Mycobacterium tuberculosis (Mtb) is a major cause of morbidity and mortality worldwide. Slow progress has been made in lessening the impact of tuberculosis (TB) on human health, especially in parts of the world where Mtb is endemic. Due to the complexity of TB disease, there is still an urgent need to improve diagnosis, prevention, and treatment strategies to control global spread of disease. Active research targeting avenues to prevent infection or transmission through vaccination, to diagnose asymptomatic carriers of Mtb, and to improve antimicrobial drug treatment responses is ongoing. However, this research is hampered by a relatively poor understanding of the pathogenesis of early infection and the factors that contribute to host susceptibility, protection, and the development of active disease. There is increasing interest in the development of adjunctive therapy that will aid the host in responding to Mtb infection appropriately thereby improving the effectiveness of current and future drug treatments. In this review, we summarize what is known about the host response to Mtb infection in humans and animal models and highlight potential therapeutic targets involved in TB granuloma formation and resolution. Strategies designed to shift the balance of TB granuloma formation toward protective rather than destructive processes are discussed based on our current knowledge. These therapeutic strategies are based on the assumption that granuloma formation, although thought to prevent the spread of the tubercle bacillus within and between individuals contributes to manifestations of active TB disease in human patients when left unchecked. This effect of granuloma formation favors the spread of infection and impairs antimicrobial drug treatment. By gaining a better understanding of the mechanisms by which Mtb infection contributes to irreversible tissue damage, down regulates protective immune responses, and delays tissue healing, new treatment strategies can be rationally designed. Granuloma-targeted therapy is advantageous because it allows for the repurpose of existing drugs used to treat other communicable and non-communicable diseases as adjunctive therapies combined with existing and future anti-TB drugs. Thus, the development of adjunctive, granuloma-targeted therapy, like other host-directed therapies, may benefit from the availability of approved drugs to aid in treatment and prevention of TB. In this review, we have attempted to summarize the results of published studies in the context of new innovative approaches to host-directed therapy that need to be more thoroughly explored in pre-clinical animal studies and in human clinical trials.Infection by the intracellular bacterial pathogen  Mycobacterium tuberculosis (Mtb), the primary causative agent of human tuberculosis (TB), remains a prominent global health concern, despite a decline in total incident cases and mortality within the last decade [t decade . CurrentThe host response to Mtb infection can be broadly characterized as chronic granulomatous inflammation, which is thought to contain bacilli at the site of infection and prevent dissemination within and between susceptible individuals. The clinical manifestations of active Mtb infection are complex and include an aggressive cellular and humoral immune response aimed at eradicating difficult to kill bacilli as well as clearing persistent bacterial antigens , 3. HoweThe factors involved in the control of TB granuloma formation are poorly understood, but involve adaptations by both the host and pathogen to a changing microenvironment. Alveolar macrophages are thought to be among the first and most important innate host defenses at the site of primary infection in the lung and the most important effector cells encountered by Mtb following aerosol exposure. It is generally believed that the ability of bacilli to survive intracellular killing by macrophages is the initial step in the establishment of Mtb infection . Mtb parWith the establishment of Mtb infection, expansion of lesions, and progression of disease, granulomas enlarge and undergo alterations in their morphological features. These processes increase the potential for bacilli to spread from the primary site of infection, the lung, to other tissues and organs. During the progression of the inflammatory response, particularly within the relatively high oxygen concentrations in the lung, both Mtb bacilli and host cells undergo a variety of metabolic adaptations in response to immune stimulation. In vitro model systems have clearly demonstrated how limiting oxygen levels alters Mtb metabolism and drug antimicrobial susceptibility , 9. HoweAnother hallmark of progressive TB disease, aside from organized granuloma formation, is the development of lesions with central necrosis and cavitation. The appearance of lesions with these morphotypes represents an important transition toward not only irreversible tissue damage in some patients but also the establishment of extracellular populations of Mtb. Extracellular Mtb populations are further isolated from immune effector cells and are functionally tolerant of antimicrobial drug treatment. Most granulomatous lesions share fundamental structural similarities, but differences in the rate of progression and transformation accounts for granuloma heterogeneity within and between patients, equating to variable disease progression and response to treatment. Shown in humans and recently in animal models, heterogeneous patterns of disease evolve as a result of differences in Mtb virulence, host immune status, and the absence of communicable and non-communicable TB risk factors –15. WithAfter the release of intracellular bacilli from infected cells, extracellular organisms become attached or enmeshed within a complex matrix composed of host- and pathogen-derived macromolecules. The attachment and colonization of Mtb within these complex extracellular polymeric substances has been equated to biofilm formation, similar to other pathogenic bacteria. However, this concept remains controversial , 16, 17.Currently, two major frameworks exist around the discussion of the role of the granuloma in TB pathogenesis and treatment. The granuloma is either considered a critical component of the protective cellular immune response, serving a vital role in pathogen containment, or is considered detrimental, contributing to the clinical manifestations of active TB disease and persistence of Mtb. The protective view of granuloma formation stems from the predominance of macrophages and T and B lymphocytes at the site of infection, which are thought to have important effector functions against intracellular Mtb. These cellular infiltrates along with fibrous encapsulation are thought to form a mechanical and functional barrier to prevent bacilli dissemination within and between hosts . HoweverThe debate over the protective or destructive result of granuloma formation has thus driven recent investigations into the use of host-directed and specifically granuloma-targeted therapy to either promote natural immunity and healing or to limit the tissue damaging consequences of advanced granuloma formation. Excessive pro-inflammatory responses are unfavorable in the early stages of infection because they result in extensive tissue damage prior to the development of Mtb-specific adaptive immunity. This leads to unregulated inflammation and further tissue damage, creating a microenvironment which promotes the persistence of non-replicating bacilli. In contrast, killing of intracellular bacilli by macrophages as well as maintaining a balanced inflammatory response early during infection contributes to the establishment of more effective adaptive immune protection. Studies have suggested that tight regulation of the cytokines interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) is important in coordinating protective granuloma formation –29. StudThe cytokine TNFα in particular has a dose-dependent effect on lesion morphology . The absHistorically, treatments specifically targeting the TB granulomas consisted of surgical-based techniques, practiced most commonly in the pre-antibiotic era. TB patients that were considered candidates for surgical intervention generally shared in common lesions with extensive tissue damage often with lesion necrosis or cavitation. Collapse therapy was among the first interventional adjunctive granuloma-targeted therapies with the potential to improve patient survival and overall TB treatment outcomes. Collapse therapy encompassed many procedures, including pneumothorax , pneumoperitoneum , phrenic crush , thoracoplasty , and extrapleural lucite pack  . These tSurgical lung re-sectioning of portions or entire lung lobes referred to as a lobectomy is also a common adjunctive treatment, particularly in patients that fail to respond to conventional antimicrobial therapy. The overall goal of removing entire lung lobes is to physically reduce the bacterial and lesion burden and therefore regions of the lung that were most refractory to antimicrobial treatment. Similar to collapse therapy, surgical debulking of damaged tissue aids in re-establishing vascular perfusion and healing of more normal tissue to improve drug treatment responses and patient survival. The development of technological advances including video-assisted thoracoscopic approaches enable more efficient and selective removal of lesions while at the same time reducing the incidence of postoperative complications . With thCorticosteroids were explored as a potential host-directed therapeutic based on their anti-inflammatory properties. Recent meta-analysis indicates that 17 % of TB-associated mortality can be reduced with the use of corticosteroid therapy. However, when sensitivity analysis was conducted by the same authors on cases of pulmonary TB alone, there was no significant reduction in mortality when corticosteroids were used . Prospec14C labeled INH, and others more recently using advanced mass spectrometry-based techniques, demonstrate that antimicrobial drug penetration is severely impaired in TB lesions with necrosis or cavitation [The discovery of the anti-TB drug isoniazid (INH) led to some of the first studies suggesting that poor antimicrobial treatment responses were in part due to poor drug penetration . These svitation , 62. As vitation . The hetvitation . In the vitation , 13. Thivitation . The usevitation .The inability of antimicrobial drugs to reach therapeutic levels within granulomatous lesions not only accounts for poor treatment responses but also has the potential to promote the development of drug-tolerant Mtb phenotypes and contribute to the emergence of drug-resistant strains of Mtb. The failure of antimicrobial drugs to reach therapeutic concentrations may have a similar effect to improper dosing or patient non-compliance. Therefore, an additional host-targeted therapy aimed at promoting granuloma vasculature perfusion also has the potential to improve drug treatment responses. Since oral or parenterally administered drugs must first reach therapeutic blood levels, there is a direct correlation between peripheral blood drug levels and what can be achieved within the TB lesion. As a consequence, lesion vascular integrity and blood perfusion have the potential to significantly impact therapeutic effectiveness of antimicrobial drugs. Granuloma targeted therapy can be designed to either promote or inhibit new vessel formation (angiogenesis) depending on the stage of disease and the overall treatment objective. The potential of granuloma therapy targeting blood vasculature is best illustrated from the advances made in the treatment of cancer. Although no live pathogen is associated with cancer development in this case, the gap in knowledge regarding immunological and metabolic changes that occur within the granuloma microenvironment necessitates comparisons to be drawn from this area of study.Inhibition of tumor microvasculature serves as the basis of numerous cancer-based therapies designed to inhibit tumor-associated new vessel formation, which contributes to the growth and spread of neoplastic cells. Since tumor metastasis requires an intact blood supply, and neoplastic cells require the expression of proangiogenic factors to grow and spread, one approach to cancer therapy is to restrict growth and metastatic potential by starving tumors of oxygen and nutrients through inhibiting angiogenesis . DespiteMycobacterium avium-infected mice [M. avium showed alteration of lung vasculature in conjunction with changing tissue architecture, resulting in marked reductions in vessel density within the granuloma center. Additionally, expression of angiogenic factor mRNA is down regulated during mycobacterial infection. Vascular endothelial growth factor (VEGF), a potent inducer of angiogenesis which also increases endothelial cell vascular permeability, has been shown to be increased in the serum of individuals with active pulmonary TB when compared to both inactive TB infection and healthy individuals [A notable similarity between the tumor and granuloma microenvironment is localized hypoxia and the development of central lesion necrosis. In both cases, the development of central necrosis is in part due to the inability of tumor and granuloma vasculature to keep up with expansible growth of the lesions relative to the more normal surrounding tissue. In addition, the disruption of lesion vasculature is also due in part to the procoagulant state associated with chronic inflammation , 68. As ted mice . Wild-tyividuals , 73. Theividuals . Althougividuals . Based oMycobacterium marinum zebrafish embryo infection model. It was demonstrated that macrophages specifically induced the growth of new vessels in the inflammatory environment. However, inhibiting VEGF proved to be beneficial by reducing vascular leakage, and modulating angiogenic factor signal pathways altered oxygen availability for mycobacteria [M. marinum infection models and has been shown to reduce bacterial burden, reduce vascular leakiness, decrease Mtb dissemination, and also increase the effectiveness of first line anti-tubercular drug rifampicin [The association between granuloma formation and angiogenesis was also shown in the bacteria . Similarbacteria . Reducedbacteria . Pazopanbacteria . Pazopanfampicin . The prefampicin . While tfampicin . While tfampicin , 81. DesWhile therapies inhibiting angiogenesis have shown promise in reducing the rate of granuloma formation and severity, the opposite approach could also be beneficial. Rather than limit angiogenesis in an area of the granuloma that already has restricted access to host blood supply, there is the potential that increasing angiogenesis and promoting the blood supply will allow for more efficient drug penetration and increase the access of host immune cells to the granuloma . One stuSimilar to other inflammatory lesions, TB granuloma formation is characterized by an altered metabolic state both at the cellular and systemic level. The metabolic profile of cells that infiltrate the site of Mtb infection share similarities with the changing metabolic profile of the Mtb organism itself. The nature of the granuloma as an inflammatory lesion makes the metabolism of surrounding immune cells extremely important in the context of clearance of Mtb infection. The complex relationship between host-pathogen metabolism and Mtb infection has been recently reviewed . AlteredIn response to cytokine- or antigen-mediated stimulation, immune cells undergo a metabolic shift from oxidative phosphorylation to glycolysis Fig. . Rather A similar metabolic shift occurs within the TB granuloma and additional environmental changes such as a reduction or lack of oxygen within the necrotic granuloma increase the level of complexity. Although glycolysis is less energy efficient, glycolysis is active under aerobic or anaerobic conditions, enabling immune cells to function even under low oxygen condition . The chrM. marinum, the utilization of a tyrosine kinase inhibitor proven successful for antitumor treatments, imatinib, successfully reduced bacterial load, reduced lesion pathology, was effective against rifampicin-resistant M. marinum strains, and worked synergistically with rifampicin to reduce bacterial burden. Furthermore, when imatinib was utilized in a murine Mtb infection model, similar decreases in bacterial load were observed [Tyrosine kinases, key host signaling molecules, which play a role in mycobacterial entry and survival within macrophages, have recently been implicated as targets for host-directed therapeutics. Host tyrosine kinases function as regulators of phagosomal acidification, lysosomal mobility, and autophagy in macrophages and inhibition results in increased acidification of monocyte-derived macrophages . In an iobserved . A murinobserved . The utiMetformin is one of the safest and most widely prescribed drugs in the world and, when combined with dietary changes and exercise, helps in maintaining more normal blood glucose levels in patients with type 2 diabetes. Metformin, a biguanide class of anti-diabetic drugs, lowers blood glucose levels via inhibition of gluconeogenesis in the liver by targeting the mitochondrial complex I, thus limiting dysregulation in kinase signal pathways and altered lipid metabolism . AlthougMacrophages within the granuloma also have altered lipid metabolism that develops over the course of infection, as reflected by a foamy phenotype resulting from accumulated cytoplasmic lipid droplets and lipid storage organelles , 107. ThAs the macrophage becomes glucose deprived, Mtb begins to utilize host lipids as a primary source for metabolic function and alters glycolytic pathways to function under reduced oxygen tension , 115. MtAdvanced glycation end products (AGEs) are modified proteins, which have chemically reacted with sugar residues, such as glucose, thus significantly altering protein structure and function , 123. AGRecent research has revealed that AGEs play a role in Mtb infection and TB disease progression. RAGE is known to be constitutively expressed in the lung and it has been shown that levels of RAGE are elevated in mice infected with Mtb as well as human cells stimulated with Mtb antigens , 130. HoRAGE has been linked to autophagy and the establishment of neutrophil extracellular traps (NETS) in the context of adenocarcinoma . NETs coCurrently, there are no FDA approved or available drugs that specifically prevent the formation and accumulation of AGEs in humans or animals. Aminoguanidine has received the most attention to date as a potential anti-glycation therapeutic and is considered the gold standard for anti-AGE activity. Aminoguanidine showed efficacy in animal models of AGE accumulation and entered into clinical trials in 1996. Despite one phase III clinical trial in which aminoguanidine was shown to reduce the progression of diabetic retinopathy and lower low-density lipoprotein (LDL) and triglyceride levels, a further trial was discontinued due to both a lack of efficacy and safety concerns. Aminoguanidine is reported to result in several side effects including the following: flu-like symptoms, gastrointestinal disturbances, and anemia, and is not currently being investigated for the treatment of AGEs . BesidesOur laboratory has shown that first generation bis-2-amnoimidazole (B-2-AI)-derived compounds exhibit potent anti-glycating activity in vitro and are ×100 more active as anti-glycating compounds than aminoguanidine . One leaAnother approach to therapeutically targeting TB granulomas, besides limiting the destructive inflammatory response, is to promote tissue healing. The processes related to the resolution and repair of damaged tissues occur simultaneously with TB granuloma formation. The rate and extent of granuloma regression is determined by the balance between the mediators of tissue destruction and the factors that oppose or regulate those mediators . Much atThe role MMPs play in the pathogenesis of a wide variety of communicable and non-communicable diseases such as cardiovascular disease has driven the development of synthetic inhibitors that have potential as adjunctive therapies in the treatment of TB. Since MMPs are zinc-dependent, specific inhibitors have been designed to target or chelate the zinc ion as either broad spectrum or selective inhibitors and several are in various stages of clinical trials. The antimicrobial drug doxycycline at subantimicrobial doses has been shown to inhibit MMPs and currently is the only drug that is clinically available for this purpose. Recent studies have demonstrated that neutrophil-derived MMP-8 was highly expressed in NETs within cavities of TB patients and that doxycycline was effective at limiting collagen destruction by Mtb-mediated MMP-8 . DoxycycPromoting tissue repair and healing can also be accomplished by therapeutic modulation of extracellular matrix lysis as a strategy to limit the unfavorable consequences of Mtb infection. Much is known about the relative expression and immunomodulatory functions of the major cytokines and chemokines associated with Mtb infection in humans and animals. The cytokine network is complex and reflects the simultaneous and dynamic interplay between immune cell stimulation and down regulation and thus the balance between disease progression and resolution . The expCurrent opinion on the functional role of the TB granuloma and subsequent approaches to host-directed therapies which target the granuloma are still evolving. Mtb-host interactions within the granuloma remain complex and it is still unclear if the granuloma is protective or destructive to the host. Further, very little is known regarding how a severe necrotic lesion would respond to therapy in comparison to developing lesion at an earlier stage of disease. This review sought to discuss multiple approaches to host-directed therapy in the form of angiogenic, metabolic, AGE, and extracellular matrix modulators. Further research is needed to gain more information about the efficacy of these therapeutic approaches. Also, in vivo animal models will need to be utilized in order to assess the overall effectiveness of the host-directed therapies discussed here as adjunctive treatment with conventional antitubercular agents. The heterogeneity of lesion presentation and range of lesion severity within an individual will likely have an effect on host-directed therapeutic options as well. The need to develop therapies to be applied in conjunction with current antimicrobials remains apparent. This review sought to summarize and explore current as well as novel avenues for host-directed therapies targeting the TB granuloma. Finding effective ways to balance the granulomatous inflammatory response will be on the forefront of the development of host-directed therapies for TB.


Symmetric/asymmetric C-H stretching modes of a 16-Mercaptohexadecanoic acid monolayer are clearly observed as Fano-like anti-resonance peaks within a broad plasmonic absorption of the metamaterial. Spectral analysis using Fano line-shape fitting reveals the underlying resonant interference in plasmon-molecular coupled systems. Our metamaterial approach achieves the attomole sensitivity with a large signal-to-noise ratio in the far-field measurement, thus may open up new avenues for realizing ultrasensitive IR inspection technologies.The emerging field of plasmonic metamaterials has introduced new degree of freedom to manipulate optical field from nano to macroscopic scale, offering an attractive platform for sensing applications. So far, metamaterial sensor concepts, however, have focused on hot-spot engineering to improve the near-field enhancement, rather than fully exploiting tailored material properties. Here, we present a novel spectroscopic technique based on the metamaterial infrared (IR) absorber allowing for a low-background detection scheme as well as significant plasmonic enhancement. Specifically, we experimentally demonstrate the resonant coupling of plasmonic modes of a metamaterial absorber and IR vibrational modes of a molecular self-assembled monolayer. The metamaterial consisting of an array of Au/MgF Light absorption, which is one of the most fundamental light-matter interactions, is an essential phenomenon in a variety of the optical applications, such as photovoltaic cells and thermal management134678910111314151618Infrared (IR) absorption spectroscopy of molecular vibrations is of importance in material/medical science and security detection, since it provides essential information of the molecular structure, composition, and environment. For the versatile applications in prompt/easy-to-use IR inspection technologies, direct (far-field) detection of extremely small amounts of molecules is required. Although IR absorption spectroscopy offers fairly large signal intensity for practical applications, IR detection of picomole-level specimens, e.g., a monomolecular film, is still challenging. Surface-enhanced IR absorption (SEIRA) has been extensively studied and dramatic improvements of the sensitivity by several orders of magnitude were demonstrated using tailored plasmonic nanostructures202122Recent efforts to reach atto/zeptomole sensitivity of SEIRA, however, have focused on hot-spot engineering to improve the near-field enhancement and its spatial and spectral mode overlapping between the plasmons and molecular vibrations24252 gap layer. The Au ribbon width, w, and unit cell dimension, Λ, were 1.5 μm and 3 μm, respectively. The surface structure was purposely designed to exhibit an anomalous IR absorption at ~3000 cm−1, which spectrally overlapped with C-H stretching vibrational modes. The fabrication process started with electron beam evaporation of a 200-nm Au film onto a SiO2 substrate with a 5-nm Cr adhesion layer. Using a standard photolithography process, an array of one-dimensional (1D) micro-ribbon structures was patterned on the Au film surface. The surface structure was then obtained after 30-nm MgF2 and 50-nm Au films deposition and liftoff process. 2 and its SEM image, demonstrating uniform surface structures over a large chip area.−1. Due to the presence of a thick Au film on the substrate, there is no transmittance and an incident IR beam is either reflected or absorbed by the surface structure. Since the excitation of plasmon modes of the metamaterial manifests as absorption dips in the reflection spectrum, we have evaluated the reflection spectrum of the metamaterial normalized by that of a bare Au reference sample.The absorption property of the metamaterial absorber was characterized by using a Fourier-transformed infrared spectrometer (FTIR) equipped with a variable angle reflection accessory . −1 (f = 30–150 THz), in addition to several minor absorptions. To identify the underlying plasmon modes for these absorptions, a set of numerical simulations was carried out using the finite-element-method (FEM). In the calculations, the refractive index of SiO2 was set at 1.45 and the empirical values were used for Au and MgF227ω/c)sinθ = (π/Λ)l, where ω is the angular frequency, c is the speed of light, and l is integer. On the other hand, the frequencies of the major absorptions, labeled by m = 1, 2, and 3, are independent of the incident angle, and they are attributed to the localized plasmon resonances of the Au/MgF2/Au structure.y and Ez) of the major absorption dips at θ = 80°. Note that all figures were vertically enlarged 3 times for clarity. For an incident p-polarized light, localized plasmons are resonantly excited in the upper Au ribbon with dipole orientation along the ribbon width. The bare localized plasmons then interact with their own mirror images in the thick Au film, and the plasmon hybridization forms two new eigenmodes, symmetric and asymmetric onesSince the net electric dipole moment of such magnetic modes is zero in the quasistatic limit, the mode excitation is naturally weak and strongly depends on the retardation effect induced by the oblique incidence. In the case of m = 2 , since t−1. The m = 2 mode of the metamaterial, which spectrally overlapped with these vibrational modes, was employed to form a plasmon-molecular coupled system. The self-assembling process of a molecular monolayer started by immersing the metamaterial into a 16-MHDA ethanol solution with a concentration of 10−3 MSince the metamaterial absorber offers not only tailored plasmonic enhancement but also significant background suppression, such a unique surface environment can provide sensitive IR detection scheme of extremely small amounts of molecules. Here, we explore low-background resonant SEIRA by utilizing the resonant coupling between the plasmonic modes of a metamaterial absorber and IR vibrational modes of a molecular SAM. 16-Mercaptohexadecanoic acid , shown in the inset of ωpl = 2921.9 cm−1 and the dip became pronounced as the incident angle increased. With the molecules in the vicinity of the surface structure, their vibrational modes resonantly coupled with the plasmonic modes of the metamaterial. This in turn produced distinct Fano-like anti-resonant peaks within a broad absorption of the metamaterial, and this resonant coupling process depended on the incident angle. The signal intensities reached their maxima around θ = 40° while no clear vibrational signal was obtained at θ < 30° or θ > 70°. When θ < 30°, anti-resonant peaks were too weak to detect because of the weak excitation of the plasmonic mode of the metamaterial. At θ > 70°, on the other hand, the molecular vibrational modes were excited directly by the incident IR light, rather than by the resonant coupling process, and these competing processes rendered the signals weak.ωvib is the resonant frequency, γ is the damping constant (HWHM), and F is the Fano parameter to describe the degree of asymmetry. The spectral line-shapes of the experimental result were well re-produced by the Fano fitting curves with the corresponding parameters [bottom in F = 0) to asymmetric one (F = −0.05) due to the nature of the Fano resonance. Using the SAM packing density of 21.4 Å2/molecule, the sensitivity was estimated to be ~1.8 attomoles within the diffraction-limited IR beam spot in the FT-IR reflection measurementThe net vibrational excitation of molecules can be extracted by the baseline correction to divide the measured reflectance spectrum by the line-shape of the plasmon resonanceIn conclusion, a novel spectroscopic technique based on metamaterial-enhanced IR absorption of a molecular SAM was proposed and demonstrated. The low-background detection scheme with tailored plasmonic enhancement by the metamaterial absorber achieved the attomole sensitivity of direct IR absorption spectroscopy. Spectral analysis using Fano line-shape fitting revealed the underlying resonant interference in plasmon-molecular coupled systems. Our metamaterial approach may open up new avenues for realizing ultra-sensitive IR inspection technologies.How to cite this article: Ishikawa, A. and Tanaka, T. Metamaterial Absorbers for Infrared Detection of Molecular Self-Assembled Monolayers. Sci. Rep.5, 12570; doi: 10.1038/srep12570 (2015).


The epoxyeicosatrienoic acid (EET) neutralizing enzyme soluble epoxide hydrolase (sEH) is a neuronal enzyme, which has been localized in both the cytosol and peroxisomes. The molecular basis for its dual localization remains unclear as sEH contains a functional peroxisomal targeting sequence (PTS). Recently, a missense polymorphism was identified in human sEH (R287Q) that enhances its peroxisomal localization. This same polymorphism has also been shown to generate weaker sEH homo-dimers. Taken together, these observations suggest that dimerization may mask the sEH PTS and prevent peroxisome translocation. In the current study, we test the hypothesis that dimerization is a key regulator of sEH subcellular localization. Specifically, we altered the dimerization state of sEH by introducing substitutions in amino acids responsible for the dimer-stabilizing salt-bridge. Green Fluorescent Protein (GFP) fusions of each of mutants were co-transfected into mouse primary cultured cortical neurons together with a PTS-linked red fluorescent protein to constitutively label peroxisomes. Labeled neurons were analyzed using confocal microscopy and co-localization of sEH with peroxisomes was quantified using Pearson’s correlation coefficient. We find that dimer-competent sEH constructs preferentially localize to the cytosol, whereas constructs with weakened or disrupted dimerization were preferentially targeted to peroxisomes. We conclude that the sEH dimerization status is a key regulator of its peroxisomal localization. Historisurvival .Endogenous sEH exhibits a dual cytosolic and peroxisomal subcellular distribution , resultiHowever, the regulation of sEH subcellular distribution in neurons has not previously been investigated. This is an important cell type to understand the mechanism of sEH subcellular distribution given previous studies identifying sEH as a stroke risk factor ,13. FurtPeroxisome localization of sEH has recently been shown to reduce infarct size in response to stroke . TherefoThe investigation conformed to the Association for Assessment and Accreditation of Laboratory Animal Care AAALAC Accreditation (approved July 2013) and the Office of Laboratory Animal Welfare . The Oregon Health and Science University Animal Care and Use Committee approved all experiments.sEH cDNA containing sEH dimerization mutants were amplified using primers that created a 5’-EcoRI and 3’-NotI restriction site for in-frame subcloning into a vector expressing enhanced green fluorescent protein (GFP) driven by elongation factor α (EF1α) promoter . PCR med2. Neurons were cultured in Neurobasal medium without phenol red (Invitrogen), supplemented with 2% B27 (Invitrogen), 1% Glutamax (Invitrogen) and 1% penicillin/streptomycin (Invitrogen). Cultures consisted of >90% microtubule-associated protein 2 (MAP2)-positive and <10% glial fibrillary acidic protein (GFAP)-positive cells on day 10 in vitro (DIV 10).Primary cortical mouse neurons were prepared from C57/BL6 (Charles River) mouse brains at E16, as previously described . CorticeGFP-sEH and RFP-SKL plasmids were purified with a Maxi Kit (Qiagen) according to manufacturer’s protocol and diluted to a final concentration of 900 ng/μL. Lipofectamine 2000 (Invitrogen) was mixed with 900 ng of each plasmid in accordance with the manufactures instructions. All transfections were done on DIV 7 using 1000 μl of supplemented Neurobasal medium for 1 hour, which is then changed to fresh maintenance medium. Cells were fixed 72h after transfection on DIV 10 in 4% paraformaldehyde. Hoechst (Invitrogen) staining of nuclei was performed with 3.2 μM final concentration in PBS for 90 seconds. Slides were then washed and mounted with Fluoromount G (Southern Biotech).Human Embyronic Kidney (HEK) cells were maintained and transfected as previously described . RecombiImages were acquired on a Zeiss LSM 510 microscope using a Plan Apochromat 63x/1.4NA oil objective lens unless otherwise noted in figure legend. GFP was excited with a 488-nm laser and detected at 500–530 nm. RFP was excited at a 543-nm laser and detected beyond 560 nm. Laser intensities were optimized for each individual image to take advantage of the full dynamic range of signal. As expected with a transient transfection, each condition contained a mixed population of dim and bright cells. The depth of each neuron was captured with 2048 x 2048 pixel (68 x 68 μm) resolution centered on the nucleus with 16-bit depth coverage. At least 20 images were analyzed for each experimental groups. The specific number of images analyzed for each group is presented in each figure. Images were acquired from at least 3 independently transfected coverslips representing 2 to 5 independent neuronal cultures .Images were analyzed for co-localization with IMARIS (Bitplane) using the co-localization module unless otherwise noted in figure legend. For all GFP-sEH groups, a single representative optical section was used to measure Pearson’s correlation coefficients. Raw images were thresholded equally in the GFP  and RFP channel , and then masked in the green channel at (5.69%) before calculating coefficients between the GFP (sEH) and RFP-SKL (demarcating peroxisomes) signals.ZEN software (Zeiss) and Photoshop (Adobe) were used to create images presented in manuscript unless otherwise noted in figure legend. Presented images are maximum intensity projections of a 2 μm sub-stack highlighting a dendritic process and part of the neuronal cell body from the full view images used for analysis. While raw images were analyzed for co-localization, for presentation purposes brightness and contrast have been adjusted in the representative figures.Data is presented as mean ± sem of Pearson’s correlation coefficient values. To determine if there was a difference in co-localization between wild-type GFP-sEH (GFP-sEH-WT) and different mutant groups, one-way analysis of variance (ANOVA) was performed, followed by Student-Newman-Keuls post-hoc analysis. The number of cultures, coverslips, and images are presented in We employed an N-terminal GFP fusion strategy to preserve the C-terminal PTS of sEH that allows us to track the effects of dimerization on the localization of human sEH. This approach parallels a previously study of sEH distribution using heterologous expression of human polymorphisms in Chinese hamster ovarian (CHO) cells  Fig 1A)Fig 1A).Western blot analysis of HEK cell lysates transfected with either GFP-sEH-WT recombinant sEH or GFP proteins was used to confirm expression fusion of GFP-sEH fusions. We found that HEK cells transfected with GFP-sEH-WT produced only a single band that demonstrated immunoreactivity for both anti-sEH and anti-GFP antibodies, similar to the expected size of 90 kD for a GFP-sEH fusion protein .We visualized the localization pattern of sEH by transfecting either GFP or GFP-sEH-WT into DIV 7 primary cortical neurons. The cells were allowed to express each protein for 72 hours to reach a dynamic equilibrium of localization before the cells were fixed on DIV 10. In order to obtain high-resolution images, neurons were imaged using laser-scanning confocal microscopy. We found that cells expressing GFP exhibited the expected diffuse staining pattern, whereas neurons transfected with GFP-sEH-WT had a dual staining pattern that is both diffuse and punctate , consistThe addition of C-terminal –SKL peptide to fluorescent proteins has been extensively used to label peroxisomes in living cells –20. HereHaving confirmed that our GFP-sEH-WT construct exhibited dual cytosolic and peroxisomal distribution, we next mutated the PTS of sEH in order to artificially enhance or prevent sEH peroxisomal translocation using PCR mutagenesis. We chose to either prevent sEH from translocating to peroxisomes by deleting the PTS (GFP-sEH-ΔPTS) or enhance sEH peroxisomal translocation by replacing the C-terminus of sEH with the highly efficient canonical PTS (GFP-sEH-SKL).We found that deletion of the C-terminal PTS of sEH (GFP-sEH-ΔPTS) results in a diffuse pattern similar to neurons transfected with GFP . In contThese distinct localization patterns allowed us to develop and validate a quantification system for the distribution of sEH between the cytosol and peroxisomes using Pearson’s correlation coefficient (PCC) by comparing the distribution GFP-sEH-WT and the peroxisome labeling protein RFP-SKL. PCC is an established measure of co-localization between two signals that ranges from -1 (perfect exclusion) to 1  . ImportaOur GFP-sEH transfection model coupled with PCC quantification of sEH peroxisomal localization allowed us to test the hypothesis that dimerization regulates the distribution of sEH between the cytosol and peroxisomes. Previously, a human polymorphism in sEH (R287Q) was shown to partially disrupt the homodimerization of sEH . This spWe previously identified and characterized additional systematic mutations of amino acids forming an essential dimer-stabilizing salt-bridge (E254 and R287) that control the dimerization state of sEH . We furtWe found that dimer-disrupting mutations significantly increase peroxisome localization as measured by PCC from 0.43±.03 (GFP-sEH-WT) to 0.67±.02, 0.63±.02, and 0.69±.02 for GFP-sEH-R287Q, GFP-sEH-R287E and GFP-sEH-E254R respectively . InteresIn contrast to disrupting dimerization, rescuing sEH dimerization, by combining the E254R and R287E mutations (GFP-sEH-E254R+R287E), results in a dual diffuse and punctate pattern in which the punctate signal co-localizes with peroxisomes .A previous study presented evidence that in addition to dimerization, expression level of sEH may also be a contributing factor in its subcellular distribution . To deteSoluble epoxide hydrolase is a promising therapeutic target for stroke, with interesting biology, protein structure and subcellular localization. Previous work with a human polymorphism of sEH (R287Q) suggested a relationship between dimerization of sEH and its subcellular localization . Here weHere, we find that two different constructs of sEH previously shown to form dimers (GFP-sEH-WT and GFP-sEH-R287E+E254R) co-localize with a peroxisome marker less than three sEH constructs , which disrupt dimerization. We believe this to be a compelling evidence that dimerization plays a central role in the regulation of sEH subcellular localization. Interestingly, there was no increase in peroxisomal localization of GFP-sEH-R287E and GFP-sEH-E254R compared to GFP-sEH-R287Q, despite the fact that R287E and E254R mutations were shown to disrupt dimerization more significantly than the R287Q polymorphism. While, the R287Q polymorphism was measured to have a ratio of 60% dimer and 40% monomer, almost no dimer formation was detected for E254R and R287E mutations ,15. Thisin vitro findings [A previous study showed that in addition to dimerization, expression level may contribute to the subcellular distribution of sEH within cells. This conclusion was based on the observation that a stable cell line of CHO cells expressing low levels of GFP-sEH-WT did not have evidence of puncta indicative of peroxisome localization paired with the observation that CHO cells sorted by flow cytometry for the low expression of GFP-sEH-WT also did not have any evidence of puncta indicative of peroxisome localization. To test whether expression of sEH contributes to the subcellular distribution of sEH within primary cortical mouse neurons we transfected neurons with different amounts GFP-sEH and co-localized GFP-sEH to a RFP peroxisome marker. Our results suggest that expression level does not affect the subcellular distribution of GFP-sEH-WT or GFP-sEH-R287Q. The discrepancy in our results compared to the previous publication may simply reflect a biological difference between CHO cells and mouse primary cortical neurons. Alternatively, the previous paper was able to observe cells with a 10-fold difference in expression which is a larger dynamic range then we were able to obtain in our study. This study has a number of limitations. First, this study extrapolates the dimerization status of each sEH construct based on previous findings . Given tfindings .Our findings in primary neurons strengthen the model of sEH peroxisomal translocation which proposes that in its dimerized form the endogenous PTS of sEH and the peroxisome transport protein peroxin 5 (Pex5) have unfavorable binding conditions resulting in a majority of sEH to be localized to the cytosol. However, as a monomer, the binding conditions of SKM and Pex5 become more favorable resulting in more efficient translocation into peroxisome .de novo by microsomal cytochrome P450 epoxygenases or released from the plasma membrane into the cytosol. In both cases, they are further metabolized into their diol metabolites in the cytosol by the action of sEH.Generally, protein localization is a key determinant of function, and protein mis-localization is associated with multiple diseases . The preOn the other hand, the role sEH plays in peroxisomes remains unclear. It is known that peroxisomes play a key role in the metabolism of reactive oxygen species (ROS) within the cell . Under sIn conclusion, this work described here suggests that disrupting sEH dimerization results in enhanced translocation of the enzyme to peroxisomes. This places new emphasis on the role that sEH plays in peroxisomes especially in the context of the clinical phenotypes associated with R287Q human polymorphism ,25–27. O


The terrestrial cycling of Si is thought to have a large influence on the terrestrial and marine primary production, as well as the coupled biogeochemical cycles of Si and C. Biomineralization of silica is widespread among terrestrial eukaryotes such as plants, soil diatoms, freshwater sponges, silicifying flagellates and testate amoebae. Two major groups of testate (shelled) amoebae, arcellinids and euglyphids, produce their own silica particles to construct shells. The two are unrelated phylogenetically and acquired biomineralizing capabilities independently. Hyalosphenids, a group within arcellinids, are predators of euglyphids. We demonstrate that hyalosphenids can construct shells using silica scales mineralized by the euglyphids. Parsimony analyses of the current hyalosphenid phylogeny indicate that the ability to “steal” euglyphid scales is most likely ancestral in hyalosphenids, implying that euglyphids should be older than hyalosphenids. However, exactly when euglyphids arose is uncertain. Current fossil record contains unambiguous euglyphid fossils that are as old as 50 million years, but older fossils are scarce and difficult to interpret. Poor taxon sampling of euglyphids has also prevented the development of molecular clocks. Here, we present a novel molecular clock reconstruction for arcellinids and consider the uncertainties due to various previously used calibration points. The new molecular clock puts the origin of hyalosphenids in the early Carboniferous (∼370 mya). Notably, this estimate coincides with the widespread colonization of land by Si-accumulating plants, suggesting possible links between the evolution of Arcellinid testate amoebae and the expansion of terrestrial habitats rich in organic matter and bioavailable Si. Phaeodactylum tricornutum have orthologs in the genomes of other eukaryotes, including Opisthokonts, Viridiplantae and other “Chromalveolates” eolates” . These oeolates” , suggestThere are several groups of unrelated testate amoebae. The two most prevalent and abundant in terrestrial environments are the euglyphid and the arcellinid testate amoebae. Both inhabit the same environments—bodies of fresh water, soils, peatlands and other humid microhabitats—and have approximately the same sizes, with the majority of species being between 30–300 µm long or wide. However, the two groups are vastly divergent genetically and historically. Euglyphids include about 800 species and are in the super group Rhizaria . These ovia silica deposition vesicles . Others (Nebela  and relaArgynnia ), as weluereusia .kleptosquamy . Microscopic observations were made using an Utermöhl chamber  on an Olympus IX81 inverted microscope equipped with oil immersion Differential Interference Contrast optics (20×–40×–60×–100×). All images were recorded by an Olympus DP-71 camera.Samples of kleptosquamy, with possible states present, absent or unknown.We have performed ancestral state reconstructions on the topologies from molecular reconstructions of two recently published phylogenies . Each rePaleoarcella athanata (type specimen HUPC #62988), described in Centropyxis genus  was set to the split between Padaungiella lageniformis and Hyalosphenia elegans with soft bounds at 736–100 mya; origin of genus Arcella (“node D”), calibrated the clade containing A. hemisphaerica and A. vulgaris WP with soft bounds at 105–100 mya. We did not include the fourth calibration point suggested by Fiz-Palacios and colleagues (Lesquereusia–Difflugia divergence) because the Lesquereusia SSU rDNA is not available. Fortunately, Fiz-Palacios and colleagues (estbranches (part of multdivtime package (baseml (part of the PAML package (Molecular clock reconstructions (MCR) were performed using PhyloBayes 3.3 . We usedlleagues  have teslleagues . The use package ), with i package ), followPadaungiella lageniformis was isolated while preying upon a specimen of Euglypha sp. The Euglypha cytoplasm had been almost completely ingested at the stage of isolation  are able to re-use scales obtained from euglyphids and three others  do not. We have performed a parsimony based ancestral reconstruction of character states in both topologies available  were shArgynnia . Conside lineage . Other f a whole .To determine the origin of hyalosphenids, we generated a dated tree for the arcellinids . In ordeOur reconstruction stands in sharp contrast with another recent molecular clock reconstruction of the arcellinids . The twoNebela collaris) had to be fed with fast-growing species of euglyphids, such as Euglypha hyalina . For instance, one cannot say with certainty whether a hyalosphenid is able to construct the shell without any euglyphid scales. This caveat undermines the interpretation of klepstosquamy as ancestral in the group, for this reason, we clearly establish that our working hypothesis is that scaled euglyphids appeared before hyalosphenids.Kleptosquamy can be inferred as an ancestral character state in hyalosphenids , i.e., tScutiglypha from diatomaceous earth demonstrate that modern genera have existed for at least 15 million years (Scutiglypha (Euglyphidae). Older records of shells are much more difficult to interpret, as the conditions of shell preservation make the separation between arcellinids and euglyphids ever more difficult: because of intense convergence, pseudopods would be the only reliable way of separating arcellinids and euglyphids, but these are usually not preserved in the fossil record . This is in marked contrast to the recent reconstruction of arcellinid history by Arcella + Netzelia clade, as well as the soil dwelling Trigonopyxis + Bullinularia clade have diversified after the mid-Devonian. In contrast to the preceding periods, when plant cover was restricted to moist habitats, the Late Devonian and the Carboniferous saw the diversification of plants that were well adapted to life on land, with deeper roots and soil forming capabilities  The root systems of the Late Devonian/Carboniferous plants are thought to have promoted silicate weathering ng e.g., , were abng e.g.,  and the ng e.g., .To move forward from here, we need to (i) better understand Si metabolism across protist groups, including deeper understanding of physiological aspects; (ii) obtain reliable fossil evidence for the appearance and diversification of Si-metabolizing lineages; (iii) improve the molecular clock by expanding the molecular database, using appropriate numerical models and carefully checking the reliability of the fossil record, (iv) improve constraints on the contribution of Si-precipitating organisms to the cycling of Si and C in terrestrial systems. The combination of these efforts is challenging, but can be met with a combination of approaches including molecular phylogeny, biogeochemistry, and paleontology.10.7717/peerj.1234/supp-1Supplemental Information 1Alignments, trees and command lines used in molecular clock reconstructions.Click here for additional data file.


Escherichia coli strains from the feces of beef cattle on 14 Japanese farms. In the present study, the genetic backgrounds and phylogenetic relationships of 45 AMR isolates were characterized by the chromosome phylotype, AMR phenotype, AMR genotype, and plasmid type. These isolates were classified into five chromosome phylotypes, which were closely linked to the farms from which they were isolated, suggesting that each farm had its own E. coli phylotype. AMR phenotype and plasmid type analyses yielded 8 and 14 types, all of which were associated with the chromosomal phylotype and, thus, to the original farms. AMR genotype analysis revealed more variety, with 16 types, indicating both inter- and intra-farm diversity. Different phylotype isolates from the same farm shared highly similar plasmid types, which indicated that plasmids with AMR genes could be transferred between phylotypes, thereby generating multi-antibiotic-resistant microorganisms. This ecological study demonstrated that the chromosome phylotype was strongly correlated with the farm from which they were isolated, while the AMR phenotype, genotype, and plasmid type were generally correlated with the chromosome phylotype and farm source.The emergence of multiple-antibiotic-resistance bacteria is increasing, which is a particular concern on livestock farms. We previously isolated 1,347 antimicrobial-resistant (AMR)  Escherichia coli, Enterococcus spp., Salmonella spp., and Staphylococcus spp.  is a serious concern in human medicine and public health, and the emergence of multiple-antibiotic-resistant bacteria constitutes a global problem. The most commonly encountered AMR bacteria are cus spp. . Intesti factors  and have factors . Commensg humans , and canhttp://www.maff.go.jp/nval/tyosa_kenkyu/taiseiki/index.html). The use of antibiotics in feed averaged 171 tons per year from 2000 to 2005 . However, the amount of veterinary antimicrobial agents (therapeutic and feed additive) sold per food-producing animal weight  in Japan increased from 132 mg kg−1 to 153 mg kg−1 from 2005 to 2010 .Bacterial AMR profiles, such as the number of antimicrobial agents, were previously shown to be strengthened when antimicrobial agents were used under selective pressure , 36. The to 2010 . The useE. coli isolates from the feces of beef cattle on 14 farms in three Japanese regions    and asse Kyushu) , 24, 40.E. coli  drug-inactivating enzymes, (ii) reduced membrane permeability, and (iii) antibiotic efflux pumps, or are caused by mutations in antibiotic target sites . AMR genE. coli , 15, 18.sistance , 35; howE. coli isolates from five different beef cattle farms in Japan. These isolates were resistant to nine or more antimicrobial agents and were selected from the 3,147 isolates obtained in our previous study  digestion of whole genomic DNA, followed by PFGE (see below for PFGE conditions) (Phylotypes were determined by restriction fragment length polymorphism (RFLP) analysis with ditions) . RFLP prE. coli were isolated from beef cattle fecal samples in Hokkaido, Chubu, and Kyushu, Japan , cefazolin , ceftiofur , dihydrostreptomycin , kanamycin , gentamycin , bicozamycin , oxytetracycline , colistin , chloramphenicol , nalidixic acid , enrofloxacin , and trimethoprim  . The antimicrobial resistance breakpoint as the minimum inhibitory concentration for antimicrobial agents was used according to the CLSI criteria. E. coli ATCC 25922, Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, and Pseudomonas aeruginosa ATCC 27853 were used as quality control strains.Antimicrobial susceptibility was determined using the agar dilution method in accordance with the Clinical Laboratory Standard Institute (CLSI) guidelines ; ampicilμg mL−1) , 43. TheblaTEM, blaSHV, blaOXA, blaCTX-M, and blaCMY; (ii) resistance to aminoglycosides : strA, strB, aadB, aacC2, aac(3)-IV, aphA1, aphA2, aadD, aphA3, aphA/aph(3′)-Id, aphA7, aphAI-IAB, Kn, and Kan; (iii) resistance to tetracycline (OTC): tetA, tetB, tetC, tetD, tetE, tetG, tetY, and tetS; (iv) resistance to CP: catI, catII, catIII, floR, and cmlA; and (v) resistance to TMP: dhfrI, dhfrV, dhfrVII, dhfrIX, dhfrXIII, dfrA12, dhfrXVII, and dhfrXII.We assessed the isolates for the following AMR genes , 34, 43:® Flexi DNA Polymerase  was used for the PCR detection, and amplification was performed under the following conditions: 30 s at 95°C, followed by 30 cycles of 95°C for 15 s, an appropriate annealing temperature .The presence of AMR genes were determined by PCR with the total DNA of each of the 45 strains as the template using primers reported in previous studies  was used for the PCR detection, and amplification was performed under the following conditions: 30 s at 95°C, followed by 30 cycles of 95°C for 15 s, an appropriate annealing temperature (described in At least 18 types of plasmid incompatibility have been reported in equences  and idenequences  6). GoT. GoTE. c610 1.6–1.7 according to the Pulse Net Protocol . Plugs were digested with S1-nuclease  for 10 min at 37°C. Electrophoresis was performed with pulse times from 1 to 25 s, voltage 6 V cm−1, angle orientation 120°, and a run time of 14.5 h in 1% SeaKem Gold agarose  with 0.5× Tris-borate-EDTA buffer at 14°C. Low-range PFG markers  were used as size markers. (ii) PFGE for RFLP: Agarose plugs were prepared with a cell suspension at A610 1.00. Plugs were digested with 50 U XbaI for 2 h at 37°C. Electrophoresis was performed with pulse times of 2.2 to 63.8 s, voltage 6 V cm−1, angle orientation 120°, and a run time of 18.0 h in 1% SeaKem Gold agarose with 0.5× Tris-borate-EDTA buffer at 14°C. Lambda Ladder PFG markers (New England Biolabs) were used as size markers.PFGE was performed with a CHEF-DRII system  as follows. (i) PFGE for large plasmids (>16 kb): Agarose plugs were prepared with a cell suspension at AE. coli strains isolated from the feces of beef cattle in Hokkaido, Chubu, and Kyushu, Japan . An RFLP profile could not be obtained for isolate SA1-4-1 (resistant to nine agents) despite repeated trials. This may have been because of the ease of lysis and instant DNA degradation of the isolate. Therefore, the dendrogram was built with the profiles of 44 isolates ; the remainder were resistant to 11 agents (11-AMR isolates). The AMR phenotypes (AMR susceptibility) for 13 antibiotics were determined by the agar dilution method. All 45 isolates were resistant to ABPC, DSM, OTC, NA, and TMP . ResistastrA (an aminoglycoside resistance gene), which was detected in all 45 isolates. The other major AMR genes (detected in over 30 isolates) were strB (44 isolates), blaCMY (39 isolates), aacC2 and tetB (35 isolates), and blaTEM (31 isolates). The least common AMR gene was dhfrI (8 isolates). Isolates were divided into 16 genotype groups based on the combination of resistance genes  were not assessed because resistance to these drugs is derived from DNA gyrase mutations rather than by specific resistance genes and enzymes. Of the 40 AMR genes tested, 17 were detected; each isolate contained 7–10 genes. The other 23 AMR genes were not detected. The AMR genes and genotype grouping are listed in ce genes . The 11-AMR genotype groups IX and XI correlated with phylotypes I and V-a, respectively. However, the other AMR genotype groups did not match the phylotype groups. Each phylotype group included 3–5 AMR genotype groups.Plasmid sizes were determined by conventional gel electrophoresis and PFGE with appropriate DNA size markers. All isolates carried plasmids of various sizes, ranging between 2 and 774 kb. A total of 210 individual plasmids were identified and divided into 58 classes by size . All isoblaTEM was distributed in IncFIB, F, FIA, A/C, and I1. The other example, tetA, was distributed in IncFIB, F, N, A/C, and the unidentified group.Southern hybridization was performed with 17 AMR gene-specific probes against PFGE-separated plasmid DNA in order to identify the AMR genes on each type plasmid. The number of AMR genes on individual plasmids ranged from 1–9. Various combinations of AMR genes were observed, as listed in The 210 detected plasmids were classified into 92 groups by size, incompatibility type, and AMR genes . Twenty-E. coli and the plasmid type, the phylotype dendrogram and plasmid profiles were displayed in parallel , AMR phenotype (pattern of multiple-antibiotic resistance), AMR genotype (the presence or absence of 40 AMR genes), and plasmid type (incompatibility type), and attempted to link these traits in order to characterize the mechanism by which multiple-antibiotic resistant E. coli are distributed in livestock farms.In the present study, we analyzed the phenotypic and genotypic traits of 45 multiple-antibiotic resistant E. coli were classified into five chromosome phylotypes . Many previous studies on E. coli phylotype analyses have so far shown that the cross-contamination of phylotypes over a wide area frequently occurs as a result of animal transport . This negative relationship may be related to the correlation between the replication genes of the incompatibility plasmid and the tetracycline resistance determinant  was similar to that in phylotypes II and III . The plaE. coli strains from five beef cattle farms and demonstrated their chromosome phylotype diversity at the inter-farm level. We also showed inter- and intra-farm level diversity in the AMR genotype. This may have been due to the cross-contamination of AMR genes mediated by animal transportation. The phylotype diversity of AMR strains between livestock farms has been reported previously (i.e. 8-AMR or less) will help define the distribution of AMR genes, their mechanisms, and route of dispersal. The results of our study provide fundamental information of the prevalence and diversity of AMR strains for epidemiological studies and important information for identifying or predicting the emergence of multi-antibiotic resistance microorganisms.In the present study, we investigated 45 multiple-antibiotic-resistant eviously , 42. The


Beckwith-Wiedemann syndrome (BWS), a congenital overgrowth disorder with variable expressivity and a predisposition to tumorigenesis, results from disordered expression and/or function of imprinted genes at chromosome 11p15.5. There are no generally agreed clinical diagnostic criteria, with molecular studies commonly performed to confirm diagnosis. In particular, methylation status analysis at two 11p15.5 imprinting control centres (IC1 and IC2) detects up to 80% of BWS cases (though low-level mosaicism may not be detected). In order to evaluate the relationship between the clinical presentation of suspected BWS and IC1/2 methylation abnormalities we reviewed the results of >1,000 referrals for molecular diagnostic testing.Out of 1,091 referrals, 507 (46.5%) had a positive diagnostic test for BWS. The frequency of tumours was 3.4% in those with a molecular diagnosis of BWS. Previously reported genotype-phenotype associations with paternal uniparental disomy, IC1, and IC2 epimutation groups were confirmed and potential novel associations detected. Predictive values of previously described clinical diagnostic criteria were compared and, although there were differences in their sensitivity and specificity, receiver operating characteristic (ROC) analysis demonstrated that these were not optimal in predicting 11p15.5 methylation abnormalities. Using logistic regression, we identified clinical features with the best predictive value for a positive methylation abnormality. Furthermore, we developed a weighted scoring system  to prioritise patients presenting with the most common features of BWS, and ROC analysis demonstrated superior performance  compared to previous criteria.We suggest that this novel tool will facilitate selection of patients with suspected BWS for routine diagnostic testing and so improve the diagnosis of the disorder. CDKN1C account for most cases of this disorder. Disordered IGF2/CDKN1C function may be caused by multiple mechanisms, including paternal uniparental disomy (pUPD), cytogenetic abnormalities , imprinting centre (IC) mutations/deletions and epimutations, and CDKN1C mutations (see [H19/IGF2 intergenic DMR and KvDMR1) that are coincident with the distal and centromeric imprinting centres  [IGF2 and silencing of H19 expression . A further 50% of individuals with BWS have a loss of methylation at IC2 (KvDMR1); usually the paternal IC2 allele is unmethylated and the maternal allele is methylated, but in these cases both alleles are unmethylated [CDKN1C is imprinted and preferentially expressed from the maternal allele, and a loss of maternal allele methylation at IC2 is associated with loss of maternal allele CDKN1C expression and hence a marked reduction in CDKN1C growth suppressor activity [CDKN1C mutations is small (approximately 5%) in sporadic cases, mutations may be detected in about 50% of familial BWS cases [Beckwith-Wiedemann syndrome , a congenital overgrowth disorder with a predisposition toward tumorigenesis, results from abnormal expression/function of imprinted genes from the chromosome 11p15.5 imprinted gene cluster . Only a ons  were referred for diagnostic testing. The most frequent indications for referral were: macroglossia (52.3%), anterior abdominal wall defects , hemihypertrophy (41.4%), pre- or postnatal macrosomia (38.7%), ear creases/pits (36.4%), neonatal hypoglycaemia (29.8%), facial naevus flammeus , organomegaly (17.0%), polyhydramnios (10.1%), maxillary hypoplasia (9.6%), congenital heart defects (8.3%), and embryonal tumours (3.1%). The median number of clinical features at referral was three (range 1 to 13). Of those with a single clinical indication , the most frequent was hemihypertrophy (present in 65.5% of those with a single feature). In those with two or more clinical indications for referral, the most frequent combinations of features were macroglossia and ear creases/pits (37.9%).CDKN1C mutations [Overall, 507/1,091 (46.5%) individuals tested had an abnormal methylation profile at IC1 and/or IC2. Forty-seven (4.3%) had hypermethylation at IC1 only, 321 (29.4%) had isolated IC2 hypomethylation, and 135 (12.4%) had abnormal IC1 and IC2 methylation from pUPD. Of those with an aberrant IC1 methylation profile, three (0.3%) individuals had a copy number abnormality , while of those who had an aberrant IC2 methylation profile, one (0.1%) individual had a copy number abnormality (deletion). Thus, among patients with a positive molecular diagnosis, 63.3% had evidence of an IC2 epimutation, 26.6% pUPD, 9.3% IC1, and 0.8% had a copy number abnormality .In general, the greater the number of clinical features of BWS present at referral, the greater the likelihood of a positive molecular diagnosis. Thus, 79.3% of those with five or more relevant clinical findings had a positive diagnostic test, and 65.2%, 45.5%, 26.4% and 12.4% of those with four, three, two and one clinical indications, respectively. Of those with isolated hemihypertrophy and no other clinical features, 12.9% had a positive diagnostic test.Individual clinical features associated with a greater than average frequency of a positive diagnostic test for methylation abnormalities were: FNF 73.1%), diastasis recti (72.4%), organomegaly (72.3%), macroglossia (72.2%), polyhydramnios (71.6%), and exomphalos (70.0%), whereas the presence of hemihypertrophy was associated with a lower frequency of a positive diagnostic test (38.2%)  had an embryonal tumour at referral ). Eight individuals had Wilms’ tumour , six a hepatoblastoma , one an adrenal cortical carcinoma (pUPD), and one a rhabdomyosarcoma (IC2). Of these, one individual had both a Wilms’ tumour and a hepatoblastoma (IC1). Interestingly, a further 17 mutation negative referrals had embryonal tumours, with the number of clinical features ranging from one to nine (median three).P < 0.001 and P < 0.001, respectively), macroglossia in the IC2 subgroup (88.2%) compared to pUPD  and IC1 , FNF in IC2 (44.5%) vs pUPD  and IC1 , ear creases/pits in IC2 (61.7%) vs pUPD  and IC1 , and exomphalos in IC2 (49.2%) vs pUPD  and IC1 . Conversely, there were reduced frequencies of organomegaly and embryonal tumours in the IC2 subgroup  compared to pUPD  and IC1 .We compared the frequency of different clinical features of BWS between four molecular subgroups; (a) pUPD, (b) IC1 hypermethylation, (c) IC2 hypomethylation, and (d) a normal methylation profile , the differences between IC1 (31.9%) and IC2 were not statistically significant (P = 0.279). Similarly, for diastasis recti, increased frequencies in the IC1 subgroup (21.3%) relative to IC2  were evident, but there were no significant differences between pUPD (10.4%) and IC2 (P = 0.074). There were no significant intergroup differences in frequencies for macrosomia, neonatal hypoglycaemia, polyhydramnios, prognathism, maxillary hypoplasia, congenital heart defects, or inguinal hernias.Whilst there was a significant increase in the frequency of umbilical hernias in pUPD (34.8%) in comparison to IC2  for a positive molecular diagnosis. Given that a score of 3.0 equates to a probability of 0.49, we settled for a minimum score of 3.5 (probability of 0.59). Thus, our simplified scoring system, with few and easy to distinguish features, has positive and negative predictive values of 78.4% and 79.6%, respectively  have a significantly higher frequency of exomphalos than in patients with pUPD or IC1 epimutations , whereas the risk of Wilms’ tumour is higher in the latter two groups. In this current study, an analysis of >500 patients with pUPD, IC1 and IC2 epimutations identified additional potential genotype-phenotype associations. Thus, isolated IC2 epimutations have a significantly increased association with macroglossia and FNF, whilst the pUPD and IC1 subgroups have a greater association with organomegaly than in IC2.Previously, we and others have reported epigenotype-phenotype associations in BWS ,19,23-25CDKN1C/IC2 deletions – though each of these findings is rare [CDKN1C mutations will not be detected, these are infrequent in sporadic cases, and only about 15% of BWS patients have a positive family history [CDKN1C mutation analysis in familial cases or cases with the highly suspect clinical features of macroglossia and exomphalos) [We further identified the clinical features that are most predictive of a positive molecular genetic test for abnormal methylation at the two 11p15.5 ICs. The most common molecular abnormality in BWS is loss of maternal allele methylation at IC2 (found in the 50 to 60% of cases with IC2 epimutations and in the 20% with pUPD) followed by gain of maternal allele methylation at IC1 (found in 5 to 10% of cases with IC1 epimutations and in the 20% with pUPD) . IC1 and history . Hence, mphalos) .CDKN1C mutations are the most frequent abnormality [CDKN1C mutation analysis [CDKN1C mutations, 2.9% (17/584) of referrals with a normal ‘BWS methylation assay’ result were later found to have a CDKN1C mutation . Furthermore, the challenge that detection of low-level mosaicism brings to BWS diagnostics must be considered [As genetic testing becomes less expensive and more accessible, testing is frequently requested by non-specialist clinicians, and it becomes increasingly useful to have criteria for identifying those patients who are most likely to have a positive test. Traditionally, clinical diagnostic criteria are used to select patients for molecular investigations. However, in the case of BWS clinical diagnostic criteria might be satisfied but molecular testing might be negative and, conversely, a molecular abnormality might be detected in a patient who does not satisfy clinical diagnostic criteria – such as in isolated hemihypertrophy. Here, we have focused on identifying a set of clinical criteria that are most predictive of a positive ‘BWS methylation assay’. Initially, we evaluated different sets of proposed clinical diagnostic criteria and, although some performed better than others, none was strikingly superior. We therefore used logistical regression analysis to identify those features most predictive of a positive ‘BWS methylation assay’ test and then developed a weighted clinical scoring system that was demonstrated to outperform previously reported sets of clinical diagnostic criteria -20. Hencormality , a scorianalysis . Similarnsidered . Thus, gnsidered , and thensidered .vice versa, thus improving the overall diagnosis of the disorder. Moreover, in view of the tumour risk to a missed diagnosis of BWS, a molecular diagnostic or specialist genetic referral may still be warranted in individuals with borderline scores (3.0). Indeed, as testing becomes increasingly less expensive, the probability threshold for a BWS methylation abnormality can be reduced in order to improve overall diagnosis at the cost of a less-specific scoring system. Nevertheless, multicentre studies of even larger datasets will enable further refinement of our scoring system – in particular the incorporation of rarer clinical features that would increase the prior probability of BWS  and also allow bespoke predictions for specific BWS molecular subtypes.Although our scoring system focuses on improving the diagnostic rate of BWS, in comparison to current clinical diagnostic criteria, for any given sensitivity it has the highest specificity and In summary, we suggest that this novel tool will facilitate selection of patients with suspected BWS for routine diagnostic testing and so improve the diagnosis of the disorder.We systematically collected clinical and molecular data on children and adults with suspected BWS referred to a molecular diagnostic testing service between April 2003 and October 2013. Clinical data were collected by a standardised questionnaire completed by the clinician who ordered the molecular assay and by inspecting clinical notes. Not all the clinical data requested was available, which is reflected by the variable number of clinical features. Consent for diagnostic testing was provided by the patient or by the parent/guardian for children. The collection of clinical and molecular data to evaluate the diagnostic testing service was approved by the Birmingham Women’s Hospital Research and Development Office.DNA was isolated from peripheral blood lymphocytes or tissue samples by standard procedures. Methylation analysis was performed in the West Midlands Regional Molecular Genetics Laboratory using a methylation-sensitive multiplex ligation-dependent probe amplification (MS-MLPA) kit  and/or pyrosequencing, with both methods giving comparable results ,27. A caP = 0.05. Data were analysed using statistical packages R v3.0.2 (http://www.r-project.org/) and SPSS v20 .We utilised logistic regression in order to determine the predictive power for a positive methylation assay test of current BWS clinical diagnostic criteria Table . We deveAUC: area under the curve; BWS: Beckwith-Wiedemann syndrome; FNF: facial naevus flammeus; IC: imprinting centre; MI: methylation index; MS-MLPA: methylation-sensitive multiplex ligation-dependent probe amplification; pUPD: paternal uniparental disomy; ROC: receiver operating characteristic.The authors declare that they have no competing interests.AI carried out the data analysis and co-wrote the first draft of the manuscript; GK, RPD, JB and DL provided clinical data for analysis; CH and FMacD undertook/supervised molecular epigenetic studies; PN supervised the data analysis; EM conceived and supervised the study and data analysis and co-wrote the first draft of the manuscript. All authors read, critically revised and approved the final manuscript.


Molecular beam techniques are a key to many experiments in physical chemistry and quantum optics. In particular, advanced matter-wave experiments with high-mass molecules profit from the availability of slow, neutral and mass-selected molecular beams that are sufficiently stable to remain intact during laser heating and photoionization mass spectrometry. We present experiments on the photostability with molecular libraries of tailored oligoporphyrins with masses up to 25 000 Da. We compare two fluoroalkylsulfanyl-functionalized libraries based on two different molecular cores that offer the same number of anchor points for functionalization but differ in their geometry and electronic properties. A pentaporphyrin core stabilizes a library of chemically well-defined molecules with more than 1600 atoms. They can be neutrally desorbed with velocities as low as 20 m/s and efficiently analyzed in photoionization mass spectrometry. Copyright © 2015 John Wiley & Sons, Ltd. Compleme≃ 200 fm. Both dev≃ 200 fm.–8 Here, neutral molecules. The analyte itself can act as its own matrix if it is sufficiently absorptive and photostable. The resulting beam velocities can be smaller by an order of magnitude than in common MALDI studies.[Several methods have been developed to launch massive and fragile molecules: electrospray ionization (ESI) an studies.,14The de studies.–17 Large studies.,19 – witmonomer with long fluoroalkylsulfanyl chains[oligomers in the attempt to enhance the molecular stability at even higher mass.Molecules can also be tailored to facilitate volatilization and photoionization. The covalent tagging of biomolecules with ionizable chromophores has already been implemented before,–24 and ayl chains,26 was sλdB = h/mv). We compare two molecular libraries[Applications in quantum interferometry will profit from a molecular library, i.e. a molecular ensemble with well-defined mass separation.,4 At a flibraries as shownlibraries The centlibraries1 and 2, we devised an experiment as shown in Fig.1 in 1.5 ml of pure diethyl ether or 7.5 mg of library 2 in 1.5 ml pure tert-butyl methyl ether. Droplets of this solution without any matrix are deposited on a clean stainless steel plate and dried in air. The N2 laser beam  is focused onto the sample plate under an angle of 30°. During desorption, the sample plate is laterally shifted to ensure that each laser shot hits a fresh sample spot.In order to assess the VUV photoionization properties of library 2 excimer laser beam , focussed into a rectangular shape of 8 ± 1 mm2. The ions are detected by a linear time-of-flight mass spectrometer  in Wiley McLaren[The neutral molecular beam is ionized by the VUV light of an Fy McLaren configur1 consists of eight well-resolved peaks  minus the fluorine atom that is substituted in order to attach the chain. The characteristic abundance of all peaks in this mass spectrum agrees well with MALDI spectra.Figure1 to C10. The mass difference between neighbouring peaks of 457 ± 18 Da corresponds to the molecular weight of a single chain. This signal is attributed to the clustering of fluoroalkylsulfanyl chains in the expanding plume. The C1 chain is abundant during synthesis and was not eliminated.The post-ionization mass spectrum also shows a distribution of low masses, the most prominent one denoted by C2 is shown in Fig.n = 50. Individual peaks can still be clearly resolved up to 25 000 Da. The characteristic distribution after laser desorption/photoionization is similar to MALDI measurements immediately after synthesis.The result of a similar experiment with library λdB = 200 fm, we are aiming for neutral particle beams with a momentum of less than 25 000 Da × 40 m/s.Slow molecules are essential for many experiments, such as beam deflection,32 or ad1 and 2. For a velocity analysis, we select three of the most prominent mass peaks of Fig.Here, we measure the molecular velocity distribution by varying the delay time between the desorbing and the ionizing laser pulse. Figure1, the neutral part of the distribution is best fitted with a Gaussian distribution centred at around v ≃ 90 m/s. There is still a substantial signal at 40 m/s  and n = 40 (library 2). The absence of any significant deviation over time suggests that library 1 is stable over its transit time to the detector. The same is true for library 2, in spite of its double number of chains. Even if there was some fragmentation in free flight – which we do not see in Fig.In Fig.1) is attributed to photoinduced fragmentation. Interestingly, this is not observed for library 2, because it has a larger number of fluoroalkylsulfanyl chains and more relaxation channels.[Figurechannels. Thus, itchannels.,37v < 45 m/s. This matches the initial de Broglie wave requirement λdB ≥ 200 fm. Fluoroalkylsulfanyl-functionalized porphyrins therefore fulfil important expectations for future matter-wave studies. The synthesis of such compounds is demanding, but the concept of fluoroalkylsulfanyl-functionalized particles has certainly not yet reached its limits. It will be intriguing to explore ways to launch and detect neutral mass-specified particles even more complex than those presented here.We have studied the volatilization and post-ionization properties of two new molecular libraries with different core conjugation but similar chains. Both can be efficiently launched at low velocities and photoionized at a laser wavelength of 157 nm even at masses up to 25 000 Da. For the most abundant library element, we still find a substantial signal of neutral particles with velocities


EHP, I want to provide an update to our readers and authors about recent events and changes. My first priority is to thank our associate editors and reviewers, who donate their valuable time and expertise to ensure that peer review is fair and rigorous, and who help EHP publish the most credible and important environmental health research. One measure of their contribution is reflected in the increase in EHP’s impact factor from 7.98 in 2014 to 8.44 in 2015. Over the past 12 months, EHP continued to receive about 120 new submissions per month. We sent about 30% forward for peer review, with two-thirds of those becoming accepted after peer review, for a final acceptance rate of about 20%.Approaching the first anniversary of my becoming Editor-in-Chief of EHP website at http://ehp.niehs.nih.gov/editorial-board/.Special thanks go out to the following associate editors who completed terms of service in 2016: Michelle Bell, Douglas W. Dockery, Bruce Fowler, Donald A. Fox, Eliseo Guallar, Lizbeth Lopez-Carillo, Chensheng (Alex) Lu, and Kyle Steenland. We have added about 20 new associate editors to balance the expertise needed for submissions in the diverse fields that make up the environmental health research community, especially in areas of growth such as epigenetics, metabolomics, climate change, and social factors. The full list of current associate editors can be found on the EHP. In calendar year 2015, a total of 874 reviewers completed at least one review for EHP. We thank them all mightily and extend special appreciation to those who went above and beyond the call of duty to review four or more papers: Sara Adar, Adrian Barnett, Xavier Basagaña, Michael Bates, David Bellinger, James Bonner, Joseph Braun, John Bucher, Jessie Buckley, Jonathan Chevrier, Marc Edwards, Suzanne Fenton, Kelly Ferguson, Francesco Forastiere, Rebecca Fry, Ulrike Gehring, Ghassan Hamra, Jaime Hart, Gerard Hoek, David Hondula, Kazuhiko Ito, Peter James, Margaret Karagas, Marianthi-Anna Kioumourtzoglou, Seth Kullman, Christopher Lau, Robert Laumbach, Johanna Lepeule, Matthew Longnecker, Thomas Luben, Thomas McKone, Hua Naranmandura, Tim Nawrot, C. Arden Pope III, Craig Rowlands, Jason Sacks, Jonathan Samet, Ellen Silbergeld, Gina Solomon, Massimo Stafoggia, David Stieb, Matthew Strickland, Adam Szpiro, Susan Teitelbaum, George Thurston, Marie Vahter, Jennifer Weuve, and Kai Zhang.Many, many scientists serve as peer reviewers for EHP’s Operations Manager, Shaun Halloran. Since joining EHP in the spring of 2015, Shaun has brought his extensive experience in journal management and production to our team. He shepherded us through an important transition to a new manuscript processing system this past spring and helped us institute a number of features designed to improve peer-reviewer performance and efficiency. For example, we now ask reviewers to complete an online checklist to ensure that all aspects of scientific ethics, rigor, reproducibility, and transparency have been evaluated. We believe this is especially helpful for new reviewers and ensures consistency across all reviewers. We also instituted a more detailed expertise classification system to assist associate editors in finding qualified reviewers—please log in to Editorial Manager at http://www.editorialmanager.com/ehp/ and update YOUR expertise—and a new checklist for authors to make sure that all required materials accompany each submission.I also want to acknowledge EHP’s Science Editor, Jane Schroeder, we have also modified our process for preliminary review of all submissions, which includes consistent use of criteria for quality, relevance, and importance, in consultation with an associate editor when needed. Together, the goal of the EHP editorial team is to improve submission and peer-review processes up front and thereby obviate the need for additional revisions at the end of the process. In addition, I am very pleased to announce that we have eliminated a backlog of papers that had been awaiting a final internal review, which will substantially reduce the time to advance publication.In collaboration with EHP is fully online, we are also relaxing historically strict word limits (see EHP’s revised Instructions to Authors at http://www.ehponline.org/instructions-to-authors/). For each type of article, we now provide recommended word limits for the main text only, excluding references, tables, and figure legends. This change is made in the spirit of rigor and reproducibility, emphasizing the need to include in the main text key methods and results to ensure that the design, limitations, and primary conclusions are clear, while using supplemental material for background details and results of secondary analyses that in the past might have been reported as “data not shown.”Given that In summary, we have made peer review more effective and efficient, and have substantially reduced the time to final decision, without sacrificing careful and thorough peer review.EHP Advisory Committee in the coming months, we will begin to address important issues that are under intense discussion in the scholarly publishing community. These include transparency and ethical issues related to open data and data access, as well as a growing interest in the online posting of preliminary manuscripts before they are submitted to a journal for peer review. Discussions about these issues at the 2016 Council of Science Editors meeting (which Shaun and I attended) stressed the need for clear guidelines and sustainable infrastructure to support such efforts.As I convene a new EHP and to receiving your best papers!With these and other challenges ahead, I am looking forward to another successful and productive year for