A comprehensive MCD spectral analysis coupled with a molecular field model of a σπ − d exchange analogous to sp-d interactions in Diluted Magnetic Semiconductors (DMS) renders an enhanced Zeeman splitting and a modified g-factor of −4 for the electrons that mediate the interaction. These studies define an experimental tool for identifying electronic states involved in spin-dependent exchange interactions in organic materials.The origins of spin exchange in crystalline thin films of Copper Octabutoxy Phthalocyanine (Cu-OBPc) are investigated using Magnetic Circular Dichroism (MCD) spectroscopy. These studies are made possible by a solution deposition technique which produces highly ordered films with macroscopic grain sizes suitable for optical studies. For temperatures lower than 2 K, the contribution of a specific state in the valence band manifold originating from the hybridized lone pair in nitrogen orbitals of the Phthalocyanine ring, bears the Brillouin-like signature of an exchange interaction with the localized  Elucidating these exchange mechanisms is very important for magneto-optics and spintronics applications121415Metal phthalocyanines (MPcs) are often presented as the archetype of small molecule organic semiconductors because they are employed in the vast majority of organic optoelectronics applications12d9 configuration of the Cu2+ ion results in a single unpaired electron spin located in a bg1 orbital. Magnetization measurements reveal the presence of an antiferromagnetic coupling between Cu ions, characterized by a Curie -Weiss temperature of −2.6 K10π electrons19202122A comprehensive study of spin exchange interactions in these materials must necessarily start with Copper Phthalocyanine (CuPc), the most studied and best understood member of this family in terms of its electronic and magnetic properties78σ+) and left (σ−) circularly polarized light in a sample, MCD ~ (ARCP − ALCP), in the presence of a DC magnetic field parallel to the k-vector of incident light (Faraday geometry). This difference is a measure of the change in angular momentum projection on the applied magnetic field direction for electrons that absorb the circularly polarized photons in accordance with conservation of angular momentum. Unlike calorimetry or SQUID-based magnetization techniques that probe the statistical ensemble average of all contributions to the net magnetization, MCD directly probes individual contributions of different electronic states to the exchange mechanism responsible for magnetic coupling, by spectrally resolving the net total angular momentum of individual excited states. In the presence of spin-orbit coupling, this is a measure of the electron spin polarization. Furthermore, constant advancements in high magnetic fields/low temperature technologies and state-of the art polarization optics manufacturing techniques enable MCD experiments in small molecule organic semiconductors where spin-exchange and spin-orbit coupling are orders of magnitude weaker than typical magnetic thin films28Since the first optical rotation measurements performed by Michael Faraday2526c-plane (MCD - inactive) crystalline sapphire substrates using the rectangular capillary hollow pen writing technique343536The MCD study reported here employs an organo-soluble copper phthalocyanine (Cu-OBPc), where the octabutoxy groups substituted in the non-peripheral positions of the phthalocyanine ring improve the solubility of CuPc in common organic solvents see  inset, e303233μm. The image contrast originates in the different orientation of the crystalline axes in neighboring grains. In crystalline Cu-OBPc, molecules pack tightly along a particular direction further referred to as the stacking axis1718A typical polarized microscope image of the resulting Cu-OBPc film is shown in MCD and absorption measurements were carried out with the sample mounted in a 10 Tesla (T) Oxford superconducting magnet (Spectromag) equipped with a He3 insert for reaching sub-kelvin sample temperatures. In these crystalline thin films, the molecules stack edge-on with their stacking axis parallel to the substrate. The inset in 00 at 736 nm corresponds to the au1(π) → eg(π*) transition at the HOMO-LUMO gap and is polarized in the plane of the molecule. Q10 at 696 nm is the contribution of vibrational overtones and Q20 located at 658 nm can be assigned as a transition from lower lying σ states in the valence band manifold (eu(σ) → eg(π*)), polarized perpendicular to the molecular plane and vibronically coupled to Q003920 spectral region. The π orbital overlap and the phonon coupling leads to a degeneracy lifting of the electronic states, resulting in a manifold of MCD-active states in that spectral region.4041eu(σ) → eg(π*) transitions (marked with blue arrows in the figure) evolves with magnetic field and temperature in a non-trivial way. The MCD recorded during a magnetic field sweep at 648 nm  displays a marked non-linear evolution with magnetic field  doped with magnetic d-shell ions, in particular Mn, Fe or Cr26484950d-shell spins are aligned with the applied magnetic field. In the case of Cu-OBPc, the MCD never saturates but rather continues to increase linearly with magnetic field at a slope identical to the diamagnetic contribution. In contrast, the MCD of Zn-OBPc at 1.5 K is almost identical to that of Cu-OBPc at 70 K. Since the two molecules have identical ligands and their valence band density of states is virtually identical, as evidenced by electron energy-loss spectroscopyd-shell localized states and the ligand, delocalized π - electrons. In fact, low temperature MCD studies on isolated CuPc molecules in an argon matrixd-shell and π electrons and spin-orbit coupling are present at low temperatures in Cu-OBPc.In order to confirm the distinct evolution of the 648 nm contribution with magnetic field, the MCD values extracted from the spectra in 4546eu(σ) → eg(π*) (648 nm) transition and the corresponding peak energy as a function of temperature at B = 10 T. Since the MCD vs. T curve in our experiment can be interpreted similarly to the temperature dependence of magnetic susceptibility in SQUID measurements, the initial increase in MCD from 0.4 K to 1.5 K, followed by a sharp decay indicates the possible presence of antiferromagnetic coupling between the Cu spins below 2 K, in agreement with previously reported results10d-shell and π electrons and the presence of spin-orbit coupling. Unfortunately, the difficulties associated with performing temperature sweeps in free space optics cryostats for T < 2 K prevented us from establishing whether the MCD vs. T curve in et al.π orbital energy originating from a temperature -dependent interplay between the spin-orbit and vibrational coupling. We can thus conclude that, the spin-orbit coupling is only effective below 50 K for this particular state in the valence band manifold, allowing the observation of an MCD signature of spin exchange at low temperatures and possibly enabling the onset of ordering of Cu spins through an indirect exchange mechanism observed in previous magnetization studiesAnother interesting observation is the MCD peak energy  → eg2(π*) (Q00) transitions. Similarly, features 2 and 3 represent the nominally forbidden eg(π) → eg1(π*) (Q10) that are present in the MCD spectra because of the phonon coupling between the au1 and eg valence band states. Features 7 and 4 are phonon replicas of Q00 and Q10 respectively. Finally, feature 1 is associated with the optically allowed eu1(σ) → eg2(π*) (formerly Q20) polarized along the stacking axis. In the low magnetic field regime  contribution in high magnetic fields is the typical signature of a Zeeman-split degenerate ground state where the higher energy spin state is gradually depopulated as the magnetic field increases. All other features simply decrease in intensity without suffering any major line shape changes throughout the entire magnetic field range.By comparing the high B-field spectra recorded at T = 300 Kau1(π), eg1(π) and eu1(σ) states in the valence band manifold.g-factor mentioned earlier. In this case, the Zeeman splitting of the eu1(σ) state is extracted from the fitted MCD lineshape of feature 1 and the corresponding fitted absorbance spectrum . In most organic molecules, electron g-factors are typically equal to −2 for all orbitals. This is still the case for the eg(π*) conduction band states in CuPc since the unpaired Cu2+ spin is expected to exclusively interact with states in the valence band manifold. Any observed enhancement in the measured g-factors will therefore be the result of exchange interactions with the valence band states. In g-factor of −6 is extracted from the low magnetic field slope, which results in a −4 valence band electron g-factor. While this enhancement is not as spectacular as the one observed in inorganic DMSs, it is consistent with the relatively low (<2 K) antiferromagnetic exchange measured in CuPc. More importantly, it holds great promise for future studies of other species such as CoPc and MnPc, where magnetization studies already indicate the presence of a much more robust magnetic order and ferromagnetism.where, σ − π mixing strongly influences the spin-orbit coupling in CuPc11d-shell electron spins (σ(π) − d exchange will predominately involve the former eu1(σ) orbitals of the ligand that are now mixed with the π states and delocalized over distances larger than the nearest neighbor distance between the unpaired Cu spins located on the bg1d- orbital. Theoretical predictions cited earlier also rely on the molecular orbitals picture of the electronic states, and point towards the eg1(π) orbital in the valence band as the state mediating the AFM coupling between Cu spins. Since the Cu spin is located on a high energy eu1(σ) orbitals which most likely hybridize with the π states in the crystalline phase. Most importantly it indicates that one can take advantage of the large transition dipole of the optically -allowed eu(σ) → eg(π*) transition to optically create spin polarized electrons.The discovery of a spin-exchange coupling in Cu-OBPc from MCD experiments presented here has a threefold significance. First, it proves that spin polarized mobile  electrons can inherently exist in organic systems and identifies which of these mobile electrons are accessible with an applied magnetic field or circularly polarized light. Second, it provides a better understanding of the origins of antiferromagnetic coupling between Cu spins reported in literature. Third, it provides experimental evidence that on spins . In the σ orbitals of the lone nitrogen pair of the Pc ring bears the Brillouin-like signature of spin exchange with the d-shell unpaired Cu spin system. In analogy to the DMS molecular-field model of a temperature and magnetic field dependent electron g-factor, the strength of the exchange mechanism between the delocalized ligand electrons and the d-shell localized spins is reflected in an enhanced g-factor of −4 at low temperatures for electrons occupying a specific ligand orbital. This study directly and unambiguously identifies the delocalized electron state responsible for mediating the indirect exchange mechanism between Cu spins at low temperatures.In conclusion, we performed variable temperature, variable field MCD Spectroscopy studies of Cu-OBPc polycrystalline thin film for temperatures ranging 0.4 K to 300 K and magnetic fields up to 10 T. Evolution of seven MCD -active states identified in the bandgap region of the absorption spectrum was monitored as a function of temperature and magnetic field using a spectra fitting routine. One of these states, originating in the et al.From a broader perspective, these measurements constitute an interdisciplinary approach to probing magnetism in organic semiconductors that bridges quantum chemistry to condensed matter physics. Small molecule semiconductors such as the CuPc investigated here belong to an interesting intermediate regime where electrons are neither completely localized nor quasi-free and a hybrid model that accounts for longer range interactions and the role played by phonon coupling in the organic crystalline phase is perhaps necessary to fully describe all aspects of electronic and magnetic behavior. Many of these interesting phenomena were previously hidden by the overwhelming defects and disorder traditionally omnipresent in organic thin films. This problem is now circumvented by a whole new range of deposition techniques that improve the long range ordering in films to a large degree through careful fine control of deposition parameters. The combination of better deposition control and a magneto-optics technique that can directly address specific excitonic states in the bandgap manifold could lead to the practical realization of engineered thin films with radically different behavior such as the two-dimensional tilted porphyrins lattices that support topologically protected exciton states proposed by Yuen-Zhou Copper(II) 1,4,8,11,15,18,22,25-octabutoxy-29H,31H-phthalocyanine (Cu-OBPc) powder was purchased from Sigma-Aldrich and further purified by recrystallization in THF and ethanolc-plane cut sapphire (MCD-inactive) substrates using a rectangular capillary hollow pen writing technique developed at the University of Vermont by Headrick et al.34Both of these Pc derivatives show increase in solubility in common organic solvents after purification. Crystalline thin films were deposited on ac) while an optical chopper operating at 266 Hertz measured the background signal (Vdc)A ~ Vac/Vdc) and the overall transmittance (Vdc) were recorded using standard lockin amplifier techniques. The cryostat probe allowed for varying the sample temperature from 0.4 K to 80 K. For temperatures larger than 100 K we reproduced the experiment in the 25 T Split-Florida Helix magnet at the National High Magnetic Field Laboratory equipped with a nitrogen cooled custom sample holder.MCD and Absorption measurements were carried out with the sample mounted in a 10 T Oxford superconducting magnet (Spectromag) equipped with a He3 insert for reaching sub-kelvin sample temperatures. The quasi -monochromatic output (bandwidth = 2 nm) of an Oriel 1/4 meter monochromator equipped with a 300 Watt xenon lamp was modulated into left and right circularly polarized light using a piezo-elastic modulator at a frequency of 50 kilo Hertz .


The signature was then used to identify factors that activate or suppress PPARα in an annotated mouse liver/primary hepatocyte gene expression compendium of ~1850 biosets. In addition to the expected activation of PPARα by fibrate drugs, di(2-ethylhexyl) phthalate, and perfluorinated compounds, PPARα was activated by benzofuran, galactosamine, and TCDD and suppressed by hepatotoxins acetaminophen, lipopolysaccharide, silicon dioxide nanoparticles, and trovafloxacin. Additional factors that activate  or suppress (infections) PPARα were also identified. This study 1) developed methods useful for future screening of environmental chemicals, 2) identified chemicals that activate or suppress PPARα, and 3) identified factors including diets and infections that modulate PPARα activity and would be hypothesized to affect chemical-induced PPARα activity.The nuclear receptor family member peroxisome proliferator-activated receptor α (PPARα) is activated by therapeutic hypolipidemic drugs and environmentally-relevant chemicals to regulate genes involved in lipid transport and catabolism. Chronic activation of PPARα in rodents increases liver cancer incidence, whereas suppression of PPARα activity leads to hepatocellular steatosis. Analytical approaches were developed to identify biosets  in a genomic database in which PPARα activity was altered. A gene expression signature of 131 PPARα-dependent genes was built using microarray profiles from the livers of wild-type and PPARα-null mice after exposure to three structurally diverse PPARα activators . A fold-change rank-based test (Running Fisher’s test (p-value ≤ 10 Coordinated efforts are underway by a number of agencies that regulate chemicals to define networks of adverse outcome pathways (AOP) , 2. AOPsSuppression of the ability of PPARα to activate fatty acid catabolism genes can lead to the buildup of fat in hepatocytes. Fatty liver disease is the most common liver disease in humans and encompasses a spectrum of hepatic steatosis which can progress to an inflammatory state (steatohepatitis) sometimes leading to cirrhosis and hepatocellular carcinoma . Fatty lThe ability to accurately predict PPARα activation or suppression would be useful to evaluate the potential for diverse factors including chemicals to contribute to liver cancer or steatosis by affecting PPARα. In the present study, a screen of a gene expression compendium was carried out to identify chemicals and other factors that affect PPARα activation. To facilitate screening, a gene expression signature used to predict PPARα activation or suppression was developed by identifying genes that were consistently altered by three PPARα activators in wild-type but not PPARα-null mice. Coupled with a fold-change rank-based similarity test, the signature was found to be very accurate in predicting activation of PPARα and was used to screen a mouse liver compendium of ~1850 comparisons to find chemicals and other factors that affect PPARα. Our screen identified chemicals, diets and infections that activate or suppress PPARα and could potentially contribute to liver cancer or steatosis, respectively.Fig. 1. A screen for PPARα modulators required 1) a gene expression signature of PPARα-dependent genes, and 2) an annotated database of gene expression profiles  of statistically filtered genes. The PPARα signature is a list of genes with associated fold-change values that reflect average differences in expression between control and chemically-treated mice and that require PPARα for these changes. A commercially available gene expression database provided by NextBio (www.nextbio.com) facilitiated screening. The NextBio database contains over 110,000 lists of statistically filtered genes from over 15,700 studies carried out in 16 species . Each list (bioset) is compared to all other biosets in the database using a fold-change rank-based statistical test called the Running Fishers test which allows an assessment of the overlap in regulated genes and whether those genes are regulated in a similar or opposite manner. In this study, ~1850 biosets from mouse liver, mouse primary hepatocytes or hepatocyte-derived cell lines were evaluated. Available information about each bioset was extracted from NextBio and used to populate a database of information about the experiments. Each bioset was further annotated using information derived from the GEO submission and/or the original publication. Importantly, each bioset was annotated for the type of factor  and the name of the factor  examined. To assess PPARα activation or suppression, the PPARα signature was uploaded to the Nextbio database and compared to all biosets in the database using the Running Fishers test. Results of the test were exported and used to populate the annotated compendium with p-values of each comparison. Test results were used to determine the accuracy of predictions and to further examine those biosets that achieved statistical significance. A number of predictions were tested in independent studies. Additional analyses were carried out using an independently-derived database of biosets used for PCA, determination of the relationship between Running Fisher’s p-value and behavior of genes in the signature, and machine learning classification.The methods used in this study are outlined in Three animal studies carried out as part of this investigation.Study 1. Perfluorononanoic acid (PFNA) and perfluorohexane sulfonate (PFHxS) in wild-type and PPARα-null mice.PPARαtm1Gonz/J, stock #003580) and wild-type mice  were purchased from The Jackson Laboratory  and maintained as an inbred colony on the 129/Sv background at the U.S. EPA, Research Triangle Park, NC. Randomized animals were housed 4 per cage and allowed to acclimate for a period of one week prior to the conduct of the study. Food  and filtered distilled water were provided ad libitum. Animal facilities were controlled for temperature (20–24°C), relative humidity (40–60%), and kept under a 12 hr light-dark cycle. PPARα-null and wild-type male mice at 6–9 months of age were dosed by gavage for 7 consecutive days with either 0, 3, or 10 mg/kg PFHxS, or 1 or 3 mg/kg PFNA  in water. PFHxS was kindly provided by 3M Corp . Four biological replicates consisting of individual animals were included in each dose group used for microarray analysis. Dose levels reflected exposures that produce hepatomegaly in adult mice without inducing overt toxicity. All dosing solutions were freshly prepared each day. At the end of the dosing period, animals were euthanized by CO2 asphyxiation and tissue was collected from the left lobe of the liver for preparation of total RNA. Livers were removed 24-hrs after the last dose and were rapidly snap-frozen in liquid nitrogen and stored at -70°C until analysis.The animals study was carried out at the Reproductive Toxicology Facility of the EPA in Research Triangle Park, NC. Studies were approved by the U.S. EPA ORD/NHEERL Institutional Animal Care and Use Committee. The facilities and procedures used followed the recommendations of the 1996 NRC “Guide for the Care and Use of Laboratory Animals”, the Animal Welfare Act, and the Public Health Service Policy on the Humane Care and Use of Laboratory Animals. Animals were housed in fully-accredited American Association for Accreditation of Laboratory Animal Care (AAALAC) facilities. PPARα-null mice  in wild-type and PXR-null mice.ad libitum. Mice were housed 2 to 4 per cage. All mice were treated once a day i.p. with either vehicle (corn oil) or PCN at 400 mg/kg/day for 4 days. Livers were removed 24-hrs after the last dose. Portions of the livers were rapidly snap-frozen in liquid nitrogen and stored at -70°C until analysis.The animal study was carried out at the University of Kansas Medical Center  and was approved by KUMC Institutional Animal Care and Use Committees. Animals were housed in fully-accredited American Association for Accreditation of Laboratory Animal Care (AAALAC) facilities. Eight-week-old adult C57BL/6 mice were purchased from Jackson Laboratories . PXR-null breeder pairs were engineered and backcrossed into the C57BL/6 background . Adult mStudy 3. 12 treatment study in male and female mice.p-dioxin (TCDD) and PCB-153 were purchased from NeoSyn . Six-week-old male and female B6C3F1 mice (B6C3F1/Crl) were purchased from Charles River Laboratories . Upon arrival, the mice were acclimated for one week prior to initiation of the study. The animals were weighed and randomized into treatment groups to ensure the mean body weight in each group was approximately the same. Animals were housed three per cage except male mice which were housed individually in shoe box cages and segregated by treatment group. Alpha-dri cellulose bedding  was used. Animals had access to reverse osmosis water  and pellet NIH-07 certified feed  ad libitum. The animal room was kept within the standard temperature and humidity parameters (64 to 79°F with 30 to 70% relative humidity) and standard light cycle (0700–1900 hrs). The animal exposures were similar to those employed in a previous study P-treated mice approached significance (data not shown). Recent reports have shown that AhR activators TCDD and B[a]P when given to mice result in disturbances of lipid homeostasis that led to steatosis   in BALB/eatosis . The pattern of PPARα suppression somewhat paralleled the strain susceptibility to APAP-induced damage (C57 > SMJ >DBA > SJL) with strains most susceptible having the most significant PPARα suppression at 6 hrs. PPARα-regulated pathways have been shown to be targets of APAP treatment [Transcriptional responses to acetaminophen (APAP)-induced liver injury were examined in 4 strains of mice for 3 or 6 hrs . APAP sureatment .Fig. 8B), consistent with a number of other reports of suppression by LPS [The antibiotic trovafloxacin (TVX) has caused severe idiosyncratic hepatotoxicity in humans, whereas the structurally similar antibiotic levofloxacin (LVX) has not. Mice co-treated with TVX and lipopolysaccharide (LPS), but not with LVX and LPS, develop severe hepatocellular necrosis . In a ren by LPS , 53 . The pattern of suppression was somewhat consistent with the ability of the particle to cause liver damage, with smaller nanoparticles causing the greatest damage at equivalent doses [Mice exposed intraperitoneally for 6 hrs to particles consisting of 3 different sizes of silicon dioxide caused suppression of PPARα  as well as Coxiella burnetii, Burkholderia pseudomallei, Ehrlichia chaffeenis and Streptococcus pneumonia (data not shown). Suppression of PPARα was usually more severe with longer infection times. As Francisella tularensis and Yersinia pestis are gram negative bacteria, the effects of exposure to LPS (derived from gram negative bacteria) were evaluated. In addition to the bioset derived from female mice with significant suppression of PPARα , six of the 7 biosets in which transcriptional effects of bacterial LPS in the liver were examined in other studies showed suppression of PPARα , with two biosets exhibiting significant suppression including one discussed above  while 2 others approached significance. For the three biosets which exhibited significant suppression of PPARα after LPS exposure, 43 of the PPARα signature genes exhibited consistent behavior for suppression, i.e., decreased expression of genes positively regulated by PPARα and increased expression of genes negatively regulated by PPARα . In particular, there were strong increases in expression of three genes that were negatively regulated by PPARα . A small set of genes  were regulated in a manner similar to that in the signature across the treatments indicating that not all of the signature genes respond predictably to LPS exposure. Although there is at least one study showing the effects of infection on PPARα activation [The 91 biosets from the livers of mice exposed to infectious agents were examined for effects on PPARα. A number of biosets derived from mice following infection showed suppression of PPARα. The species whose infection suppressed PPARα included tivation , the divS1 File.The effects of diet on PPARα are discussed in A signature-based approach was used to identify chemicals, infections, and diets that have an impact on PPARα activity. To create and test the PPARα signature, we capitalized on microarray comparisons from the livers of wild-type and PPARα-null mice after exposure to either chemicals or synthetic triglycerides. Genes that were consistently activated or repressed in a PPARα-dependent manner were identified using a stringent set of criteria including 9 statistical tests and a number of filters ultimately resulting in a final list of 147 probe sets representing 131 genes. Most of these genes have been shown to be responsive to PPARα activators based on published studies annotated in the Comparative Toxicogenomics Database (CTD). Many of the genes included those involved in lipid homeostasis, as expected.Fig. 3F) which was superior to predictions using a number of well known machine learning classification algorithms (S1 File). The signature was able to clearly distinguish between chemical activators of PPARα and activators of other xenobiotic-activated transcription factors . The methods used here have the additional advantage that factors that suppress PPARα can be simultaneously identified. The signature will be a useful starting point for identification of a smaller subset of genes that can be used to reliably predict effects in high-throughput screens of environmentally-relevant chemicals or drugs. Although these genes are applicable for screening chemicals in mouse liver or mouse hepatocyte-derived cell lines, it may be possible to use the signature in other tissues that exhibit relatively high expression of PPARα including heart, kidney, and muscle.To screen for factors that lead to alterations of PPARα, we compared the signature to a gene expression compendium of annotated biosets using the fold-change rank-based nonparametric Running Fisher’s algorithm , analogoFig. 10A, B. Direct binding to PPARα by chemicals is the most widely recognized mechanism by which PPARα is activated. Typical activators of PPARα usually possess a bulky hydrophobic structure and an acidic group or a structural moiety that can be metabolized to an acidic group. The signature accurately predicted activation by compounds containing these structural features including hypolipidemic drugs, perfluorinated compounds, and synthetic triglycerides metabolized to fatty acid activators . A number of other compounds that do not possess these structural properties were found to activate PPARα. These atypical activators included benzofuran, galactosamine, the Nrf2 activator, CDDO-Im, AP20187 and the AhR activator, TCDD . We hypothesize that activation of PPARα by these chemicals is indirect, possibly through perturbation of fatty acid metabolism resulting in increases in endogenous activators of PPARα including fatty acids and eicosanoids. There is growing evidence that TCDD perturbs lipid metabolism and can increase levels of prostaglandin metabolites [S1 File). Future efforts could focus on determining linkages between activation of PPARα and downstream events including lipid metabolism, cell fate and under long-term activation conditions, liver cancer.Comparison of the PPARα signature to the liver gene expression compendium identified diverse factors that affect PPARα. A summary of the factors identified in our study which led to activation or suppression of PPARα is shown in abolites  that actabolites . Other fPpara gene itself. However, decreases in Ppara gene expression do not consistently determine decreases in PPARα activation . The NF-kB subunit p65 (Rela) and the AP-1 subunit (Jun), both important mediators of inflammatory responses in multiple tissues, bind directly to the PPARα protein and inhibit activation at a PPRE [S1 File) that are all known to cause a cascade of effects including increases in liver injury, infiltration of inflammatory cells, secretion of cytokines, and induction of inflammatory mediators. The chemicals included acetaminophen, LPS, trovofloxacin, and nanoparticles. A subset of the high fat diets caused PPARα suppression presumably, because steatosis had progressed to steatohepatitis, although this hypothesis needs to be confirmed independently. A number of infectious agents associated with liver damage and inflammation also caused suppression of PPARα. Future studies could focus on the quantitative relationships between level of PPARα suppression and hypothesized phenotypic consequences of suppression of fatty acid catabolism and transport that can lead to steatosis and under prolonged exposure conditions, steatohepatitus.Mechanisms by which chemicals or other treatments decrease PPARα activity are enigmatic. Decreases in activity could come about through decreases in the expression of the t a PPRE , 61. In t a PPRE . In vivoA number of agencies that regulate chemicals have initiated programs to define the universe of AOPs affected by chemical exposure , 2. ThesS1 FileContains 1) treatment, dose, vehicle, and time of sacrifice of 12 treatment study in male and female mice; 2) classification accuracy of machine learning models used to predict PPARα activation; and 3) effects of diets on PPARα.(DOCX)Click here for additional data file.S2 FileContains 1) list of probesets and genes in the PPARα signature; and 2) description of biosets that activate or suppress PPARα discussed in this study.(XLSX)Click here for additional data file.


Numerourvention , 6.Recent work from our lab has built upon these findings by demonstrating that the inhibin βA transcript is translationally regulated by hnRNP E1 during TGFβ-induced EMT, and the consequent establishment of Activin A autocrine and paracrine signaling is capable of altering the tumor microenvironment and promoting a permissive niche at both the primary tumor site and at secondary metastatic foci . Based oin vivo. These observations come in marked contrast with the observation that despite its ability to promote migration and invasion, Activin A alone is not capable of inducing a complete mesenchymal transition nor does this ligand appear to enhance the transition induced by TGFβ. This lack of EMT induction may be due to deficient Smad2/3 activation or the requirement of parallel non-canonical pathways activated by TGFβ, but not by Activin A, that are required alongside Smad2/3 signaling to induce an EMT. Inhibin βA can therefore be classified as a factor that promotes the invasive phenotype associated with EMT induction and a recently established member of a cohort of ‘EMT signature’ genes regulated by TGFβ at the translational level [The functional significance of inhibin βA upregulation was demonstrated by enhanced cell migration and invasion of mammary epithelial cells when treated with recombinant Activin A. Furthermore, silencing of inhibin βA attenuated the invasive phenotype al level . Thus, tMechanistically, the RNA binding protein hnRNP E1 binds to the 3′-UTR of these mRNAs and regulates their translation in a TGFβ-dependent manner. This represents an unusual case of agonist- or stimulus-dependent upregulation of translation through a 3′-UTR element. Thus, the elucidation of this post-transcriptional regulatory pathway is of note in that it not only identified ‘EMT signature’ genes, but also provided mechanistic information as to how they control TGFβ-mediated EMT. Our data demonstrate that phosphorylation of hnRNP E1 is the trigger for the reversal of translational silencing, resulting in a temporal and spatially controlled increase in protein expression. In the dephosphorylated state hnRNP E1 mediates translational silencing, whereas its phosphorylation, in response to TGFβ, relieves translational silencing and allows transition to the mesenchymal phenotype. These findings may have significant implications towards potential prognostic and clinical applications. If in fact the EMT transition is reflective of the metastatic process, then one might predict that the phosphorylation status of hnRNP E1 may be indicative of metastatic progression and the prognosis of patients.


INK4a is more highly expressed in the human body during senescence, studies on its relevance to handgrip strength among old adults, are relatively sparse. We enrolled 205 community-dwelling old adults aged 65 years and older without specific medical conditions. Handgrip strength of the dominant hand was measured. Low handgrip strength was defined as the lowest quartile of handgrip strength among the participants. RNA was extracted from peripheral white blood cells. Use quantitative polymerase chain reaction to estimate the p16INK4a mRNA expression level. The average handgrip strength was 25.22 ± 8.98 kg, and gender difference was observed. In the linear regression model, the p16INK4a mRNA expression level was significantly negatively associated with handgrip strength in men but not in women. The β coefficient, representing the change of handgrip strength for each increment in the p16INK4a mRNA expression level, was −0.208 (p = 0.024) among old men. The negative association remained after additional covariates adjustment. In the multiple logistic regression model among old men, the odds ratio (OR) of low handgrip strength was 1.246 (p = 0.032). In this study, we observed the p16INK4a mRNA expression level was negative associated with handgrip strength among community-dwelling old men.Although many studies have shown that p16 Low muscle mass with poor physical performance is a crucial problem in geriatric population. A large body of evidence has suggested that poor muscle function could result in increased morbidity, disability, and mortality in long-term follow-up. Handgrip strength is a unique representative performance marker of overall muscular strength used to predict the health outcome in the elderly123INK4a expression in relation to grip strength. The expression of p16INK4a has been shown to increase with age in human subjects. p16INK4a induces permanent growth arrest termed “cellular senescence.” Association studies have suggested that altered regulation of p16INK4a expression may result in human age-associated phenotypes such as type 2 diabetes89101213INK4a expressing cellsIn this study, we intended to investigate an ageing-related molecular marker, p16INK4a expression level and handgrip strength in old persons. This study aims to investigate the connection between aging biomarker and handgrip strength among community-dwelling old adults.Few studies have examined the association between p16This is a cross-sectional, observational study. We conduct this study from one medical center  in northern Taiwan during May, 2012 to April, 2013. People aged 65 years and older who lived in the community of Taipei City eligible for an annual routine geriatric health check-up were the source population. They are functionally independent and relatively healthy in general. All of the participants are Taiwanese older adults. Participants who had cognitive impairment, chest pain and bone pain during exercise, congestive heart failure, regular hemodialysis, or treatment for malignant disease were excluded. At first, trained investigator will screen the subjects by medical record reviewing to find out whether they have the exclusion conditions mentioned above. If they were eligible for this study, the trained investigator will invite them to participate and provide inform consent. The patients’ basic demography, health condition, smoking status, and alcohol consumption and physical activities were reviewed by using a structured questionnaire. Ever smoking in life was defined as a positive smoking status. Alcohol intake was defined as drinking alcohol at least once every week, and was dichotomized. The presence of hypertension was defined according to a self-reported doctor’s diagnosis, the use of antihypertensive medications, or an average blood pressure of ≥140/90 mm Hg. Diabetes mellitus was defined as a self-report of a physician’s diagnosis, a fasting plasma glucose level of ≥126 mg/dL, or the use of diabetic medications . Medical histories of coronary artery disease, stroke, chronic obstructive pulmonary disease (COPD), or arthritis were ascertained through participants’ self-report. The scores of the brief symptom rating scale (BSRS) and Alzheimer dementia 8 questions (AD-8) were obtained by using a structured questionnaire.Twenty milliliters of peripheral blood was collected from the participants. After the screening, there were 210 old adults entered our study. There were three participants with incomplete handgrip strength and gait speed measurement (n = 3), two participants with missing data of gait speed test (n = 2). Finally, 205 participants contributed to the final analysis. The participants provided written informed consent before participation. The protocol was approved by the Institutional Review Board of Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan. (TSGHIRB 100-05-257). The methods were carried out in accordance with the relevant guidelines and regulations, including any relevant details.The handgrip strength of the dominant hand was measured three times with an analogue isometric dynamometer , and the average value was calculated. Low handgrip strength was defined as the lowest quartile of handgrip strength among the participant. Walking time was measured for all participants over a 15-foot distance as fast as they can. Gait speed was calculated as the distance (m) divided by walking time (sec).INK4a with the LightCycler®TaqMan®Master kit to perform quantitative polymerase chain reaction (qPCR) by using Roche LightCycler . The 36B4 gene was used as an internal control. The logarithm of the estimated ratio of p16INK4a repeat cycle number to a 36B4 cycle number (p16INK4a/36B4 mRNA ratio) [Ct(p16INK4a assay)/Ct(36B4 assay)] indicates the relative expression ratio of p16INK4a mRNA. Ct is the fractional cycle number for a threshold fluorescence level to be reached during qPCR. Each assay was performed only once. Using the RNA extracted from Hep3B as the template, the amplification efficiency of p16INK4a and 36B4 were respectively 1.89 and 1.92 (close to 2.0)The total RNA was extracted from peripheral white blood cells with TRIzol reagent  and was reverse-transcribed into cDNA. By using Roche Universal Probe library No. 34 (cat. no. 04687671001) as a specific probe, we measured p1636B4 mRNA ratio. Multi-variates logistic regression analyses between the p16INK4a/36B4 mRNA ratio and low handgrip strength were conducted further. By using an extended-model approach for covariate adjustment, three models were investigated: model 1 = age and health behaviors ; model 2 = model 1 plus chronic diseases ; model 3 = model 2 plus body mass index, white blood cell count, hemoglobin, albumin, BSRS, AD-8 scores and gait speed. All analyses were conducted by using Statistical Package for Social Sciences version 14.0 software .The continuous variables of participants’ characteristics are represented as mean ± standard deviation. Categorical variables are expressed as case numbers with percentage. We used multi-variates linear regression to determine the change of handgrip strength for each increment in the p16/p < 0.001). Men had higher levels of hemoglobin, and more male participants had COPD and arthritis than the females .The characteristics of the study subjects as a whole are summarized in p < 0.001), which is much stronger than other covariates. We also conduct interaction analysis and it revealed a significant interaction between gender and p16INK4a/36B4 mRNA ratio for handgrip strength (p = 0.004). Based on these findings, we performed further gender stratified analyses. In the linear model, the p16INK4a/36B4 mRNA ratio was significantly associated with handgrip strength in men but not in women in a negative manner  among old men. After additionally adjusting for other covariates in Models 2 and 3, the β coefficient was −0.232 (p = 0.023) and −0.236 (p = 0.024), respectively.Initially, we performed a combined gender analysis in the linear regression model. The β coefficient between gender and handgrip strength was 0.754, 0.714, 0.684 from model 1 to model 3  for low handgrip strength was 1.211  after controlling for age and health behaviors (Model 1). After controlling other covariates in Models 2 and 3, the association between p16 INK4a mRNA expression level and low handgrip strength remained unchanged; the ORs were 1.230  and 1.246 , respectively.We subsequently used a logistic regression model to analyze the associations between p16icipants . For eacINK4a mRNA expression level was indicated as a senescence marker in human subjects. Handgrip strength decreased gradually with aging. This study demonstrated the negative relation between the p16INK4a mRNA expression level and handgrip strength among community-dwelling old men. To our best knowledge, this is the first study to examine the association between the p16INK4a mRNA expression level and handgrip strength among old adults.The p16INK4a with aging has been reported to limit the regenerative capacity of pancreatic β-cells17INK4a, which was linked to senescence of neural stem cellsINK4a induces a permanent growth arrest termed cellular senescence. A common variant of single nucleotide polymorphisms close to the p16INK4a genetic region is strongly associated with poor physical function in persons aged 65–80 yearsINK4a expression may result in human age-associated phenotypes such as type 2 diabetes89101213INK4a expression with aging has an impact on human health and leads to morbidity.The elevated expression of p16INK4a expression level and the degenerative tissue or aging organ system; however, studies about the p16INK4a mRNA expression level in muscle tissue as well as functional performance in human subjects were relatively sparse. Different methods were used in functional measurements, such as self-reported questionnaires or short physical performance battery scores; however, the results were inconsistent across studiesINK4a mRNA expression level implies that aging is occurring in the human body. The negative correlation between the p16INK4a mRNA expression level and handgrip strength in our study suggested that the muscle tissue of the upper arm, which is responsible for handgrip strength, degenerates and produces worse performance during the process of aging. This is the first study to explore the relation between the p16INK4a mRNA expression level and handgrip strength in the elderly population. The possible mechanism of p16INK4a expression resulting in muscle loss and subsequently leading to functional decline merits further investigation.The aforementioned studies focus on the relevance between p16INK4a mRNA expression level, an aging biomarker, and handgrip strength with adjustment for multiple covariates that had been reported in previous studies. We observed that the p16INK4a mRNA expression level was negatively associated with handgrip strength in old men. This negative relation also existed among old women; however, it did not reach statistical significance.A number of factors are known to be implicated with muscle strength decline, including a sedentary lifestyle5INK4a mRNA expression level and handgrip strength in different sex groups might be because the mean age of men was higher than that of women, resulting in a relatively low level of p16INK4a mRNA expression in the female participants. Second, comorbidities such as diabetes, obesity, stroke, and COPD were much more prevalent in men than in women, and all of these illnesses accelerate the aging process, leading to an increase in the p16INK4a mRNA expression level. In addition, previous studies disclosed that handgrip strength is additionally influenced by body mass index in men but not in womenINK4a expression level and decrease in handgrip strength.The discrepancy in statistical significance on the negative association between the p16INK4a mRNA expression level was associated with lower handgrip strength. This result indicates the potential role of the p16INK4a mRNA expression level of peripheral white blood cells on poor physical performance estimation, especially the upper limbs. Second, handgrip strength is an easy-to-measure indicator of physical function in clinical practice; regular measurement and comparison to detect aging process related functional decline is important. Moreover, for each increment in the p16INK4a mRNA expression, the odds of low handgrip strength increased 24.6% in men after controlling potential covariates. Although p16INK4a mRNA was obtained from peripheral white blood cells in our study, this association in men may provide a clue for clinical and basic mechanistic research.Our findings have some clinical applications. First, a higher p16INK4a mRNA expression level and handgrip strength could not be established. Second, our study enroll old adults aged 65 years and older who live in the community are relative healthy and ambulatory. Although we try to adjust multiple covariates in the regression model to minimize the possible confounding effects, this observation may not be applied to the elderly population with severe illness leading to the limited generalizability to other population. Third, the estimated power for two-sample comparison of means of p16INK4a/36B4 mRNA ratio between male group with and without low handgrip strength was 62%. However, even though our study had a few sample size and unsatisfied statistical power, the p16INK4a/36B4 mRNA ratio was significantly associated with handgrip strength in men. In addition, the p16INK4a mRNA expression level was measured from peripheral white blood cells, not from muscle cells. Also, we did not estimate the participants’ muscle mass; thus, the distribution of p16INK4a mRNA expression level in different muscle types could not be analyzed in this study. Furthermore, few inflammatory biomarkers were measured in this study; however, the relevant comorbidities mentioned in previous studies were included.There are several limitations in our study. First, this is a cross-sectional study that enrolled only a few participants at only one medical center; moreover, a causality between p16INK4a mRNA expression level is negatively associated with handgrip strength among community-dwelling old men. Further investigations of the causality between the p16INK4a mRNA expression level of peripheral white blood cells and muscle mass, as well as physical performance, are necessary.In this study, we found that the p16How to cite this article: Kao, T.-W. et al. Examining how p16INK4a expression levels are linked to handgrip strength in the elderly. Sci. Rep.6, 31905; doi: 10.1038/srep31905 (2016).


Schistometopum thomense. Variation in pigmentation, morphology and size of this taxon over its c. 45 km island range is extreme, motivating a number of taxonomic, ecological, and evolutionary hypotheses to explain the observed diversity. We conducted a population genetic study of S. thomense using partial sequences of two mitochondrial DNA genes (ND4 and 16S), together with morphological examination, to address competing hypotheses of taxonomic or clinal variation. Using Bayesian phylogenetic analysis and Spatial Analysis of Molecular Variance, we found evidence of four geographic clades, whose range and approximated age (c. 253 Kya – 27 Kya) are consistent with the spread and age of recent volcanic flows. These clades explained 90% of variation in ND4 (CTφ = 0.892), and diverged by 4.3% minimum pairwise distance at the deepest node. Most notably, using Mismatch Distributions and Mantel Tests, we identified a zone of population admixture that dissected the island. In the northern clade, we found evidence of recent population expansion (Fu's sF = −13.08 and Tajima's D = −1.80) and limited dispersal . Color assignment to clades was not absolute. Paired with multinomial regression of chromatic data, our analyses suggested that the genetic groups and a latitudinal gradient together describe variation in color of S. thomense. We propose that volcanism and limited dispersal ability are the likely proximal causes of the observed genetic structure. This is the first population genetic study of any caecilian and demonstrates that these animals have deep genetic divisions over very small areas in accordance with previous speculations of low dispersal abilities.Islands provide exciting opportunities for exploring ecological and evolutionary mechanisms. The oceanic island of São Tomé in the Gulf of Guinea exhibits high diversity of fauna including the endemic caecilian amphibian,  Islands are self-contained natural laboratories and provide the backdrop to many of Darwin's and Wallace's observations that culminated in the theory of evolution pers. obs. 1 Aug 2012) – São Tomé is one of three oceanic islands in the Gulf of Guinea that rose along the Cameroon Line roughly 13 Mya 2, with the highest peak at 2024 m. Over the course of its existence, volcanic activity has reshaped topography Notable for its high endemism in both flora and fauna – including approximately 80 plants a.k.a., caecilians – for which very little intraspecific information exists The presence of salt-intolerant taxa on truly oceanic islands has garnered particular interest because mechanisms accounting for presence imply dispersal across marine environments, but direct dispersal is assumed to be limited by physiology. However, recent phylogenetic studies have suggested the existence of delivery mechanisms, in particular oceanic currents, which carry flotsam Schistometopum thomense (Barboza du Bocage) Schistometopum gregorii, occurs on the far eastern coast of continental Africa (Kenya and Tanzania) Schistometopum ephele Taylor S. thomense  S. thomense, recognizing that criteria used to designate S. ephele Taylor were expressions of sexual dimorphism and of possible clinal variation from north to south on the island, and confirming that the third taxon – S. brevirostre – had been described without knowledge of the initial description S. thomense may have limited dispersal ability receives further support from the finding that this species exhibits Bergmann's rule; Measey and Van Dongen S. thomense, examining genetic variation at the species level offers indirect means to understand microevolutionary processes acting within this fossorial species.The caecilian amphibian S. thomense, we investigated the distribution of genetic diversity using mitochondrial DNA (mtDNA). In particular, we were interested in whether previously proposed taxonomic  or clinal variation  hypotheses could better explain the high phenotypic variation exhibited by caecilians on São Tomé In order to assess population structuring of Schistometopum thomense on the island of São Tomé was was granted by Hon. Arlindo Carvalho, Director General, Ministry of Environment, Republic of São Tomé and Príncipe. Ethics clearance for this study was granted by the Office for the Protection of Human and Animal Subjects, San Francisco State University (Protocol # A3-014).Permission to collect, sample and export Schistometopum thomense (Barboza du Bocage) is an elongate, limbless, burrowing amphibian of the Order Gymnophiona  on the island of São Tomé (Schistometopum gregorii (MW 03231) was used as an outgroup in phylogenetic analyses.In order to assess genetic and chromatic variation within the range of São Tomé . SpecimeSão Tomé . Specime2, 0.033 U Bioline Taq, 10 µL template DNA), and used the temperature profile: 94°C for 7 min., 40 cycles of 94°C for 30 sec., 46°C–50°C for 30 sec., 72°C for 1 min., followed by 72°C for 7 min. Non-hot-start reactions were conducted in 10 µL volumes  and used the temperature profile: 94°C for 7 min., 25 cycles of 94°C for 30 sec., 50°C for 30 sec.-1 min., 72°C for 1 min., followed by 72°C for 7 min. In addition to ND4, approximately 600 base pairs of the small ribosomal subunit 16S were amplified using the primers 16sar and 16sbr Whole genomic DNA was extracted from liver tissue, using either phenol-chloroform μL volumes , incubating at 37°C for 15 min., 80°C for 15 min., then cooling to 4°C. Based on visual strength of bands in an agarose gel, 0.5–1.0 μL Wizard Prep product or 1.0–4.5 μL of ExoSAP-IT reaction product were then used in cycle sequencing reactions.PCR product was cleaned using the Promega Wizard PCR Preps DNA Purification System  following manufacturer's protocol, or with ExoSAP-IT , using the following reaction conditions: 7.5 http://www.ncbi.nlm.nih.gov/BLAST/) in GenBank Cycle Sequencing was performed on either a Perkin Elmer 9600, Perkin Elmer 9700 or BioRad thermocycler, following ABI prism instructions for either Big Dye version 2.0  or Big Dye version 3.1  , depending on which reagent was used. For all samples, amplified products were sequenced in both the 3′ and 5′ directions, using the original PCR primers. Nucleotide order was read by an ABI-PRISM 3100 Genetic Analyzer . Base calls and editing were resolved by eye using Sequencher versions 3.1 and 4.2 . Confirmation of ND4 as the amplified product was made with BLAST searches . Because SAMOVA performs poorly with datasets populated by low sample-size locations, disjunct sampling localities represented by one individual only were excluded and neighboring and/or remaining low sample size locales (n≤2) were grouped with proximate larger populations, creating a dataset of 136 individuals from 16 sample sites (SAMOVA sample sites represented in green and blue and lumped populations circled by dashed red lines in hπsF and Tajima's D) to determine whether any group might be out of mutation drift equilibrium FS and Tajima's D tests are sensitive to frequencies of alleles within a sample and will generate significantly negative values when an excess of rare frequency alleles exists, a condition that is expected after recent population expansion. In the instance that a group returned significant values of either of these tests, mismatch distribution analyses were run t = τ/(2u), in which t is generation time, τ is the age of the expansion in mutational units (estimated in the model of demographic expansion) and u is the sum of the per nucleotide mutation rate for the region sequenced S. thomense, generation time was set at two years based on reported ages at first reproduction for captive females u was estimated from mutation rates of ND4 in amphibians that were available in the literature .To further examine relationships among groups, the full ND4 dataset , the geometric mean of the mutation rate (U) for the markers sequenced was used: T = t/Uμ) per nucleotide site per million years were estimated from amphibian rates for ND4 and 16S in the literature  light yellow, no brown flecks, 2) dark yellow, no brown flecks, 3) dark yellow, light to moderate brown flecking, 4) dark yellow, heavy brown flecking. Colors were scored by two of us (RES and GJM) from live animals or from images of animals (after anesthesia). Any disagreements in scores were resolved by reviewing images of specimens concerned.We followed Nussbaum and Pfrender S. thomense (n = 138) as a nominal response variable (category 4 serving as the reference) in a multinomial logistic model implemented in PROC GLIMMIX of SAS v 9.2 c scores To test whether color variation was best explained by genetic lineages and/or clinal variation, we treated color coding of ND4-sequenced Schistometopum thomense, respectively, collected at 24 sample locations across the island of São Tomé  and 538 additional base pairs of mitochondrial 16S rRNA from 138 and 26 individual nhaplo = 57) revealed two deeply divergent and geographically-concordant evolutionary lineages on the island  of the ND4 haplotype dataset  – within population variance explained only 6.34% of the total island variance. Likewise, variance among populations within groups – described by φSC, which was lower (0.412) – explained only 4.45% of total island variance . We found φCT for this SAMOVA continued to rise marginally (<0.01) when the number of possible groups was increased, but adding groups did not significantly change φCT , but not for the Central+Eastern group.In order to provide a more historical interpretation of matrilineal populations, a second SAMOVA was run, for which the four admixed populations were split into their component Northern and Central+Eastern subsets. As expected, this analysis identified the Northern, Western, Central+Eastern, and Southern groups, generating a nge φCT . As illuh) and nucleotide diversity (π) – was highest in the Western and Northern lineages, slightly less variable in the Central+Eastern lineage, and least variable in the Southern lineage  allowed greater insight into lineage ages. Two distinct divergence periods were identified: one between 200–250 Kya and the other between 20–30 Kya. The first divergence between Northern and other lineages produced the highest point for divergence (HPD) at 253 Kya (95% HPD = 136–435 Kya), and the lineage on the west coast appears to have split very close to this time (average HPD = 215 Kya: 95% HPD = 101–354 Kya). Most recently, the lineage in the extreme south diverged from the lineage in the central and eastern areas around 27 Kya (95% HPD = 2–242 Kya). Although 95% HPDs were overlapping for all of these events, the highest point for the HPD for each of the divergence events was used as an indication of the most likely divergence scenario.IMa2 analysis of a composite ND4 and 16S dataset , allowing an additional calculation of time since the inferred demographic expansion in the North of São Tomé: ca. 167.5 Kya . The Central+Eastern and Western groups also showed conformity to a pattern of recent population expansion based on mismatch distribution tests, but for neither of these groups were Tajima's D or Fu's Fs significantly negative. The Bayesian phylogram shows the same relative ‘ages’ of the lineages and the haplotypes within: apparent ancestral haplotypes were collected from the west of the island . Our AICc ordering of multinomial logistic regressions selected the global model – retaining the explanatory variables of genetic group, geology, latitude and altitude – as the best model , ranking more than 21 AICc units above the next best model and accounting for virtually all the weight among our candidate model set. Despite retaining all explanatory variables, the best model only produced significant Type III effects for genetic group  and latitude . Examination of 95% confidence limits surrounding β estimates indicated that most support for these covariates came from the comparison between dark yellow, unflecked morphs (category 2) to dark yellow, flecked morphs  and number of individuals in each response category (ncolor = ∼35) relative to total model parameters (k = 27), our ability to detect significant effects may have been restricted. For the geology covariate, sample size may have limited the power to detect an effect in some levels, e.g., only one specimen resided on older basaltic lava flows 3–8 Mya , coupled with genetic data, show that S. thomense, as currently recognized, is highly structured for mtDNA and that this structure is concordant with northern, western, eastern+central and southern geographic groupings on the island. Although, a north-south cline is not rejected by our morphological analyses , our mitochondrial genetic results show that this species exhibits a narrow zone of admixture between the two most divergent clades (∼4% minimum uncorrected pairwise distance between ND4 lineages) and ages this split at approximately 0.25 Mya. We require nDNA sequencing to ascertain whether the admixed area contains hybrids between the two mtDNA lineages. However, the mixed nature of color forms in these areas suggests that this is the case. Admixed zones have been found for other amphibians which have arrived on islands Our results suggest that both genetic clades and clinal variation contribute to the strikingly high differences in coloration that led Taylor S. thomense arrived on São Tomé by a single or multiple colonization events remains inconclusive, given the relatively recent estimates of population divergence (relative to island age) and of volcanic flows. Multiple independent colonization events have been suggested for geckos Zosterops spp. and Speirops spp., respectively) Whether The most recent eruptions have been placed at 36 Kya, in geologic samples collected from the islet of Rolas  Ichthyophis sp. from the Western Ghats of India) suggests that caecilians are not necessarily philopatric pers. obs.). An explanation for these two very different results for caecilian taxa may stem from the Indian study being based on a very small sample (n = 18) using relatively undifferentiated mtDNA markers (12S and 16S) in a phylogeographic context Amphibians have been classically described as highly philopatric S. thomense on São Tomé given the neutral behavior of the mutations observed, the geographic congruence of haplotype pattern, and the generalized concordance with morphological data.We emphasize provisional acceptance of our results given potential bias posed by use of a two-gene mtDNA dataset. Because (1) natural selection influences mutations in functional genes, (2) the coalescence of gene lineages to cladogenic events may be slow to reflect organismal phylogeny or gene lineages may diverge within a taxonomic line and insinuate a taxonomic division when one does not exist, and (3) – for most taxa – mitochondrial phylogenies track only the evolutionary history of matriarchal lines, we highlight the fact that cladogenic results reported here require substantiation with nDNA. However, we note that single-gene mtDNA histories have proven informative in many studies Schistometopum thomense on São Tomé has a strongly differentiated population structure shown by mtDNA, something that was hitherto unknown for any species in this amphibian Order. Our study confirms previous hypotheses concerning clinal latitudinal variation S. thomense and S. ephele), although applying genetic barcoding standards to 16S Abstract S1Resumo. Abstract in Portuguese.(DOCX)Click here for additional data file.Table S1Specimen Data.(DOCX)Click here for additional data file.


The influence of temperature on the formation of products is optimized to grow WS2 nanowires covered with nanoflakes. Current-voltage and resistance-temperature measurements carried out on random networks of the nanostructures show nonlinear characteristics and negative temperature coefficient of resistance indicating that the hybrids are of semiconducting nature. Bottom gated field effect transistor structures based on random networks of the hybrids show only minor modulation of the channel conductance upon applied gate voltage, which indicates poor electrical transport between the nanowires in the random films. On the other hand, the photo response of channel current holds promise for cost-efficient solution process fabrication of photodetector devices working in the visible spectral range.In this work, WS Since the band gap values of TMDCs are comparable to that of Si (1.1 eV), transistor structures with similar low voltage operation are expected. As demonstrated lately, extremely high on/off channel current ratios  are recently getting an enormous attention due to the their two-dimensional graphene-like layered structure and semiconducting behavior12+) in the layered material to make the adjacent sulfide layers more separated or expanded  by which the van der Waals interaction is weakened. Then a mechanical force is applied (e.g. sonication) to separate the intercalated layers that become suspended in the solvent in the form of mono or few-layered flakes1620Chemical1112142 nanowires and nanowire-nanoflake hybrid materials by a simple single-step sulfurization from hydrothermally grown WO3 nanowires. The geometry of the prepared nanostructure is novel, as the flakes reside directly on the surface of nanowires and are a direct continuation of the nanowire crystal structure. This direct crystallographic interconnection between two dimensional and one dimensional structures hold the possibility to exhibit new optical and electrical properties. The obtained semiconducting hybrid nanomaterials display semiconducting behavior with direct band-to-band transition suggesting applications in field-effect transistors and particularly in photodetector devices without the need for manipulating individual nanoparticles.Both mechanical and chemical exfoliation methods are tedious, and the immobilization of individual single TMDC flakes on chips for testing or to produce devices for commercial purposes is cumbersome. Accordingly, robust methods to fabricate nanoflakes of TMDCs would help in the practical exploitation of these fascinating materials. In this work, we discuss the synthesis of WS3 nanowires were synthesized by the hydrothermal method as described in our earlier work2WO4·2H2O  and 22.49 g of Na2SO4  were dissolved in 600 mL distilled water and the pH = 1.5 was adjusted using 3 M HCl solution. After 10 min stirring, the solution was transferred to an autoclave (Parr Series 4520 Bench Top Reactor with PTFE lining) and stirred (35 rpm) for 48 h at 180 °C under autogenic pressure. The product suspension was collected, centrifuged , washed with distilled water and ethanol and then dried at 60 °C for 24 h.WO2 nanomaterials were obtained by a sulfurization of the WO3 nanowires. Sulfur powder  and WO3 nanowires (0.1 g) were placed in two different alumina boats at the center of a tubular reactor . Before sulfurization, the reactor was evacuated to a base pressure of ~1 Torr and filled up with N2 at 1 bar. The evacuation and flushing steps were repeated three times to minimize O2 in the reactor. Sulfurization is performed for a period of 10 min at 500, 600, 700 or 800 °C in a flow of 400 sccm N2. Structural characterization. The crystal phase and microstructure of the obtained material is studied by the means of X-ray diffraction , micro-Raman spectroscopy (Horiba Jobin-Yvon Labram HR800 at λ = 488 nm), as well as scanning  and transmission electron microscopy (FEI Tecnai G2 20 X-Twin at 200 kV acceleration) techniques.WS2 nanowire-nanoflake hybrid field-effect devices were fabricated on Si/SiO2 substrate . The source and drain Au microelectrodes (300 nm thick Au sputtered on 45 nm Ti adhesion layer) were defined by photolithography. The WS2 nanowire and nanoflake hybrids were dispersed in acetone and deposited on the electrodes by drop casting. Temperature dependent current-voltage analysis was performed by probing the chips in a Linkam TMS 94 heating stage in air atmosphere using a computer-controlled Keithley 2636A source meter. The transfer characteristics were measured in a bottom-gated FET arrangement, while the optical properties were analyzed by illuminating the channel of the transistor devices using a standard collimated RGB light-emitting diode .WS2.The flakes are linked to the nanowires and look like being partially peeled off from the surface rather than subsequently attached to the nanowires. Although the growth mechanism for WO3 nanowires is now well understood and has been discussed in literature, the formation of WS2 nanowire-nanoflake hybrid is not yet clear. Shortly, one dimensional WO3 forms as follows: Initially a WO3 crystal nucleus is formed, which is then followed by WO3 primary prism particle formation due to the presence of Na+ and/or K+, which is present due to the formation of NaCl as byproduct242 nanoflake formation however is less clear. It is believed that any minor mechanical stress caused by the transformation of oxide to sulfide might induce easy sliding and fragmentation of the layered WS2 sheets. As demonstrated earlier, similar but nanomechanical cleavage of MoS2 layers from a single crystal using an ultra-sharp W tip may be achievedThe cleaned product of the hydrothermally synthesized nanowires  shows a 3 nanowires are of the WO3 hexagonal crystal phase [PDF 01-075-2187]. In the course of sulfurization however, the diffraction pattern changes particularly when the reaction temperature is increased suggesting the conversion of WO3 to WS2. Comparing the relative intensities of the most intensive reflections, it may be concluded that the WO3 phase dominates until 500 °C, while at 600 °C and above the hexagonal WS2 [PDF#841398] phase appears as the major constituent of the samples, with a complete oxide to sulfide transformation occurring at 800 °C. In the course of the reaction, oxygen in the anionic sites of the WO3 crystal is replaced by sulfur resulting in the formation of WS3, which then decomposes to WS2 as WS3 → WS2 + 1/8 S8X-ray diffraction indicates the hydrothermally synthesized WO2 structure. Structures were also examined by SAED technique .Since complete transformation of the oxide to sulfide takes place only for the samples treated at 800 °C, the discussion will focus only on this product. High-resolution transmission electron microscopy was performed to observe the morphology and the layer separation in detail . The meaechnique , where dWS2. XRD  and EDS WS2. XRD  techniqu2g mode (at ~352 cm−1) and to the out-of-plane A1g mode (at ~420 cm−1) of crystalline WS2. As reported recently, the intensity ratios and peak frequencies of the WS2 Raman modes can give information about the number of layers282 flakes though, the peak ratios of Raman spectra do not allow the exact determination of layer numbers, as the most prominent change in peak ratios happens at structures having 4 layers or less, as it was discussed by earlier papers. As a result of this, the determined intensity ratio of 0.5 of the peaks for the in-plane E2g1 and out-of-plane A1g phonon modes can merely confirm that the prepared WS2 structure is indeed multilayer, which result is also in good agreement with our TEM observations.Peaks in the micro-Raman spectrum of the sample synthesized at 800 °C  can be amode at ~52 cm−1 a2 interface, the contacts between the nanohybrids in the random network are also contributing to the contact barriers. Resistance versus temperature measurements (2-probe) carried out in air between 22 °C and 150 °C  = R0·eE/kT) vs. T−1) gives an excellent fit for our dataset with a reasonable barrier of 0.29 eV obtained from the slope of the fitting parameter, thus thermally activated conduction is a plausible mechanism. Note, the variable range hopping model for 3-dimensional conduction (in which ln (σ) is linearly proportional to T−1/4, where σ is the conductivity)30Current-voltage curves measured by 2 and 4-probe setups display slightly nonlinear current-voltage characteristics . Since td 150 °C  show a d2 nanohybrid materials to or knowledge have not been studied yet. In order to investigate the electrical characteristics of the nanohybrid networks, bottom-gated field-effect devices are prepared on a Si chip of both high and lower synthesis temperature materials. By sweeping the source-drain voltage (Vds) between the Au electrodes the current is measured in the WS2 channel, while a gate voltage (Vg) through the conductive Si/Al/Au substrate is applied. Materials prepared at lower temperatures (500 and 600 °C) exhibited no field effect behavior  and dISD/dVG is the transconductance (~2.2 × 10−10 A/V) estimated from the I–V slopes measured at different gate voltages 372 from MoO3 and S precursors (0.02 cm2V−1s−1)2 from WO3 and S precursors (0.01 cm2V−1s−1)2 materials exhibit higher carrier mobilities due to the smaller number of crystal interconnections as opposed to the smaller nanowire materials where charge needs to pass through a higher number of junctions. Such examples are single crystal WS2 with d ≈ 20 μm crystal size (100 cm2V−1s−1)2 films (9–15 cm2V−1s−1)2 (80 cm2V−1s−1)2 nanotubes (50 cm2V−1s−1)2 nanoparticles instead of thick high density random networks between the electrodes, which will result in a smaller number of crystal interconnections between the electrodes.The measured value is comparable with results found for MoS2V−1s−1) of WS2 in the channel is at least an order of magnitude higher than in the devices with high-density WS2 nanohybrid networks supporting our hypothesis regarding the performance-limiting effect of excessive numbers of contacts in the films.To verify this assumption, transistors having only a few nanowire-nanoflake hybrid particles connecting the source and drain electrodes were fabricated. The corresponding current-voltage characteristics as shown in 3 after sulfurizationOptical measurements of powder samples were carried out to analyze further the properties of the samples. By using a spectrophotometer system  equipped with an integrating sphere, we measured total reflectance within the 400–1100 nm spectral range from the scattering powders mounted on a microscope slide with double-sided Scotch tape. Then we calculated the optical absorption, from which the band gap of the materials synthesized at various conditions was derived. From the Tauc plot for direct interband transitions  we foundThe photoelectric response of high density network FET devices is studied by illuminating the chips with light-emitting diodes (LEDs) of different color. As displayed in 2 nanowire-nanoflake hybrid materials by a simple sulfurization of WO3 nanowires at elevated temperatures. Field-effect transistors are demonstrated using bottom gated chips with channel made of random networks of the hybrid material drop casted between source-drain electrodes. The same structures were also applied as light detectors with good response to visible photons. The performance of both FET and photodetector devices is limited by the large contact resistance between the hybrid nanomaterials in the random network, which may be overcome by using optimized device structures having only a few nanowire-nanoflake hybrid particles in the channel. The work presented here suggests that nanowire-nanoflake hybrids of WS2, which can be synthesized by using simple chemical methods in large quantities, are competitive counterparts of few layer chalcogenides in electrical and optoelectronic applications.We have synthesized layered semiconducting WSHow to cite this article: Alene, G. et al. A novel WS2 nanowire-nanoflake hybrid material synthesized from WO3 nanowires in sulfur vapor. Sci. Rep.6, 25610; doi: 10.1038/srep25610 (2016).


We previously reported that remote limb ischemic conditioning  during acute stroke confers neuroprotection, possibly due to increased cerebral blood flow (CBF). Vascular cognitive impairment (VCI) is a growing threat to public health without any known treatment. The bilateral common carotid artery stenosis (BCAS) mouse model is regarded as the most valid model for VCI. We hypothesized that RLIC  will augment CBF during chronic cerebral hypoperfusion (CCH) and prevent cognitive impairment in the BCAS model. BCAS using customized microcoil was performed in C57/B6 male mice to establish CCH. A week after the BCAS surgery, mice were treated with RIPostC-therapy once daily for 2 weeks. CBF was measured with laser speckle contrast imager at different time points. Cognitive testing was performed at 4-week post-BCAS, and brain tissue was harvested for biochemistry. BCAS led to chronic hypoperfusion resulting into impaired cognitive function as tested by novel object recognition (NOR). Histological examinations revealed that BCAS triggered inflammatory responses and caused frequent vacuolization and cell death. BCAS also increased the generation and accumulation of amyloid beta protein (Aβ), resulting into the loss of white matter (WM) and myelin basic protein (MBP). RIPostC-therapy showed both acute increase as well as sustained improvement in CBF even after the cessation of therapy for a week. RIPostC improved cognitive function, inhibited inflammatory responses, prevented the cell death, reduced the generation and accumulation of Aβ, and protected WM integrity. RIPostC is effective in the BCAS model and could be an attractive low-cost conventional therapy for aged individuals with VCI. The mechanisms by which RIPostC improves CBF and attenuates tissue damage need to be investigated in the future.The online version of this article (doi:10.1007/s12975-014-0374-6) contains supplementary material, which is available to authorized users. In the The pathological hallmark of VCI is white matter (WM) damage from ischemia in the periventricular regions and centrum semiovale , 5. The Remote limb ischemic conditioning (RLIC) is the simple, inexpensive, and safe use of repetitive inflation-deflation procedure of a blood pressure (BP) cuff on the arm or leg to protect distant organs such as the brain, heart, and kidney from ischemic injury . A numbeThere are a number of proposed animal models for VCI. In a recent review of all mouse models for VCI and vascular dementia , Bink anN = 5), an untreated BCAS/VCI-induced group (with RIPostC sham procedure), i.e., BCAS group (N = 7), and a VCI-induced RIPostC-treated BCAS + RPostC-group (N = 8). The outcome measures were blinded. Cognitive function and CBF changes were considered as the primary outcomes to calculate the sample size of 5 in each group to provide >80 % power at α = 0.05, as reported by us earlier [The Institutional Animal Care and Use Committee of Georgia Regents University (GRU) approved the experimental procedures as per the National Institute of Health guidelines. C57BL/6 J wild-type male mice (10 ± 1 weeks old) were procured from Jackson Laboratory  and were housed in GRU’s AAALAC accredited facility. In these experiments, we use the term “remote ischemic postconditioning” (RIPostC) as we start the conditioning 1 week after the VCI procedure for 2-week period. We randomized 20 mice into 3 groups: a sham-operated group for procedures of BCAS and therapy /(Tn + Tf), where Tn is the time spent by the mouse with the novel object and Tf indicates the time spent with the familiar object.Please see the 42 were performed by using anti-MBP and anti-Aβ antibodies , respectively. ELISA assay for Aβ42 in the brain tissue was performed using Aβ42 selective commercial kit and following the manufacturer’s protocol . Real-time quantitative PCR was performed as reported by us [At day 28, brain tissues were harvested after perfusion sacrifice, and the two hemispheres were immediately separated. One of the hemispheres was fixed in the chilled buffered formalin (10 %) for neuropathology and immunostaining, while the other one was snap frozen in liquid nitrogen for tissue biochemistry. Hematoxylin and eosin (HNE) staining was performed to estimate cell death and score the severity of pathology as reported by us . Luxol fed by us .p < 0.05).CBF was compared using repeated measure ANOVA only between the BCAS and BCAS + RIPostC therapy groups because the Sham group did not undergo any occlusive procedure, which can affect CBF. Other data were compared between all three groups using ANOVA. Wherever mentioned, Means with different letters are significantly different while “ns” stands for “no significant” difference (p = 0.03) in CBF after 7 days of RIPostC therapy in the BCAS + RIPostC group when measured the same day (day 14 post-BCAS) and 1 h after the treatment (161 ± 10 vs 192 ± 20 PU). We further continued the RIPostC therapy for 1 week more up to day 21 post-BCAS, and then discontinued it for the next 1 week. When measured at day 28, we again found significantly (p = 0.0001) increased CBF in the BCAS + RIPostC group as compared to the BCAS group (193 ± 23 vs 265 ± 23 PU). This demonstrates a sustained effect on the CBF even after the cessation of RIPostC therapy. We performed NOR test, which reliably evaluates the nonspatial working memory of the frontal subcortical region [Tn (p = 0.0004) as well as DI (p < 0.0001) in comparison to the Sham group. This indicates that BCAS causes impairment of discriminative ability of the mouse toward novelty of an object. RIPostC therapy in the BCAS + RIPostC group significantly restored the Tn (p = 0.0336) and DI (p = 0.0256) scores as compared to the BCAS group. In conclusion, the data demonstrate that BCAS results in the loss of working memory while RIPostC attenuates this deficit.Figure l region . The resChronic cerebral hypoperfusion triggers inflammatory responses, which impair microvascular function, increase generation of Aβ and decrease its clearance, and cause neurodegeneration deeper into the WM , 19–21. 42 (4-fold increase) in the brain of BCAS group as compared to the Sham group (p < 0.0001). RIPostC therapy for 2 weeks decreased the Aβ42 content (2-fold decrease) in BCAS + RIPostC-group as compared to BCAS group (p = 0.0051). Since Aβ plaque is a hallmark of dementia and AD-type pathology, we next investigated the expression and accumulation pattern of Aβ in the different regions of the brain. At 28-day post-BCAS, we did not find major plaque formation, but the expression of Aβ was increased after BCAS in every region of the brain  RIPostC therapy significantly increased the CBF in a sustained fashion and improved cognitive function. CBF remain increased for at least 1 week even after the cessation of long-term RIPostC therapy  is a European multicenter collaboration with the aim to predict disability in the patient aged 65–84 years with leukoaraiosis . These pN = 20), but the study is randomized, blinded, and adequately powered. The effects observed were robust, however, indicating that large sample sizes are not needed. Second, while the BCAS model is regarded as the most valid for VCI, it does not recapitulate small vessel disease, the underlying pathophysiology of VCI. While the model produces chronic hypoperfusion, it does this via obstruction of the larger blood vessels affecting microvascular flow but the pathology is itself not intrinsic in the smaller vessels. Moreover, the disease process has a defined start whereas in humans, it is insidious with slow onset. Third, we treated and followed these mice for a relatively shorter period of time, 28 days. Fourth, we used young male mice, and VCI is a disease of the elderly. Fifth, in this translation report, we did not investigate the mechanism of protection by RIPostC. We now have ongoing work to test RIPostC therapy in aged mice of both sexes; MRI to see WM changes, and study the possible mechanism of vascular protection by long-term RIPostC therapy.Our study has several limitations. First, our number of animals is small Below is the link to the electronic supplementary material.