Comparison of Beta-lactamase Genes in Environmental Rahnella Isolates

Antibiotic resistance is a growing problem in the field of healthcare. Antibiotics are becoming less effective as species of bacteria adapt and share resistance mechanisms. If transmission of mechanisms can be better understood at the molecular level, inhibitors could be developed to lessen the likelihood of antibiotic resistance. In this study, Rahnella spp were isolated from environmental sources on MacConkey plates containing 100 µg ml-1 ampicillin, and confirmed by 16S rRNA gene sequencing. Whole genomic DNA was extracted from isolates and initial amplifications were performed by polymerase chain reaction (PCR) using primers specific for 16S amplification. New primers were designed based on the sequence of a β-lactamase gene identified in a Rahnella genome. These primers provided strong amplification. The products of these amplifications were sequenced, with the predicted protein products showing high sequence similarities to a previously identified Rahnella β-lactamase gene. Individual sequences were compared and found to cluster into two distinct groups, with each being distinct from the known Rahnella β-lactamase. Additional sequence data was used to determine the full sequences of this class A beta-lactamase gene predicted to be responsible for beta-lactam resistance. Primers were produced to amplify the full gene and a High Fidelity PCR Kit by Qiagen was used to amplify the gene and furthermore, sequence the full gene. Ongoing research is being conducted to understand more about the mechanism by which the class A beta-lactamase gene confers resistance and additionally how this resistance is transferred between bacteria

LIM Kinase Regulation of Cytoskeletal Dynamics is Required for Salivary Gland Branching Morphogenesis

Coordinated actin microfilament and microtubule dynamics is required for salivary gland development, although the mechanisms by which they contribute to branching morphogenesis are not defined. Because LIM kinase (LIMK) regulates both actin and microtubule organization, we investigated the role of LIMK signaling in mouse embryonic submandibular salivary glands using ex vivo organ cultures. Both LIMK 1 and 2 were necessary for branching morphogenesis and functioned to promote epithelial early- and late-stage cleft progression through regulation of both microfilaments and microtubules. LIMK-dependent regulation of these cytoskeletal systems was required to control focal adhesion protein– dependent fibronectin assembly and integrin β1 activation, involving the LIMK effectors cofilin and TPPP/p25, for assembly of the actin- and tubulin-based cytoskeletal systems, respectively. We demonstrate that LIMK regulates the early stages of cleft formation—cleft initiation, stabilization, and progression—via establishment of actin stability. Further, we reveal a novel role for the microtubule assembly factor p25 in regulating stabilization and elongation of late-stage progressing clefts. This study demonstrates the existence of multiple actin- and microtubule-dependent stabilization steps that are controlled by LIMK and are required in cleft progression during branching morphogenesis

Death of the TonB Shuttle Hypothesis

A complex of ExbB, ExbD, and TonB couples cytoplasmic membrane (CM) proton motive force (pmf) to the active transport of large, scarce, or important nutrients across the outer membrane (OM). TonB interacts with OM transporters to enable ligand transport. Several mechanical models and a shuttle model explain how TonB might work. In the mechanical models, TonB remains attached to the CM during energy transduction, while in the shuttle model the TonB N terminus leaves the CM to deliver conformationally stored potential energy to OM transporters. Previous studies suggested that TonB did not shuttle based on the activity of a GFP–TonB fusion that was anchored in the CM by the GFP moiety. When we recreated the GFP–TonB fusion to extend those studies, in our hands it was proteolytically unstable, giving rise to potentially shuttleable degradation products. Recently, we discovered that a fusion of the Vibrio cholerae ToxR cytoplasmic domain to the N terminus of TonB was proteolytically stable. ToxR–TonB was able to be completely converted into a proteinase K-resistant conformation in response to loss of pmf in spheroplasts and exhibited an ability to form a pmf-dependent formaldehyde crosslink to ExbD, both indicators of its location in the CM. Most importantly, ToxR–TonB had the same relative specific activity as wild-type TonB. Taken together, these results provide conclusive evidence that TonB does not shuttle during energy transduction. We had previously concluded that TonB shuttles based on the use of an Oregon Green® 488 maleimide probe to assess periplasmic accessibility of N-terminal TonB. Here we show that the probe was permeant to the CM, thus permitting the labeling of the TonB N-terminus. These former results are reinterpreted in the context that TonB does not shuttle, and suggest the existence of a signal transduction pathway from OM to cytoplasm

The infuence of the host microbiome on 3,4- methylenedioxymethamphetamine (MDMA)-induced hyperthermia and vice versa

Hyperthermia induced by 3,4-methylenedioxymethamphetamine (MDMA) can be life-threatening. Here, we investigate the role of the gut microbiome and TGR5 bile acid receptors in MDMA-mediated hyperthermia. Fourteen days prior to treatment with MDMA, male Sprague-Dawley rats were provided water or water treated with antibiotics. Animals that had received antibiotics displayed a reduction in gut bacteria and an attenuated hyperthermic response to MDMA. MDMA treated animals showed increased uncoupling protein 1 (UCP1)and TGR5 expression levels in brown adipose tissue and skeletal muscle while increased expression of UCP3 was observed only in skeletal muscle. Antibiotics prior to MDMA administration significantly blunted these increases in gene expression. Furthermore, inhibition of the TGR5 receptor with triamterene or of deiodinase II downstream of the TGR5 receptor with iopanoic acid also resulted in the attenuation of MDMA-induced hyperthermia. MDMA-treatment enriched the relative proportion of a Proteus mirabilis strain in the ceca of animals not pre-treated with antibiotics. These findings suggest a contributing role for the gut microbiota in MDMA-mediated hyperthermia and that MDMA treatment can trigger a rapid remodeling of the composition of the gut microbiome

Boundary Liouville theory at c=1

The c=1 Liouville theory has received some attention recently as the Euclidean version of an exact rolling tachyon background. In an earlier paper it was shown that the bulk theory can be identified with the interacting c=1 limit of unitary minimal models. Here we extend the analysis of the c=1-limit to the boundary problem. Most importantly, we show that the FZZT branes of Liouville theory give rise to a new 1-parameter family of boundary theories at c=1. These models share many features with the boundary Sine-Gordon theory, in particular they possess an open string spectrum with band-gaps of finite width. We propose explicit formulas for the boundary 2-point function and for the bulk-boundary operator product expansion in the c=1 boundary Liouville model. As a by-product of our analysis we also provide a nice geometric interpretation for ZZ branes and their relation with FZZT branes in the c=1 theory.Comment: 37 pages, 1 figure. Minor error corrected, slight change in result (1.6

A mitochondria-targeted mass spectrometry probe to detect glyoxals: implications for diabetes

The glycation of protein and nucleic acids that occurs as a consequence of hyperglycaemia disrupts cell function and contributes to many pathologies, including those associated with diabetes and aging. Intracellular glycation occurs following the generation of the reactive 1,2-dicarbonyls methylglyoxal and glyoxal and disruption to mitochondrial function is associated with hyperglycemia. However, the contribution of these reactive dicarbonyls to mitochondrial damage in pathology is unclear due to uncertainties about their levels within mitochondria in cells and in vivo. To address this we have developed a mitochondria-targeted reagent (MitoG) designed to assess the levels of mitochondrial dicarbonyls within cells. MitoG comprises a lipophilic triphenylphosphonium cationic function, which directs the molecules to mitochondria within cells and an o-phenylenediamine moiety that reacts with dicarbonyls to give distinctive and stable products. The extent of accumulation of these diagnostic heterocyclic products can be readily and sensitively quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), enabling changes to be determined. Using the MitoG-based analysis we assessed the formation of methylglyoxal and glyoxal in response to hyperglycaemia in cells in culture and in the Akita mouse model of diabetes in vivo. These findings indicated that the levels of methylglyoxal and glyoxal within mitochondria increase during hyperglycaemia in both cells and in vivo, suggesting that they can contribute to the pathological mitochondrial dysfunction that occurs in diabetes and aging

Bertotti-Robinson type solutions to Dilaton-Axion Gravity

We present a new solution to dilaton-axion gravity which looks like a rotating Bertotti-Robinson (BR) Universe. It is supported by an homogeneous Maxwell field and a linear axion and can be obtained as a near-horizon limit of extremal rotating dilaton-axion black holes. It has the isometry $SL(2,R)\times U(1)$ where U(1) is the remnant of the SO(3) symmetry of BR broken by rotation, while $SL(2,R)$ corresponds to the $AdS_2$ sector which no longer factors out of the full spacetime. Alternatively our solution can be obtained from the D=5 vacuum counterpart to the dyonic BR with equal electric and magnetic field strengths. The derivation amounts to smearing it in D=6 and then reducing to D=4 with dualization of one Kaluza-Klein two-form in D=5 to produce an axion. Using a similar dualization procedure, the rotating BR solution is uplifted to D=11 supergravity. We show that it breaks all supersymmetries of N=4 supergravity in D=4, and that its higher dimensional embeddings are not supersymmetric either. But, hopefully it may provide a new arena for corformal mechanics and holography. Applying a complex coordinate transformation we also derive a BR solution endowed with a NUT parameter.Comment: 21 page

Multiscale Feature Analysis of Salivary Gland Branching Morphogenesis

Pattern formation in developing tissues involves dynamic spatio-temporal changes in cellular organization and subsequent evolution of functional adult structures. Branching morphogenesis is a developmental mechanism by which patterns are generated in many developing organs, which is controlled by underlying molecular pathways. Understanding the relationship between molecular signaling, cellular behavior and resulting morphological change requires quantification and categorization of the cellular behavior. In this study, tissue-level and cellular changes in developing salivary gland in response to disruption of ROCK-mediated signaling by are modeled by building cell-graphs to compute mathematical features capturing structural properties at multiple scales. These features were used to generate multiscale cell-graph signatures of untreated and ROCK signaling disrupted salivary gland organ explants. From confocal images of mouse submandibular salivary gland organ explants in which epithelial and mesenchymal nuclei were marked, a multiscale feature set capturing global structural properties, local structural properties, spectral, and morphological properties of the tissues was derived. Six feature selection algorithms and multiway modeling of the data was performed to identify distinct subsets of cell graph features that can uniquely classify and differentiate between different cell populations. Multiscale cell-graph analysis was most effective in classification of the tissue state. Cellular and tissue organization, as defined by a multiscale subset of cell-graph features, are both quantitatively distinct in epithelial and mesenchymal cell types both in the presence and absence of ROCK inhibitors. Whereas tensor analysis demonstrate that epithelial tissue was affected the most by inhibition of ROCK signaling, significant multiscale changes in mesenchymal tissue organization were identified with this analysis that were not identified in previous biological studies. We here show how to define and calculate a multiscale feature set as an effective computational approach to identify and quantify changes at multiple biological scales and to distinguish between different states in developing tissues

Interleukin-1 regulates multiple atherogenic mechanisms in response to fat feeding

Background: Atherosclerosis is an inflammatory process that develops in individuals with known risk factors that include hypertension and hyperlipidaemia, influenced by diet. However, the interplay between diet, inflammatory mechanisms and vascular risk factors requires further research. We hypothesised that interleukin-1 (IL-1) signaling in the vessel wall would raise arterial blood pressure and promote atheroma. Methodology/Principal Findings: Apoe(-/-) and Apoe(-/-)/IL-1R1(-/-) mice were fed high fat diets for 8 weeks, and their blood pressure and atherosclerosis development measured. Apoe(-/-)/IL-R1(-/-) mice had a reduced blood pressure and significantly less atheroma than Apoe(-/-) mice. Selective loss of IL-1 signaling in the vessel wall by bone marrow transplantation also reduced plaque burden (p<0.05). This was associated with an IL-1 mediated loss of endothelium-dependent relaxation and an increase in vessel wall Nox 4. Inhibition of IL-1 restored endothelium-dependent vasodilatation and reduced levels of arterial oxidative stress. Conclusions/Significance: The IL-1 cytokine system links atherogenic environmental stimuli with arterial inflammation, oxidative stress, increased blood pressure and atherosclerosis. This is the first demonstration that inhibition of a single cytokine can block the rise in blood pressure in response to an environmental stimulus. IL-1 inhibition may have profound beneficial effects on atherogenesis in man