cell protein. Current obstacles for the application of aerobic methanotrophs include incomplete understanding of their metabolism and genetics. This work studies the methanotrophic response to metals, i.e., copper and rare earth elements, with the goal of achieving better control of methanotrophic activity. The expression and activities of alternative forms of methane monooxygenases in methanotrophs are regulated by the availability of copper. The genetic regulation by copper in methanotrophs involves in a copper-chelating molecule called methanobactin (mb) produced by methanotrophs. First, the uptake mechanism of mb was investigated. mbnT, encoding for a TonB-dependent transporter, was knocked-out in Methylosinus trichosporium OB3b. The mutant was able to synthesize and secrete mb but not take it up as evidenced by significant decrease in copper uptake when grown at presence of exogenous mb. The mutant was, however, still able to take up free copper, indicating that there is (are) alternative copper uptake pathway(s) in M. trichosporium OB3b. Second, the biosynthesitic pathway of mb was investigated. Specifically, mbnN, encoding for an aminotransferase, was disrupted in M. trichosporium OB3b. mb produced by this mutant has only one of the two oxazolone rings and the C-terminal methionine was missing. This study lays the foundation for achieving fine-tuning mb structure and for enhancing its production for potential applications. In addition to copper, it was found that cerium also regulates key enzymes in methanotrophs, i.e., alternative forms of methanol dehydrogenases (MeDHs). This finding was first extended to consider the effect of other rare earth elements (REEs). It was found that lanthanum, praseodymium, neodymium and samarium also regulate the expression of MeDHs in M. trichosporium OB3b. These effects, however, were only observed in the absence of copper, indicating cross-regulation by copper and REEs. Second, the whole transcriptomic response to copper and/or cerium in M. trichosporium OB3b was studied using transcriptomic analyses. Interestingly, the largest difference in gene expression was observed when both copper and cerium were present. Many genes of the central methane oxidation pathway, the serine cycle, and the ethylmalonyl-CoA pathway were upregulated, indicating more efficient carbon assimilation. Lastly, attempts were made to elucidate alternative mechanism(s) of copper uptake in M. trichosporium OB3b. Specifically, copCD, putatively encoding for a periplasmic copper-binding protein and an inner membrane protein, respectively, were knocked out in wildtype and a mb-defective mutant of M. trichosporium OB3b. Our results showed that these genes are not critical for copper uptake nor were they basis of copper-regulation in M. trichosporium OB3b.PHDEnvironmental EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/138475/1/gwenyu_1.pd

Methylmercury uptake and degradation by methanotrophs

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

Uptake and effect of rare earth elements on gene expression in Methylosinus trichosporium OB3b

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

Capacity Analysis and Optimization in Heterogeneous Network with Adaptive Cell Range Control

As an attractive means of expanding mobile network capacity, heterogeneous network is regarded as an important direction of mobile network evolution. To increase the capacity of, for example, hot spots, a typical scenario in heterogeneous network is that the coverage areas of low power nodes (LPNs) are overlapped with macrocell. To increase the utilization of small cells generated by LPNs, cell range extension (CRE) is used to extend the coverage of the small cells by adding cell specific offset (CSO) to small cells during cell selection procedure. The value of CSO, however, needs to be set carefully. In this paper, the capacity of users in macrocells, users in small cells, and users in range extension areas is analyzed thoroughly in conditions with and without CRE. Based on the analysis, an adaptive CSO updating algorithm is proposed. The proposed algorithm updates the CSO value periodically by predicting the overall capacity and a new CSO value is selected which can give the optimal overall capacity. The proposed algorithm is evaluated by system-level simulations. Simulation results indicate that the proposed algorithm can ensure a nearly optimal performance in all tested traffic load situations

Crystal Structure of a UBP-Family Deubiquitinating Enzyme in Isolation and in Complex with Ubiquitin Aldehyde

AbstractThe ubiquitin-specific processing protease (UBP) family of deubiquitinating enzymes plays an essential role in numerous cellular processes. HAUSP, a representative UBP, specifically deubiquitinates and hence stabilizes the tumor suppressor protein p53. Here, we report the crystal structures of the 40 kDa catalytic core domain of HAUSP in isolation and in complex with ubiquitin aldehyde. These studies reveal that the UBP deubiquitinating enzymes exhibit a conserved three-domain architecture, comprising Fingers, Palm, and Thumb. The leaving ubiquitin moiety is specifically coordinated by the Fingers, with its C terminus placed in the active site between the Palm and the Thumb. Binding by ubiquitin aldehyde induces a drastic conformational change in the active site that realigns the catalytic triad residues for catalysis

Soluble Receptor for Advanced Glycation End Product Ameliorates Chronic Intermittent Hypoxia Induced Renal Injury, Inflammation, and Apoptosis via P38/JNK Signaling Pathways

Obstructive sleep apnea (OSA) associated chronic kidney disease is mainly caused by chronic intermittent hypoxia (CIH) triggered tissue damage. Receptor for advanced glycation end product (RAGE) and its ligand high mobility group box 1 (HMGB1) are expressed on renal cells and mediate inflammatory responses in OSA-related diseases. To determine their roles in CIH-induced renal injury, soluble RAGE (sRAGE), the RAGE neutralizing antibody, was intravenously administered in a CIH model. We also evaluated the effect of sRAGE on inflammation and apoptosis. Rats were divided into four groups: (1) normal air (NA), (2) CIH, (3) CIH+sRAGE, and (4) NA+sRAGE. Our results showed that CIH accelerated renal histological injury and upregulated RAGE-HMGB1 levels involving inflammatory (NF-κB, TNF-α, and IL-6), apoptotic (Bcl-2/Bax), and mitogen-activated protein kinases (phosphorylation of P38, ERK, and JNK) signal transduction pathways, which were abolished by sRAGE but p-ERK. Furthermore, sRAGE ameliorated renal dysfunction by attenuating tubular endothelial apoptosis determined by immunofluorescence staining of CD31 and TUNEL. These findings suggested that RAGE-HMGB1 activated chronic inflammatory transduction cascades that contributed to the pathogenesis of the CIH-induced renal injury. Inhibition of RAGE ligand interaction by sRAGE provided a therapeutic potential for CIH-induced renal injury, inflammation, and apoptosis through P38 and JNK pathways

Three Capsular Polysaccharide Synthesis-Related Glucosyltransferases, GT-1, GT-2 and WcaJ, Are Associated With Virulence and Phage Sensitivity of Klebsiella pneumoniae

Klebsiella pneumoniae (K. pneumoniae) spp. are important nosocomial and community-acquired opportunistic pathogens, which cause various infections. We observed that K. pneumoniae strain K7 abruptly mutates to rough-type phage-resistant phenotype upon treatment with phage GH-K3. In the present study, the rough-type phage-resistant mutant named K7RR showed much lower virulence than K7. Liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis indicated that WcaJ and two undefined glycosyltransferases (GTs)- named GT-1, GT-2- were found to be down-regulated drastically in K7RR as compared to K7 strain. GT-1, GT-2, and wcaJ are all located in the gene cluster of capsular polysaccharide (CPS). Upon deletion, even of single component, of GT-1, GT-2, and wcaJ resulted clearly in significant decline of CPS synthesis with concomitant development of GH-K3 resistance and decline of virulence of K. pneumoniae, indicating that all these three GTs are more likely involved in maintenance of phage sensitivity and bacterial virulence. Additionally, K7RR and GT-deficient strains were found sensitive to endocytosis of macrophages. Mitogen-activated protein kinase (MAPK) signaling pathway of macrophages was significantly activated by K7RR and GT-deficient strains comparing with that of K7. Interestingly, in the presence of macromolecular CPS residues (>250 KD), K7(ΔGT-1) and K7(ΔwcaJ) could still be bounded by GH-K3, though with a modest adsorption efficiency, and showed minor virulence, suggesting that the CPS residues accumulated upon deletion of GT-1 or wcaJ did retain phage binding sites as well maintain mild virulence. In brief, our study defines, for the first time, the potential roles of GT-1, GT-2, and WcaJ in K. pneumoniae in bacterial virulence and generation of rough-type mutation under the pressure of bacteriophage

Expert consensus on postoperative rehabilitation nursing of patients with head and neck cancer

The location and size of tumors, treatment methods and prognosis of patients with head and neck cancer can seriously affect their oral function and neck activity, thereby affecting daily activities such as eating, speech and upper limb movement. Early rehabilitation after head and neck cancer surgery can accelerate functional recovery, alleviate discomfort symptoms, improve quality of life, and reduce unnecessary rehabilitation or treatment measures. Developing a clinical rehabilitation nursing pathway for head and neck cancer, forming personalized rehabilitation plans, and conducting early and effective nursing interventions are currently one of the key points of clinical work for patients with head and neck cancer. At present, domestic and foreign guidelines or consensus pays less attention to the impairments of speech function, chewing and swallowing function, neck and shoulder function etc., and lacks a systematic and comprehensive rehabilitation nursing guide or consensus to provide practical guidance for the care of patients with head and neck cancer. Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine organized relevant experts from Beijing, Shanghai, Sichuan, Shaanxi, Zhejiang and Anhui to draft Expert consensus on postoperative rehabilitation nursing of patients with head and neck cancer basing on previous literature and clinical nursing skills and experiences, of which the aim is to provide guidance for those patients in the aspects of oral care, nutritional support, flap donor area care, care after tracheotomy, chewing and swallowing rehabilitation, speech function rehabilitation, neck and shoulder function rehabilitation, restricted mouth opening rehabilitation, risk identification and prevention and follow-up