A triple-isotope approach could thereby pinpoint populations or subpopulations that have distinct origins. Our results further corroborated stable isotope analysis as a powerful method to delineate animal migrations in Europe.Despite a commitment by the European Union to protect its migratory bat populations, conservation efforts are hindered by a poor understanding of bat migratory strategies and connectivity between breeding and wintering grounds. Traditional methods like mark-recapture are ineffective to study broad-scale bat migratory patterns. Stable hydrogen isotopes (δD) have been proven useful in establishing spatial migratory connectivity of animal populations. Before applying this tool, the method was calibrated using bat samples of known origin. Here we established the potential of δD as a robust geographical tracer of breeding origins of European bats by measuring δD in hair of five sedentary bat species from 45 locations throughout Europe. The δD of bat hair strongly correlated with well-established spatial isotopic patterns in mean annual precipitation in Europe, and therefore was highly correlated with latitude. We calculated a linear mixed-effects model, with species as random effect, linking δD of bat hair to precipitation δD of the areas of hair growth. This model can be used to predict breeding origins of European migrating bats. We used δ Bats are the only mammals to have conquered the aerospace by the evolution of powered flight, an ability which has allowed them to colonize almost all habitats worldwide. Some species have adapted to urbanized environments and live in close proximity to humans, and yet the feeding and roosting ecology of many bats remain enigmatic and their movements difficult to assess due to their small size and their cryptic and nocturnal habits. Like birds, bats can move to new habitats more readily than most terrestrial mammals. However, unlike birds, evidence of long-distance movement in bats was not documented until the mid-20th century, when the use of mark-recapture (banding) in bats started to yield first results More than one million bats have been banded in Europe over the past 80 years http://www.cms.int/species/eurobats/bat_text.htm). Moreover, all temperate-zone bats move between summer and winter roosts. Depending on the extent of migratory behavior, European bat species have been classified as long-distance migrants, regional migrants, and sedentary, but some species are also described as occasional migrants or vagrants As a result, little remains known about which bat species perform long-distance migrations in Europe, their migration routes, and possible population-specific differences in migratory strategies. This is a fundamental gap of knowledge in bat ecology that limits the efficiency of conservation efforts, since identification of both wintering and breeding regions is critical for the protection and long-term persistence of bat populations The discovery that the stable isotopic composition of animal tissues reflects the isotopic ratios of local water and food sources motivated scientists to use the large-scale isotope patterns present in the hydrosphere and terrestrial landscapes (isoscapes) to quantify movement magnitudes and link breeding and wintering areas of migratory animals, such as birds, butterflies, fish and elephants and even humans Lasiurus cinereus sampled throughout North America. They calculated the relationship between δD in precipitation water (δDp) and δD in bat hair (δDh) using data collected in different seasons. Because no previous studies using hair of bats of known origin had been conducted, they assumed that net isotopic discrimination between hair and local water was −25‰, as previously reported in birds. They estimated the period of hair molting as the time when the difference between δDp and δDh was closest to this value. The results supported the general observation that bats molt once per annum during summer at their breeding areas p and δDh using only data from the defined molting period, and used this regression to estimate migratory distances. Britzke et al. p and δDh for four North American bat species, one long-distance migrant and three regional migrants. As δDh in migrants do not necessarily reflect δDp of the sampling location, they collected hair during the breeding period when the bats are presumed to be sedentary and growing new hair. They found a weak relationship between δDh vs. δDp, and δDh vs. latitude for most species and sex/age categories. Moreover, the regressions were inconsistent among species and categories. Inter-individual and inter-specific differences in molting times and fidelity to the breeding areas were proposed as some of the reasons that possibly led to these inconsistent results Few studies have applied δD analysis to the study of bat migration. In a pioneering work, Cryan et al. p or some other geographical covariate  of the location of hair formation are good predictors of δDh of European bats. We chose to test this relationship using hair of sedentary bat species with a relatively broad distribution across Europe. In sedentary species that spend the summer and winter at nearby roosts (<50 km apart), the isotopic composition of hair should reflect that of local water and diet, so samples were collected at any time of the year. We developed a linear mixed effects model of δDh vs. δDp for bats of known origin with species as random effect, and further tested the relationship of δDh with other geographical covariates. It was anticipated that robust spatially calibrated regression models could later be used to estimate breeding origins of migratory bats. Since multiple isotope tracer approaches have the potential to better assign individuals to origin 13C and δ15N could help further discriminate among sampling sites that overlapped in their δD values. Based on our results, we discussed the potential and limitations of stable isotopes as a means for inferring breeding origins of European migratory bats.To apply hydrogen isotopes to the study of bat migration in Europe, it must first be firmly established that δDSampling throughout Europe was conducted by an international network of bat scientists who collected hair samples for this study during their own bat research activities. Sampling took place from 2005–2009. The bats were captured either at roosts or in foraging areas, or occasionally, hair was taken from bats found wounded or dead, e.g. dead bats found under wind turbines. All persons responsible for hair collection were qualified and experienced bat researchers who had bat capture and sampling permits issued by the competent environmental authority of their study regions.Barbastella barbastellus, which rarely covers distances of more than 20 km between summer and winter roosts Eptesicus serotinus, for which summer and winter roosts are usually less than 40–50 km apart Eptesicus isabellinus occurring in Southern Iberia E. serotinus and forming extremely philopatric maternity colonies Plecotus auritus and P. austriacus, both with seasonal movements in the range of a few kilometers We selected hair samples from five bat species reported to have sedentary habits: All stable hydrogen, carbon and nitrogen isotope analyses of bat hair were conducted at the stable isotope lab of the Leibniz Institute for Zoo and Wildlife Research (IZW) in Berlin.For δD analysis, we used the comparative equilibration method described by Wassenaar and Hobson We prepared five in-house hair standards, collected from locations that spanned the latitudinal range of bat sampling sites: sheep from Sweden (SWE-SHE), sheep from Germany (GER-SHE), sheep from Spain (ESP-SHE), horse from Spain (ESP-HOR) and goat from Tanzania (AFR-GOA). Each hair type was obtained from only one animal and came thus from a single location.n). δDn (mean ± SD) of the hair standards were: SWE-SHE: −167.9±1‰; GER-SHE: −136.8±1.5‰; ESP-SHE: −108.3±1‰; ESP-HOR: −98.7±0.5‰; AFR-GOA: −66.3±0.9‰. All hair standards had similar exchangeable hydrogen properties (8–10%).Hair laboratory standards were washed to remove dirt, dried and cleaned of surface oils by soaking for 24 hours in a 2∶1 chloroform∶methanol solution, decanted and dried again in a fume hood to remove the solvent. 50 g of each standard type were powdered using a Freezer Mill 6800 . The resulting powder was sieved to a particle diameter size of <63 µm  and blended to ensure best possible homogeneity of the standards. These five lab standards were analyzed at the Stable Isotope Laboratory of Environment Canada, Saskatoon, 5 times each using the offline steam equilibration method using the keratin references for normalization described in detail in Wassenaar and Hobson Bat hair samples were solvent cleaned in a 2∶1 chloroform∶methanol solution and dried in the same way as the standards, but were not powdered, as this would be impractical due to long processing times; this procedure is similar to that long used for feathers 2, N2, and CO. The resolved H2 sample pulse was introduced into the isotope ratio mass spectrometer . Isotopic ratios, corrected for instrumental drift, were expressed in typical delta notation as units per mill (‰) and normalized on the VSMOW-SLAP standard scale. Analytical precision based on the repeated analyses of unequilibrated powdered hair standards was ±1.02‰. All bat hair samples were analyzed in duplicate. When the difference between the two measurements of one individual was greater than 10‰, two further analyses were performed. If no outlier was detected, the mean value of the four measurements was used for the statistical analyses.The standards and bat samples were pyrolized in a high-temperature elemental analyzer  operating at 1450°C and containing a silicon carbide (SiC) tube filled halfway with glassy carbon chips. Helium was used as carrier gas with a flow rate of 100 ml/min. The gas chromatograph, operating at 70°C, separated H13C and δ15N analysis, samples were weighed to a target weight of 0.35±0.05 mg and placed into 4×6 mm tin capsules. Samples were combusted using a Flash EA 1112 Series elemental analyzer  and the carbon and nitrogen stable isotope ratios of the combusted organic material were measured using a Delta V Advantage isotope ratio mass spectrometer  operating in continuous flow mode. Carbon and nitrogen isotope ratios were reported in the delta notation in units per mill (‰) relative to V-PDB or AIR-N2 scales, respectively. Our laboratory standards (tyrosine and leucine) were calibrated with the international standards NBS 19, NBS 22, USGS 24 and L-SVEC for carbon and IAEA-N-1, IAEA-N-2 and IAEA-NO-3 for nitrogen.For δp) associated with the UTM coordinates of the hair sample locations (h and possible explanatory variables (see below) using R.2.10.1 P-value at 0.05. We first calculated a correlation matrix (Pearson's product moment correlation r) between all variables  to get a first impression of their interrelation. We then developed two different models to test for spatial dependencies, (a) for δDh from bats of known origin vs. δDp and (b) another model disentangling the relationship of δDh with geographical covariates latitude, longitude and elevation. (a) To assess the appropriate model structure, we used a likelihood ratio test (L) for comparing a fixed effects model fitted by generalized least squares (GLS) with two mixed effects models, one containing a random intercept, and one with random intercept and slope (package nlme) following Zuur et al. P-values based on L using the correction provided by Verbeke and Molenberghs h vs. δDp. (b) We then used the single spatial variables longitude, latitude and elevation including their three-way interaction and calculated a mixed effects model with species as random intercept. Before entering the independent variables, we used generalized additive models with three knots  to visually check the linearity assumption of the variables, including species as a fixed factor. Non-linear variables were included as quadratic term. For both models, we finally used the Kolmogorov-Smirnov normality test with Lilliefors correction to test for homogeneity in residuals (package nortest).We used Arc GIS 9.3.1 to extract δD of mean annual precipitation values (δDocations  from 10′13C and δ15N) to discriminate potential origins using only Eptesicus sp., i.e. the species group with the largest sample size. We removed sampling locations for which number of individuals was <4, and tested with a multivariate analysis of variance (MANOVA) in SPSS Statistics v. 19 for differences in δD, δ13C and δ15N between all remaining locations. We performed discriminant function analysis (DFA) to test whether the location of origin for a particular hair sample could be predicted based on the triple isotopic assays (performed with SPSS Statistics v. 19). DFA was used to assign samples  to groups that were defined in advance , and generated linear combinations of the variables  that were then used to separate the groups based on minimizing distances  among the samples within groups We further evaluated the utility of a triple isotope approach  ranged between 2.7 and 12.0‰  significant correlations between δDh of sedentary bats and δDp . Among the three isotopic markers, δDh and δ13C were significantly correlated; δ13C and δ15N were weakly but significantly correlated  The likelihood ratio test showed that the model containing species as random intercept performed best, indicating that adding a random effect species to the model yielded significant improvement  for predicting sample origin of Eptesicus sp., we selected five (from 21) capture locations .To test the utility of a triple isotope approach .Results of the DFA are shown in The lack of suitable tracking methods has so far hampered our understanding of migratory connectivity of European bats, information which is essential for conservation planning. We tested the utility of stable isotopes as geographic tracers of breeding origins of bats in Europe. Our test consisted of validating (ground trothing) the accuracy and precision of hair isotopic signatures for predicting the geospatial origin of bats, by using samples of resident, known-origin bats captured in their natural habitat. We then built a regression model that may serve to predict region of hair growth for migrating bats from their hair isotopic ratios.We can find various examples in the literature of different approaches to ground truthing isotopic endogenous markers for studying animal migration. One approach is to breed the animals or keep them in captivity during the period of tissue growth in several locations scattered over the geographic range of interest, providing the animals only with local water and food. Hobson et al. used this approach to study monarch butterfly migration in North America Our approach was to use samples from bat species known to have sedentary habits throughout its European distribution range. We thus followed Hobson et al. h of resident bats correlated strongly with δDp  and longitude . These results are consistent with known but weak global latitudinal patterns in plant δ13C values 15N values h  between dietary water and body tissue, their δDh changes with δDp in the same manner. In contrast to our results, Britzke et al. found significant interspecific variation in the regression equations of δDh vs. δDp, with differences in both slopes and intercepts, of four North American bat species p vs. δDh was weak. The model obtained here accounted for interspecific variation in discrimination factors and was therefore not species-specific. Yet, our analysis included only five bat species, and therefore caution should be exercised when extrapolating the model to other species.We investigated the relationship between δD13C correlated with latitude and longitude, we did not incorporate δ13C in the model because no reliable maps of δ13C plant in Europe exist that could be used for geographic assignment. The reason for this is the large variance in δ13C plant values, so that local differences generally obscure the rather weak global patterns 13C of bat hair in our study correlated strongly with δDh and therefore these two variables were not fully independent. The large dependency of δ13C and δ15N on source of diet and trophic level may further limit their predictive value for migration studies when high interspecific and interpopulation differences in diet preferences cannot be ruled out. A trophic level effect on δD of vertebrate tissues has also been suggested 13C and δ15N were unsuitable to create spatially predictive geographical models for keratin growth location of wildlife in Europe, since there are no clear geographical patterns of these two isotopes in European habitats. Nevertheless, several studies have shown that isotopic markers that do not reflect clear, predictable geographic patterns can indeed help interpretations on migratory connectivity and behavior 13C values in addition to δD values 13C, sometimes also δ15N or δ34S) to test whether different breeding populations of migratory birds stem from the same or different wintering areas 13C and δ15N) to discriminate origins of hair growth. For this analysis, we did not pool species with different dietary niches because δ13C and δ15N values are known to be influenced by diet and ecology. However, to achieve an appropriate sample size, isotopic data of Eptesicus serotinus and E. isabellinus were analyzed together, based on the assumption that these species  may be useful to evaluate whether two or more migrating bat populations that are not significantly distinctive based on δD alone have different breeding origins. This could be the case for males and females exhibiting different migratory strategies, resident and sedentary populations of the same species coexisting in an area, or bats stemming from different breeding populations gathering together at swarming sites or wintering colonies. In addition, it may eventually be possible to use combined triple isotopic values to compare the migratory populations of interest to reference isotopic data from breeding populations to find the most likely population of origin, i.e. the one with the most similar isotopic profile.In bat migration studies, measuring δh of sedentary bats of known origin with δDp was obtained which may be used for translating δDh of migratory bats into δDp of the place of origin. Incorporating sources of error (i.e. intra-population variance) into a GIS-based probability model, potential regions of origin which have δDp values matching those predicted can be identified. The use of δ13C and δ15N and possibly other isotopic assays in addition to δD may further help to identify origins through direct comparison between migrating populations of interest and their potential breeding counterparts. Stable isotope analysis therefore represents a cost-effective endogenous marker that does not require recapture of marked individuals. Being informative on a population level and relatively non-invasive, it is possibly the best technological method currently available to study bat migration in Europe.Based on our results, we determined that stable isotopes are a promising and powerful tool to estimate the origins of migratory bats in Europe. A predictive model linking δDFigure S1p, δDh, δ13C, δ15N, latitude, longitude and elevation.Correlation matrix (Pearson's r) for the variables δD The variable names are shown in the middle diagonal; r values are in the upper right half ; scatter plots are presented in the lower left half.(TIF)Click here for additional data file.Figure S2Results of the GAM used to test the linearity assumption of the variables latitude, longitude and elevation. The y-axis shows the partials (k = 3 for smoothing function) of the respective variable. The variable elevation does not show linear behavior and was therefore modeled as quadratic term.(TIFF)Click here for additional data file.Table S1Sampling locations of bat hair. Name of location, site number (refers to (DOC)Click here for additional data file.Table S2h from δDp.Results of the likelihood ratio test (L) for finding the optimal random structure of the linear mixed-effects model (LME) for predicting δD All models were estimated by REML.(DOC)Click here for additional data file.Table S3Results of the discriminant function analysis to test the utility of a triple isotope approach for discriminating sampling locations of Eptesicus sp. A. Standardized coefficients given to δD, δ13C and δ15N to maximize the difference between locations. B. Structure matrix, pooled within-groups correlations between discriminating variables and standardized canonical discriminant functions. Variables ordered by absolute size of correlation within function. *. Largest absolute correlation between each variable and any discriminant function. C. Functions at group centroids; unstandardized canonical discriminant functions evaluated at group means. D. Results of the classification in leave-one-out cross-validation, showing how many cases from each location were predicted to stem from each other location, and the total percentage of correctly classified samples per location. For all locations, 93.0% of cross-validated grouped cases were correctly classified.(DOC)Click here for additional data file.


One notable feature of the major site is the insertion of Prp20NLS Ala18 between the P0 and P1 NLS sites, noted in only a few classical bipartite NLSs. This study provides a detailed account of the binding mechanism enabling Prp20 interaction with the nuclear import receptor, and additional new information for the interaction between IMPα and cargo.The translocation of macromolecules into the nucleus is a fundamental eukaryotic process, regulating gene expression, cell division and differentiation, but which is impaired in a range of significant diseases including cancer and viral infection. The import of proteins into the nucleus is generally initiated by a specific, high affinity interaction between nuclear localisation signals (NLSs) and nuclear import receptors in the cytoplasm, and terminated through the disassembly of these complexes in the nucleus. For classical NLSs (cNLSs), this import is mediated by the importin-α (IMPα) adaptor protein, which in turn binds to IMPβ to mediate translocation of nuclear cargo across the nuclear envelope. The interaction and disassembly of import receptor:cargo complexes is reliant on the differential localisation of nucleotide bound Ran across the envelope, maintained in its low affinity, GDP-bound form in the cytoplasm, and its high affinity, GTP-bound form in the nucleus. This in turn is maintained by the differential localisation of Ran regulating proteins, with RanGAP in the cytoplasm maintaining Ran in its GDP-bound form, and RanGEF (Prp20 in yeast) in the nucleus maintaining Ran in its GTP-bound form. Here, we describe the 2.1 Å resolution x-ray crystal structure of IMPα in complex with the NLS of Prp20. We observe 1,091 Å A distinguishing feature of all eukaryotic cells is the containment of their genetic material within a stable and segregated nuclear organelle. This in turn requires the active, bidirectional transport of proteins and RNA across the nuclear envelope, a central process to a range of important cellular events including DNA replication, cell differentiation, and diseases including cancer and viral replication. In the classical nucleocytoplasmic transport pathway, the nuclear import receptor importin-α (IMPα) recognises a nuclear localisation signal (NLS) displayed on a cargo protein, and this dimer, through interaction with importin-β (IMPβ), is docked and translocated across the nuclear pore complex through interaction with nucleoporins 10–12[K/R]3/5  cNLSs) for bipartite NLSs The initial interaction of the nuclear import pathway between IMPα and the nuclear import cargo has been studied by a range of structural and functional approaches. IMPα, a large (529 residue) highly conserved macromolecule, is composed of 2 structural domains, a short basic 10 kDa N-terminal domain that binds importin-β (IBB domain), and a 50 kDa armadillo (ARM)-repeat NLS binding domain that recognises and binds NLSs of various cargo proteins The directionality of the nuclear transport process is governed by the differential localisation of Ran across the nuclear envelope  NLS . Prp20 NLS was overexpressed as a GST-fusion protein using the autoinduction method as previously described in Mouse IMPαΔIBB (residues 70–529) was overexpressed using thio-β-D-galactose (IPTG) method as outlined in Single rod shaped crystals, measuring 200×50×50 µm, were obtained in 500 mM sodium citrate pH 6–8, 10 mM DTT after 2 d, harvested with a cryoprotectant composed of 80% mother liquid and 20% glycerol, and flash frozen with liquid nitrogen. Diffraction data was collected at the Australian Synchrotron (MX2) Beamline using BLU-ICE software Diffraction images were integrated and scaled to 2.1 Å resolution in iMOSFLM, with an Rmerge of 6.7%  from excess Prp20 (<5 kDa), thus isolating a homogenous complex of equimolar concentrations.For Prp20 to maintain Ran in its nuclear, GTP bound state, it must first be translocated to the nucleus. The region within Prp20 responsible for directing this localisation has been clearly defined o–Fc and simulated annealed omit maps revealed clear density in the major and minor NLS-binding sites of IMPα, enabling residues of the Prp20 NLS to be built , because an arginine side chain at this position is too long to make optimal interactions with the IMPα side chains, arginine can still be accommodated, e.g. in the case of CBP80 360 and Glu396 within ARM repeats 7 and 8. Conversely to P1′, whilst a lysine can be accommodated at this position, an arginine side chain is able to make more favourable contacts to the IMPα minor binding site, and the Prp20 NLS therefore contains the most favoured arrangement KR motif at P1′ and P2′ positions, forming both specific side chain salt bridges, and main chain hydrogen-bonding interactions. Mutations of these residues have shown a weaker interaction to IMPα and defects in nuclear localisation in yeast cells The minor site P1′ and P2′ positions contain the ‘KR’ motif in nearly all IMPα:NLS structures solved to date, with the replacement to non-KR residues commonly resulting in cytoplasmic localisation of the protein. The P1′-binding pocket is generally defined by residues ThrOverall, our structure defines the binding mechanism of the bipartite Prp20 NLS with the nuclear import receptor IMPα. This interaction has not been described previously, and importantly, provides new structural information relating the mechanism of IMPα NLS recognition. The linker region, separating the positively charged clusters within the bipartite NLS of Prp20, whilst longer than many bipartite NLS linkers, does not perturb the ability of these clusters to interact with the major and minor sites of IMPα in a manner characteristic of other bipartite NLSs. Whilst insertion of a residue between the classical P0 and P1 disrupted the classical binding observed at P0, this was compensated by additional binding observed in the close vicinity, and highlights both the flexibility of NLS recognition contained within IMPα, and difficulty in precisely predicting NLSs.


Peroxisome proliferator-activated receptors (PPAR) are members of the superfamily of nuclear hormone receptors involved in embryonic development and differentiation of several tissues including placenta, which respond to specific ligands such as polyunsaturated fatty acids by altering gene expression. Three subtypes of this receptor have been discovered, each evolving to achieve different biological functions. The PPARs also control a variety of target genes involved in lipid homeostasis. Similar to other nuclear receptors, the transcriptional activity of PPARs is affected not only by ligand-stimulation but also by crosstalk with other molecules. For example, both PPARs and the RXRs are ligand-activated transcription factors that coordinately regulate gene expression. In addition, several mechanisms underlying negative regulation of gene expression by PPARs have been shown. It is suggested that PPARs are key messengers responsible for the translation of nutritional stimuli into changes in gene expression pathways for placental development. Coactivation of RXRα and PPARγ is believed to synergize their effects on glucose and lipid metabolism [α and PPARγ are essential for mouse placentogenesis [γ is important for mouse placenta morphology [γ and RXRα are essential for cytotrophoblast cell fusion into a syncytiotrophoblast, which is obligatory for placentation, and these expressions are deregulated in pathological placenta [Peroxisome proliferator-activated receptors (PPARs) are a family of ligand activated transcription factors belonging to the nuclear hormone receptor superfamily, which mainly regulate the expression of target genes involved in lipid and energy metabolism –3. Threend PPARγ , 5. Eachnd PPARγ . From a nd PPARγ . Therefond PPARγ , 9. PPARal cells . The PPAoduction , indicattabolism . The RXRogenesis , 14. PPArphology . In addirphology –18. Furtplacenta . So, theα, β, and γ) are nuclear hormone receptors that are known to regulate gene transcription and protein expression levels of fatty acid transport and metabolism mediating proteins through the formation of a DNA binding heterodimer complex [α is expressed at high levels in tissues that catabolize fatty acids [β/δ mRNA is ubiquitously distributed with a higher expression in the digestive tract and the placenta [γ is mostly expressed in the adipose tissue [α and PPARβ transcript levels show similar profiles during the estrous cycle [γ mRNA level is quite stable during entire estrous cycle [α, PPARβ, or PPARγ expressions have been known in uterus of various species. High levels during the luteal phase and low during the follicular suggest the association with steroids function.PPARs , a PPARγ ligand , and a peroxisome proliferator (clofibrate) are all able to induce expression of both PPARα and PPARγ [A wide variety of compounds have been identified as PPARs ligands. Among the synthetic ligands, fibrates and thiazolidinediones are PPARectively . PPARγ iet genes , 29. A Pnd PPARγ –32. Subsnd PPARγ , 34. β agonists [Ligand activated PPARs bind as heterodimers with the RXRs on PPAR response elements. A number of PPAR target genes have been characterized to date. Most of these genes are known to have roles in lipid and glucose metabolism . The endagonists , suggestagonists , 39. Intagonists . As PPARagonists , 42. Thuα, PPARβ, and PPARγ are observed, while RXRβ is not detected. Immunocytochemistry staining results also determine the presence of PPARα, PPARβ, PPARγ, RXRα, and RXRγ to be specific to the trophoblast layer of the human chorionic villi [PPARs bind to a variety of PPAR response elements (PPREs) present in the promoter regions of the responsive genes. The transcriptional regulation by PPARs requires heterodimerization with the retinoid X receptor (RXR) . Retinoiic villi . The preic villi –52. α and SIRT1 coordinately suppress genes involved in mitochondrial function [γ, including genes mediating fat storage. SIRT1 represses PPARγ by docking with its cofactors nuclear receptor corepressor and silencing mediator of retinoid and thyroid hormone receptors [γ transactivation by SIRT1 inhibits lipid accumulation in adipocytes. SIRT1 also regulates angiogenesis signaling [The NAD(+)-dependent histone deacetylase Sir2 regulates life-span in various species . Mammalifunction    and p38 MAPK activation, leading to downregulation of proinflammatory gene expression [κB, CCAAT/enhancer-binding protein (CEBP), and AP-1 are important transcription factor families that are involved in immune and inflammatory functions as well as in cell growth and differentiation. Human placenta is rich in diverse bioactive molecules, whose extract induces interleukin mRNA and protein expressions in a dose-dependent manner. For example, the IL8 promoter contains binding sites for the NFκB, AP-1, and CEBP. The IL-8 expression is inhibited by an inhibitor of JNK [Many of the anti-inflammatory effects of PPAR (NF-κB) . EPA inhactivity . Furtheractivity . In addipression . The trar of JNK , 64. α and PPARα-regulated genes. Estrogen has not been reported to be a PPAR ligand, but interactions between PPARs and ER proteins and their response elements have been described [α in skeletal muscle of rats [Interestingly, the transcriptional expression levels of fatty acid binding proteins are upregulated in males and downregulated in females . A similescribed . These i of rats .A direct relationship between the PPARs activation and the inhibition of STAT5 mediated transcription has been reported , 69. Theγ/RXRα signaling is important in human cytotrophoblast and cell fusions. A disturbed PPARγ/RXRα pathway could contribute to pathological human pregnancies. SIRT1 expression is downregulated by proinflammatory cytokines. Possessing anti-inflammatory action in human gestational tissues, the SIRT1 expression is downregulated by proinflammatory cytokines. The natural polyphenol resveratrol inhibits cytokine and prostaglandin release via the SIRT1 activation. Both mRNA and protein levels of SIRT1 are shown to decrease in placenta and fetal membranes after labor onset, which may contribute to uterine contractions associated with labor. It would be of interest to investigate the impact of different types of fatty acid, integrated into food, on ovulation capacity and fetal development. Further study of PPARs, RXRs, and SIRTs functions in placenta may indicate pathways that are common to critical processes, providing additional focus for research in important human placental diseases. In parallel, defining more specific mode of action by identifying the endogenous coactivators and modulators of these transcription factors in animal models will help to build more efficient therapeutic strategy for the diseases. Future studies using functional genomic approaches will be required to more clearly establish the complicated mechanisms by which PPARs exert their actions. Additional insight is also needed into endogenous PPAR and RXR ligands, how these molecules are formed, and how they are delivered to the nucleus in placenta. Furthermore, additional experiments are required to increase the knowledge of the way in which lipid metabolism influences reproductive functions.PPARs are lipid-activated transcription factors that have emerged as key regulators of both lipid metabolism and inflammation, and they exert positive and negative controls over the expression of a range of genes. However, the range of transcription factors affected and the molecular mechanism involved may be different for each PPAR isoforms and cell types. Furthermore, peroxisome proliferators induce numerous alterations in lipid metabolism. A comparative approach to bring together physiological and nutritional roles of PPARs across species appears critical. It is now clear that PPARs are important in the control of placental development. PPARs may play a key role in linking lipid metabolism and reproduction systems. In addition, the PPAR


Geobacter sulfurreducens requires the expression of conductive protein filaments or pili to respire extracellular electron acceptors such as iron oxides and uranium and to wire electroactive biofilms, but the contribution of the protein fiber to charge transport has remained elusive. Here we demonstrate efficient long-range charge transport along individual pili purified free of metal and redox organic cofactors at rates high enough to satisfy the respiratory rates of the cell. Carrier characteristics were within the orders reported for organic semiconductors (mobility) and inorganic nanowires (concentration), and resistivity was within the lower ranges reported for moderately doped silicon nanowires. However, the pilus conductance and the carrier mobility decreased when one of the tyrosines of the predicted axial multistep hopping path was replaced with an alanine. Furthermore, low temperature scanning tunneling microscopy demonstrated the thermal dependence of the differential conductance at the low voltages that operate in biological systems. The results thus provide evidence for thermally activated multistep hopping as the mechanism that allows Geobacter pili to function as protein nanowires between the cell and extracellular electron acceptors.The bacterium  At the most fundamental level energy transduction in all living systems depends on electron transfer (ET) reactions catalyzed by redox-active proteins, often using metal4Shewanella oneidensis, for example, produces filamentous extensions of its cytochrome-loaded outer membrane and periplasmGeobacter sulfurreducens, on the other hand, produces a conductive version of a type IV pilusGeobacter assembles several pili monolaterally, maximizing interactions with insoluble electron acceptors such as Fe(III) oxides, which are often dispersed and otherwise difficult to reachEvidence is also emerging that some bacteria can transport charges at micrometer and even centimeter distances to couple spatially separated redox reactions11121311Geobacter pilin is divergent and predicted to be optimized for ETGeobacter pilin also contains aromatic residues  and charged amino acids predicted to distribute the charges and density of states along the peptide to favor charge transport1420in vitroin vivoc-cytochromes, dried on gold electrodesin situ electrical conductivity measurements on living biofilms grown on an electrode demonstrated the thermal dependence of incoherent redox conductivityUnlike other bacterial type IV pilins, the structure of the In the present study, we isolated pili free of metals and organic redox cofactors and used scanning probe methods to evaluate the contribution of the protein fiber to charge transport. We demonstrate efficient charge transport along individual pili and the involvement of aromatic contacts of the predicted axial, multistep hopping path2 substrate and the tip of a conductive-probe atomic force microscope (CP-AFM) was positioned on regions of the pilus at various distances from the electrode edge to form the contacts for a two-point electronic transport measurement (I-V) plots. I-V responses for a pilus probed at distances between 50 to 900 nm from the gold edge and the linear fits  obtained within a ±0.6 V voltage range. Additional I-V curves are shown in I-V plots.Individual pili were deposited across the edge of gold electrodes nanofabricated onto an electrically insulating SiOsurement . The cur8 electrons per second at 100 mV) two orders of magnitude higher than the cellular rate of Fe(III) oxide respiration From the linear resistance-distance correlation  we estim2+ (1.7 ± 0.9 atoms per pilin subunit), a cation that is typically detected in pili purified from other bacteriac-cytochromes such as OmcSThe range of resistivity (0.23–0.70 Ω.cm) calculated for the immobilized and solvated pilus  is within the lowest ranges reported for moderately doped Ag and Cu nanowiresThe MD pilus model revealed a potential axial multistep hopping path involving aromatic residues The conformational arrangements in the Y27A pilus also exposed more positively charged side chains on the pilus surface . These rI-V curves acquired for the WT and Y27A pili to identify those that exhibited nonlinear features at high voltages that could result from space charge limited transport (SCLT), as described in the VC)I ~ V) regime transitions into an electronic transport regime in which I ~ V 2 I-V curves for the WT and the Y27A pili showing this type of transition. The power-law fit above the VC follows the equation I = αVb, where the exponent b is close to 2, as expected for SCLT curvesa = 1.7 × 10−9 A/V2) and the length of probing (1.6 μm) we estimated a carrier mobility of 3.2 × 10−2 cm2/Vs (see 2/Vs2/Vs)−3–10−2 cm2/Vs)We screened the 2/Vs see . This va−10 A/V2; distance, 1.1 μm) of the Y27A I-V curve (−3 cm2/Vs) one order of magnitude lower than the WT, as expected for a pilus with reduced aromatic contacts. By contrast, the effective carrier concentration, calculated as the number of conducting electrons (or holes) per unit volume, was similar in the WT (2 × 1020 cm−3) and Y27A (2.4 × 1020 cm−3) pili. This value is high compared to most nanowire systems and organic semiconductors, even after extensive doping. For example, InAs nanowires, which also exhibit SCLT, have carrier concentrations on the order of 1016 cm−3 p-type doped organic semiconductor 2,7-bis(9-carbazolyl)-9,9-spirobifluorene reaches values of approximately 1018 cm−3 at a dopant concentration of 1 mol %From the fitting parameters  could only be imaged at large sample voltages (~3 V) and low tunneling current set points  compared to CP-AFM to gain insights into the electronic structure of the WT pili and produce electron energy spectra of pili as a function of temperature. STM images of individual pili deposited on highly oriented pyrolytic graphite (HOPG) showed conducting filaments at room and cryogenic (77 K) temperatures . These ft points . At thesI-V measurements taken at fixed locations on the pilus filaments are consistent with conducting behavior of the Geobacter pili in the ±1 V voltage range and the insulating behavior of the PAK pili in the ±2 V range  temperatures , likely due to variations in the density of states at different locations on the pilus, but the gap feature was consistently observed in all. This contrasts with the room temperature dI/dV of the pili acquired in the same voltage range, which lacks the gap feature  high enough to support the respiratory rates of the cell during the reduction of Fe(III) oxides  oxides are dispersed in the environment and difficult to access, cells maximize the chances of finding the electron acceptor and disposing respiratory electrons by assembling several pili per cellThe results demonstrate that the pilus protein fiber transports charges, consistent with the predicted role of pili as protein nanowires between the cell and external electron acceptors such as Fe(III) oxides,−2 cm2/Vs) extracted from the discontinuous I-V plot (2/Vs would have been required to describe pilus charge transport according to band theory−3 cm2/Vs). The mutation also enlarged the regions of positive potential on the pilus surface  reported for cell-associated pili after charge injection with a conductive tip and visualization of the propagated charge by electrostatic force microscopyThe Y27A mutation did not substantially affect the structure of the pilus  or the nGeobacter pilus estimated from the STM data (4–5 nm) matched well the diameter of the favored MD conformer of the WT pilus in solution (4.7 nm)Geobacter pili were transversally conductive in the ±1 V voltage range by STM, and produced differential tunneling conductance plots without a full energy gap at room temperature. By contrast, PAK pili controls were insulating in the ±2 V range (Geobacter pili at the low (±100–200 mV) voltages that operate in biological systemsThe diameter of the  V range . FurtherGeobacter pili to transport electrons at μm distances is especially significant for nanotechnological applications. Recombinant techniques can be applied to mass-produce pilin derivatives, which can be self-assembled in a cell-free environment using chemical methodsGeobacter pili to bind and reductively precipitate cationic metal contaminants such as uraniumThe ability of a peptide assembly such as G. sulfurreducens strain PCA and a Y27A mutant strain (constructed as described in the 142:CO2 (80:20).2 gas to prevent contamination. The purified pili were incubated in a 2:1 (v/v) chloroform-methanol solution for 2 h at 4 °C to extract any cell-derived quinone contaminants in the chloroform phase2 gas. The dry pili samples were stored at −20 °C for short-term use or flash frozen in liquid nitrogen and stored at −80 °C for long-term use. Pili were also purified from P. aeruginosa strain K (PAK), as described in the ®, Thermo Scientific), as previously describedWT and Y27A pili were isolated as reported elsewhere−14 μg). The concentration (in ppm) of elements was used to extrapolate the number of elemental atoms per pilin.Three replicate pili samples (20 to 30 μg of pili protein) in 10 mM CHES buffer  and buffer controls (no protein) were dispensed in acid-treated glass vessels and subjected to ICP-AES quantitative elemental analysis using a Thermo Jarrell-Ash Enviro 36 Inductively Coupled Argon Plasm . The number of pilin subunits in each sample was computed for the pili protein values using the predicted pilin’s mass of 6,568.51 Da  solution of 10 mM Tris buffer (pH 7) and isopropanol and their spectral properties were characterized by UV-VIS and fluorescence spectroscopy in reference to l-tyrosine (500 μM), riboflavin (100 μM) and menaquinone (100 μM) standard solutions in Tris:isopropanol . Absorption spectra were collected with a Cary100 UV-Vis spectrometer (Varian) set to 2 nm bandpass. Fluorescence spectra were acquired with a QuantaMaster spectrometer , with 270 nm excitation and 4 nm bandpass, and corrected with the fluorescence emission baseline from a Tris:isopropanol solution. All spectra were collected at room temperature, in quartz cuvettes with a 1 cm path length (Spectrocell Inc.).2O and deposited on patterned gold electrodes nanofabricated onto a silicon chip for CP-AFM analysis, as described in the I-V) curves were collected at each position  while applying a bias voltage sweep (±1 V). I-V curves were also generated periodically on neighboring regions on the gold electrode and silicon substrate as positive and negative controls, respectively. Methods to calculate resistance values, transport rates, resistivity, conductivity, and carrier characteristics (mobility and concentration) of the pili from the I-V curves and cellular rates of Fe(III) oxide reduction for comparative analyses are described in the Pili were suspended in ddHGeobacter and PAK pili after deposition on freshly cleaved HOPG. Information about sample deposition and STM imaging and spectroscopy at room and cryogenic (77 K) temperatures is available in the Scanning Tunneling Microscopy (STM) was used to probe the topography and electronic properties of e) was as in the WT (0.1 M). The atomic coordinates of the Y27A pilus model are provided as a supplemental file (GPIL-Y27A.pdb).All the simulations were performed with the GROMACS software suite5556The VMD programHow to cite this article: Lampa-Pastirk, S. et al. Thermally activated charge transport in microbial protein nanowires. Sci. Rep. 6, 23517; doi: 10.1038/srep23517 (2016).


Sources and timing of freshwater forcing relative to hydroclimate shifts recorded in Greenland ice cores at the onset of Younger Dryas, ∼12,800 years ago, remain speculative. Here we show that progressive Fennoscandian Ice Sheet (FIS) melting 13,100–12,880 years ago generates a hydroclimate dipole with drier–colder conditions in Northern Europe and wetter–warmer conditions in Greenland. FIS melting culminates 12,880 years ago synchronously with the start of Greenland Stadial 1 and a large-scale hydroclimate transition lasting ∼180 years. Transient climate model simulations forced with FIS freshwater reproduce the initial hydroclimate dipole through sea-ice feedbacks in the Nordic Seas. The transition is attributed to the export of excess sea ice to the subpolar North Atlantic and a subsequent southward shift of the westerly winds. We suggest that North Atlantic hydroclimate sensitivity to FIS freshwater can explain the pace and sign of shifts recorded in Greenland at the climate transition into the Younger Dryas. Resolving the sources and timing of freshwater forcing during the Last Termination is key to evaluate the North Atlantic Ocean response to freshwater perturbations. Here, the authors that the North Atlantic climate system was highly sensitive to Scandinavian meltwater discharge shortly before the Younger Dryas. The conventional explanation for the YD involves a catastrophic meltwater outburst from the Laurentian Ice Sheet into the North Atlantic that triggered a widespread reorganization of the atmosphere–ocean system123456The Younger Dryas cold stadial  lake sediments, Southern Sweden. HÄ is a small ancient lake11D composition of n-C21 and n-C27-29-31 alkanes  and terrestrial (δDterr) n-alkanes are established indicators of the isotopic composition of summer precipitation δD (refs Dterr can offset δDaq owing to evaporative enrichment (ΔδDterr−aq) due to the combined effect of soil evaporation and leaf water transpiration, evapotranspiration, thus serving as an indicator of moisture availability and relative humidityWe analysed the δMethods; , which iMethods; . δD valu δD refs , 17, whi14C dates covering ∼4,000 years .The interpretation of the biomarker record is supported by quantitative summer temperature estimations based on fossil chironomids Methods; . MoreoveMethods; . After td  and reach an isotopic minimum at 12,883±35 year BP. This δDaq decline coincides with a 27‰ rise in ΔδDterr-aq, peaking at 12,883±35 year BP, and a ∼2 °C decrease in summer temperatures . The local δDaq minima and ΔδDterr-aq maxima at HÄ are synchronous with the start of the cold Greenland Stadial 1  in Western EuropeTo investigate our hypothesis, we turn to a transient simulation of the last 21,000 years performed with a coupled atmosphere–ocean climate modelIn light of our results we argue that persistent FIS ice-mass loss at the end of the AL interstadial and the resulting freshening along the continental shelf of the Nordic Seas, which resulted in an early stage of cooling in Northern Europe, have likely determined the timing of hydrological shifts in Greenland at the GI-1a/GS-1 transition. Analogously to mechanisms invoked for the onset of cold climatic phases during the last glacial cycle9In conclusion, we provide a plausible mechanism for the linkages between FIS freshwater inputs to the Nordic Seas and North Atlantic hydroclimate patterns, reconciling the timing of freshwater forcing with the major isotopic excursions recorded in Greenland ice cores at the end of the Last Termination. Altogether, we suggest a new coherent concept for the inception of GS-1 and ultimately the YD, which may be critical to gauge future climate simulations.14C dates from terrestrial plant macrofossils. 21 14C dates were transferred from a previously studied core2, resulting in total organic content records from adjacent cores to exhibit the same high resolution and identifiable lithostratigraphic patterns  were extracted from the sediments via sonication with dichloromethane: methanol (9:1) for 20 min; and subsequent centrifugation. This was repeated three times and supernatants were combined. Aliphatic hydrocarbon fractions were isolated from the total lipid extract using silica gel columns (5% deactivated) that were eluted with pure hexane. The saturated hydrocarbon fraction was separated by desulphurization over 10% AgNO3–SiO2 silica gel using pure hexane as eluent. Saturated hydrocarbon fractions were analysed by gas chromatography—mass spectrometry for identification and quantification, using a Shimadzu GCMS-QP2010 Ultra. Isotope ratios were determined using a Thermo Finnigan Delta XL mass spectrometer and all analyses were performed in triplicate. A standard mixture of n-alkanes with known δD composition  was run several times daily to calibrate the CO2 reference gas used for conversion of δD values to VSMOW scale.Fifty-four freeze-dried samples  was 0.92 and the maximum bias was 0.77 °C.Samples for chironomid analysis of the HÄ sequence were taken every 2 cm. The aim was to obtain over 100 head capsules for each sample. Studies have demonstrated that 50 head capsules are an adequate minimum to establish species diversity in a sample and to provide reliable temperature estimates362, continental ice sheets and meltwater discharge as described in details in Liu et al.We analysed the simulation of the Transient Climate of the last 21 kyr refs , 44 .Supplementary Figures 1-11, Supplementary Tables 1-3, Supplementary Discussion, Supplementary Methods and Supplementary References.age-model output; chironomid-based summer temperature data; hydrogen isotope ratios form lipid biomarkers


To enable the magnetism and keep intrinsic crystal structures, we carried out a first-principles study of the magnetic and semiconductive monolayer MoS2 adsorbed with the Mnn (n = 1–4) clusters, and bilayer MoS2 intercalated with the same clusters. Geometric optimizations of the Mnn@MoS2 systems show the complexes prefer to have Mnn@MoS2(M) pizza and Mnn@MoS2(B) sandwich forms in the mono- and bi-layered cases, respectively. Introductions of the clusters will enhance complex stabilities, while bonds and charge transfers are found between external Mn clusters and the S atoms in the hosts. The pizzas have medium magnetic moments of 3, 6, 9, 4 μB and sandwiches of 3, 2, 3, 2 μB following the manganese numbers. The pizzas and sandwiches are semiconductors, but with narrower bandgaps compared to their corresponding pristine hosts. Direct bandgaps were found in the Mnn@MoS2(M)  pizzas, and excitingly in the Mn1@MoS2(B) sandwich. Combining functional clusters to the layered hosts, the present work shows a novel material manipulation strategy to boost semiconductive ILCs applications in magnetics.The inorganic layered crystal (ILC) MoS Due to possible manipulations of electron spins and carrier intensities2 ‘pizzas’ with Mnn (n = 1–4) ‘toppings’, and bilayer MoS2 ‘sandwiches’ with Mnn (n = 1–4) clusters ‘fillings’. The manganese clusters were selected as the start clusters due to their magnetic robustness2 ‘crust’ to the bilayer ‘bread slices’. To keep the intrinsic layered structures and prevent introducing defects, the clusters were placed and adsorbed on the top or between the layers. We investigated magnetic properties and electronic structures of the cluster adsorbed mono- and bi-layers. Bonding mechanisms within Mn clusters, and between clusters and ILC hosts were studied. In addition to possible variations of magnetic moments by changing the cluster sizes, we also found that introductions of the clusters into the ILC systems will facilitate system stabilities, and operate band types.In this article, we reported on a first-principles prediction of magnetic monolayer MoSn clusters were firstly studied to give basic knowledge of the ‘toppings’ or ‘fillings’ onto or into the crusts or bread slices. More than 60 initial Mnn structures were collected for further DFT optimization. Optimized clusters geometries were depicted in n clusters evolved from 0D to 2D forms when n was tuned from 1 to 3. However, in the case of n = 4, the 3D tetrahedron cluster was found as the most stable isomer, followed by a rhombus with a relative energy of 0.17 eV higher. Our results of free Mn clusters are in agreement with the previous DFT calculations3940Investigations of the doped system’s properties debut from the geometric structures. The free Mn2 were firstly relaxed and the optimized results were shown on the left row in 2, respectively, in good agreements with other density functional theory (DFT) calculationsn@MoS2 systems are sensitive to coordination and contact positions of the Mnn clusters with respect to the ILC hosts. In what follows, we name the systems of Mnn@MoS2(M) for the Mnn cluster doped Monolayer complexes, and Mnn@MoS2(B) for the Bilayer ones. After a full optimization of all possible motifs including magnetic order effects, it is found that the Mnn@MoS2(M) prefer pizza structures. Manganese clusters maintain their initial geometries after being adsorbed on the MoS2 monolayer. However, the Mn clusters evolve to a parallel layer motif when intercalated in the bilayers. The Mnn@MoS2(B) have sandwich structures with the maximal coordination numbers to expand the interactions between the Mnn clusters and two layers of the MoS2.From the hosts’ side, the 5 × 5 supercells of single- and two-layer MoSn clusters and their hosts were studied in details. For the Mnn@MoS2(M), the most stable adsorption sites of the Mn atoms are right above the Mo atom (numbered 1 in 2 site. For the Mn4@MoS2(M), the most stable structure is constructed by the tetrahedron Mn4 cluster whose triangular face lays on the MoS2 host. The rhombic structure with four Mn atoms tiled above the Mo site is less stable with higher energy as shown in 4 cluster sandwiched between two layers of the MoS2 is unstable according to our calculation. In the figures, the dash-dot balls and lines refer to metastable positions of the Mnn clusters on the monolayer MoS2. However, in the case of inserting Mnn to bilayer MoS2 systems, the lowest energy structures exhibit odd-even alternations with the number of the Mn atoms. One Mn atom laying under the Mo atom   complexes. In this case of evenly numbered n, each Mn atom is bonded to four host S atoms and one Mo atom as Geometric arrangements of the Mnn clusters adsorbed on the monolayer and bilayer MoS2 at the most stable adsorption sites. Two kinds of hosting atoms are classified: participators with whom dopants are bonded, and spectators where no additional bonding are formed. The MoS2 honey comb structures remain unchanged after absorptions of the Mnn clusters. Very slight lattice distortions are found at the participator area near the Mnn clusters. From dS–Mo between the participator atoms of the MoS2 became larger than those of the pristine MoS2 in both the ‘pizza’ and ‘sandwich’ cases. These participator S atoms interact with the impurity Mnn clusters, weakening bonding between the S and Mo atoms. On the contrary, the bond length of spectator atoms is the same as the one of the pristine MoS2. The Mn-Mn bond lengths in ‘pizzas’ and ‘sandwiches’, except for the Mn3@MoS2(M) and Mn4@MoS2(M), are notably larger than those in the free Mnn clusters, also indicating a covalent-bond interaction between the Mn and S atoms. The Mn-Mo bond lengths in Mn2@MoS2(B) and Mn4@MoS2(B) are 2.762 and 2.800 Å, respectively. Notably, values of the farthest dMo-S in Mnn@MoS2(B) oscillate with the Mn numbers. The oscillating trend pervades to electronic and magnetic properties of the Mnn@MoS2 (B) sandwiches in the follow discussion.Ead of the Mnn clusters adsorbed on the ILC hosts were computed as follows.The adsorption energies Etotal(Mnn@MoS2) and Etotal(MoS2) represent the total energies of the lowest-energy structures of the adsorbates and pristine MoS2, respectively, and Etotal(Mnn) is the energy of the individual Mnn clusters. All adsorption energies are found largely below zero  of the Mn3@MoS2(M) for the lowest-energy structures was plotted in 3@MoS2(M). When the Mn3 cluster is adsorbed on the MoS2 slab, the DED distributes not only surrounding the Mo and S atoms in the host MoS2 but also remarkably at the intervals between the Mn and S atoms and the guest Mn clusters (see n@MoS2(M&B) and high stability of the structure due to such interactions.where zero see , indicatters see . Featurin (n = 1–4) clusters are 5, 10, 15, and 20 μB, respectively, in agreement with previous studies4041n clusters adsorbed on the ILC hosts at the lowest energy adsorption sites. Results show that magnetic moments of the impurity Mnn clusters are not quenched by the nonmagnetic host MoS2 substrate. The energetic magnetic spin state displays ferrimagnetic properties when ferromagnetic Mn clusters adsorbed on the MoS2. The Mnn@MoS2(M) pizzas prefer to have medium magnetic moments of 3, 6, 9, and 4 μB in comparison with their corresponding free Mnn clusters . The Mnn@MoS2(B) sandwiches exhibit favorable oscillatory behavior with relatively smaller magnetic moments of 3, 2, 3, and 2 μB. To reveal detailed contributions from each Mn atom in the ‘topping’ or ‘fillings’, we also studied the local spin state on the Mn atom of the Mnn@MoS2 (M&B) systems. Their magnetic moments are listed in n@MoS2(M) pizzas are in ferromagnetic states except for theses of the Mn4@MoS2(M). On the Mn4@MoS2(M) pizza, three tiled Mn atoms have “spin-up” (majority) magnetic moments and one top Mn atom has “spin-down” (minority) magnetic moments. While in the Mnn@MoS2(B) sandwiches, the Mn atoms display ferrimagnetic order as shown in n clusters may serve as an ideal system to tailor magnetic properties when introduced on or between the MoS2 ‘crust’ or ‘bread slices’. Continued experimental and theoretical studies of similar TM clusters adsorbed on MoS2 systems may lead to discoveries of new families of dilute magnetic semiconductors with tunable magnetic properties.The lowest-spin arrangements of individual clusters are all ferromagnetic from our present calculations. Magnetic moments of the Mn7 cluster absorbed on graphene exhibits a magnetic moment of 6.3 μB per cell as given by first-principles calculationsB larger than 5.0 μB in an isolated Mn7 cluster. In the case of Mn doped MoS2 studied through a combination of DFT calculations and Monte Carlo simulations, the overall magnetic moment of the supercell is 1 μB corresponding to the single excess d electron provided by the Mn atom2 monolayers were also investigated by first-principles calculations. The total magnetic moments of H-,B-, C-, N-, and F-adsorbed MoS2 monolayers were found 1, 1, 2, 1, and 1 μB, respectivelyn@MoS2 ‘pizzas’ and ‘sandwiches’ studied here. By comparing the magnetic properties between other cases and this work, more insights may be provided into the effect of the impurities types employed on a nonmagnetic layer host.It should be noted that the magnetic properties of the Mnn@MoS2(M) ‘pizza’, the isosurface spin density of the ‘pizza’ was plotted in 3@MoS2(M), the spin density is almost entirely located on the Mn3 cluster site, resulting in a robust magnetic moment of 9 μB for the Mn3@MoS2(M).Mulliken population analysis shows that the total magnetic moment of the clusters is mainly localized in the Mn atoms as tabulated in n@MoS2(M&B) complexes were plotted in 2 were also given for comparison purposes. In the monolayer, a direct bandgap was found to have energies of 1.69 eV and 1.89 eV in our GGA and Heyd-Scuseria-Ernzerhof (HSE06) calculations implemented in CASTEP package4546948495051525354n clusters inserts additional defect states within the pristine MoS2 bandgap. The valence band maximum (VBM) and conduction band minimum (CBM) are primary from the 3d orbitals of the Mnn clusters. The partial density of states (PDOS) of Mnn@MoS2(M&B) in n clusters near the VBM and the CBM. Compared with the pristine MoS2 cases, Fermi energy shifts from the VBM towards the CBM with the increase of the Mn numbers. n@MoS2. The bandgap of the pristine MoS2 is evidently reduced due to the absorptions of small TM clusters. Such a reduction can significantly affect material optical and transport properties. From the values listed in The band structures of the Mn2, there is obvious hybridization between the atomic orbitals of the guest atom Mn and host atom S. We take the PDOS plots of Mo, S, and Mn atoms of the Mn3@MoS2(M) as an example  pizza  bandgaps keep direct as their host’s. However, the bandgap turns to indirect when n = 2,3 as shown in 1@MoS2(B) ‘sandwich’, the indirect bandgap of the bilayer host was switched to a direct bandgap. The CBM and VBM are both aligned at the ĸ point. Such a direct band structure is similar to the monolayer’s ones, which have been considered as the crucial origin of the ILC unique material properties. The result indicates the bandgap of pristine MoS2 can be operated from an indirect to direct or direct to indirect bandgap by adsorbing small TM clusters like Mnn. This provides new opportunities for controlling electronic structures in nanoscale materials with novel optical behaviors.Types of bands can be switched through the present doping route. A transition from an indirect to a direct bandgap in pristine MoS1@MoS2(M), charge transfers occur from the S atoms to the Mn atoms resulting in negative charges of the Mnn clusters. For the Mnn@MoS2(B), increases of the net charge values on the Mn clusters were found, illustrating enhancements of the sandwiches structures as the successive add-on dopant. The charge transfers between the Mn clusters and host MoS2 are one reason of the reducing magnetic moment of Mnn@MoS2 from the isolate Mn clusters, while strong hybridizations of the sulfur atoms in the MoS2 with the d states of the Mn cluster atoms is counted as another.Ranging from 0.053 to −0.215 and −0.32 to −1.715 au in the ‘pizza’ and ‘sandwiches’ systems, the net charge on the impurity Mn clusters clearly shows charge transfers between the ‘toppings’ and ‘fillings’, and the S atoms in the ‘crusts’ and ‘slices’. This leads high stabilities of the ‘pizza’ and ‘sandwiches’ following the partially ionic-like bonding of the Mn−S interaction through the charge transfers. Except for the Mn3@MoS2(M) pizza case. It is clear that the relative potential energy remains unchanged within the selected time scale. The ground-state structure is stable at room temperature. Such a thermostability is in line with the experimental evidences of the Au adsorbed MoS2 monolayerThe thermodynamic stability was tested by using the Born−Oppenheimer molecular dynamics simulation implemented in the DMOL3 code at room temperature (T = 300 K). A sample of the dynamic simulations is shown in 2 complexes were revealed. Benefiting from the uniqueness of the clusters and inorganic layered crystals, it is hoped that the present work will be served as a prototype in combinations of the cluster and layered crystal sciences, and boost their applications in the semiconducting scopes.In conclusion, we have presented a new strategy of tailoring the inorganic layered crystal to the magnetic semiconductors by introducing magnetic clusters as adsorbates. Geometric optimizations show that the small clusters prefer to follow the host alignments to enhance the complex stabilities. The magnetic and electronic structures were thoroughly explored. It is found that the system magnetic properties and electronic structures can be manipulated by careful selections of the ‘pizza’ and ‘sandwich’ recipes. Moreover, switches between the direct and indirect bandgaps of the adsorbed MoSd polarization functions (DNP) were included. Generalized gradient approximation in the Perdue−Burke−Ernzerhof (PBE) functional form was chosen−5 hartree was applied on the total energy and electron density, 2 × 10−3 hartree/Å on the gradient, and 5 × 10−3 Å on lattice displacements. The 5 × 5 supercells were constructed from 75 atoms of 25 Mo atoms and 50 S atoms for the monolayer, and 150 atoms including 50 Mo atoms and 100 S atoms for the bilayer. A vacuum region of 25 Å was selected in the z-direction to exclude mirror interactions between neighboring images. The Brillouin Zone integrations were carried out on a 10 × 10 × 1 Monkhorst−Pack k-points grad for the geometry optimizations, and a 15 × 15 × 1 k-points grad for the band and density of states (DOS) properties. To elucidate system magnetic properties, we carried out a detailed calculation for each possible spin multiplicity (SM) ranging from 1 to 21 of the Mnn (n = 1–4) adsorbed MoS2 complexes.All calculations were performed by using the DMOL3 package60How to cite this article: Zhang, M. et al. Magnetic MoS2 pizzas and sandwiches with Mnn (n=1–4) cluster toppings and fillings: A first-principles investigation. Sci. Rep.6, 19504; doi: 10.1038/srep19504 (2016).


The spin conservation rules define the symmetry barrier that prevents direct reaction of diradical O2 with nonradical molecules, explaining why combustion is not spontaneous. In burning, the spin barrier is overcome when energy causes homolytic bond cleavage producing radicals capable of reacting with diradical O2 to yield oxygenated radical products that further participate in reactive propagation. Neutrophil mediated combustion is by a different pathway. Changing the spin quantum state of O2 removes the symmetry restriction to reaction. Electronically excited singlet molecular oxygen (1O2*) is a potent electrophilic reactant with a finite lifetime that restricts its radius of reactivity and focuses combustive action on the target microbe. The resulting exergonic dioxygenation reactions produce electronically excited carbonyls that relax by light emission, that is, chemiluminescence. This overview of neutrophil combustive microbicidal action takes the perspectives of spin conservation and bosonic-fermionic frontier orbital considerations. The necessary principles of particle physics and quantum mechanics are developed and integrated into a fundamental explanation of neutrophil microbicidal metabolism. Neutrophil leukocytes protect against a varied and complex array of microbes by providing microbicidal action that is simple, potent, and focused. Neutrophils provide such action via redox reactions that change the frontier orbitals of oxygen (O Considered as an organ, the collective mass of hematopoietic bone marrow in a healthy adult is greater than that of the liver. The major proportion of this hematopoietic activity is directed to producing neutrophil leukocytes. Each day a healthy human adult releases about a hundred billion neutrophils into the circulating blood . This ba et cetera, neutrophils fuse their specific  granules with their cytoplasmic membrane , thus providing the increased surface-to-volume ratio necessary for locomotion and exposing the cytokine receptors and opsonin receptors required for close endothelial contact and diapedesis  from the vascular space through the endothelial lining into the tissue interstitial space. Chemotactic locomotion to the site of infection is directed by concentration gradients of bacterial products, anaphylatoxin, cytokines, et cetera.The neutrophil serves as the principal leukocyte of the acute inflammatory response and is the primary microbicidal phagocyte of the innate and acquired immune defense systems. Accomplishing such function starts in the circulation with intricate neutrophil-endothelial contact. When stimulated by microbial products, complement activation peptides, cytokines,Contact and receptor-mediated recognition of an opsonified  microbe results in phagocytosis, formation of a phagosome, and fusion with azurophilic (a.k.a. primary) granules containing myeloperoxidase to produce the phagolysosome .2) consumption, that is, the respiratory burst. This mitochondria-independent metabolic activity is required for effective microbicidal action ħ.The principal quantum number, s. The magnitude of s is restricted to a value of 1/2. The total spin angular momentum, S, of a system is expressed by the equation S = √[s(s + 1)]ħ. The intrinsic spin with its value of (1/2)ħ (abbreviated to 1/2) is a quality of fermions without analogy in classical physics. Just as l gives rise to ml, s gives rise to the spin quantum number ms. Only two values are allowed for ms. When ms = 1/2, e− is described as spin-up (↑); when ms = −1/2, e− is described as spin-down (↓).Electrons and other fermions possess intrinsic angular momentum that is independent of orbital motion. This intrinsic quantum mechanical property, that is, spin, is described by the spin quantum number,− of an atom or molecule is defined by its five quantum numbers: n, l, ml, s, and ms. The Pauli exclusion principle states that no two electrons of a given atom can have identical quantum numbers; that is, the total wave function for a system must be antisymmetric to the exchange of any pair of electrons. For an orbit to accommodate two electrons, the electrons must have opposite spins. If one orbital e− has ms = 1/2  (↑), the other orbital e− must have ms = −1/2  (↓). As such, the total spin quantum number, S, for an orbital electron-couple is 1/2 + −1/2 = 0  (↑ ↓). Orbital coupling of the fermionic electrons results in spin-neutralization. In effect, the antisymmetric fermions combine into a symmetric boson. This concept is illustrated in 2.Each eS)|+1, where S is the total spin number. Multiplicity is a spectroscopic term and indicates the number of wave functions possible for the system; that is, singlet indicates 1, doublet indicates 2, triplet indicates 3, et cetera. In its ground (lowest energy) electronic state, atomic H has a single e− in the 1s orbital and has an S value of 1/2 or −1/2; thus |2(1/2  or  −1/2)| + 1 = 2; the multiplicity is doublet. For molecular H2 the value of S is 0; thus, |2(0)| + 1 = 1; the multiplicity is singlet. In − is represented as spin-up (ms = 1/2 = ↑) or spin-down (ms = −1/2 = ↓). The circle surrounding the σ orbital electron-couple of the HOMO of H2 symbolizes the bosonic character of the filled orbital.Multiplicity is defined as |2.Chemical reaction involves frontier orbital interaction. Frontier orbital theory focuses on the initial orbital conditions of the reactants and on reactive transition with special emphasis on the highest occupied and lowest unoccupied orbitals , 22. The2, the wave functions overlap and split producing two molecular orbitals, one with lower and one with higher energy than the original 1s atomic orbitals. The bonding orbital (σ) is lower in energy and stable, thus promoting bonding. The antibonding orbital (σ∗) is of higher energy. Populating the antibonding orbital promotes bond breaking [σ electron, its electronic excitation to the σ∗ orbital results in H2 bond cleavage.For the H atoms, the electrons of the 1s orbitals have identical energies, but with covalent bonding to form Hbreaking . ConsequThe Wigner-Witmer spin conservation rules describe reaction symmetry possibilities in terms of reactant and product multiplicities , 25. The2. With regard to orbital spin character, the two antisymmetric fermionic electrons of the atomic orbitals are coupled (condensed or combined) resulting in the bosonic electron pair of the σ orbital of H2. Since the Pauli principle requires the total wave function for any system of electrons to be antisymmetric to exchange an orbital pair of electrons, only phase-opposite electrons can occupy a given orbital state. Orbital coupling of such fermionic electrons imposes bosonic character.In 2). Ground state atomic N is a paramagnetic triradical with one electron in each of its three 2p orbitals. Note that each electron has the same ms value resulting in quartet multiplicity; that is, |2(3(1/2  or  −1/2))|+1 = 4. The orbitals of higher multiplicity states are more contracted than those of lower multiplicity. Higher multiplicity states produce greater nuclear-electron attraction and are of lower energy [Hund's maximum multiplicity rule states that the electronic configuration with highest multiplicity has the lowest energy. The greater the number of wave functions possible for a system, the lower the energy. r energy .σ and σ∗ orbitals; constructive overlap of the phase-opposite electrons of the three 2p orbitals of the two N atoms generates one σ and two π bonds of triple-bonded ground state singlet multiplicity N2. The triradical N atoms combine to produce nonradical N2. From the fermion-boson frontier orbital perspective, each N presents a complex of trifermionic SOAOs, that is, Ψ ) or −Ψ ). Consistent with S = 0) N2.Combining the atomic orbitals of the two quartet multiplicity nitrogen atoms (N) generates the filled (bosonic) (1s and 2s) 1O2∗ as depicted in 1O2∗. Note the product 1O2∗ obeys the spin symmetry rules but violates Hund's maximum multiplicity rule. As such, 1O2∗ is electronically excited (indicated by ∗) with an energy of 22.5 kcal mol−1 greater than that of triplet multiplicity ground state O2 [Frontier orbital overlap of two oxygen atoms with antisymmetric SOMO is constructive producing state O2 .1O2∗ is metastable with a relatively long half-life. The estimated four-microsecond lifetime of 1O2∗ is sufficient to allow its participation as an electrophilic reactant in dioxygenation reactions. However, such reactions are restricted to within a radius of about 0.2 microns (μm) from its point of generation [1O2∗ relaxes to its triplet multiplicity ground state (3O2) by emitting a 1270 nm (near infrared) photon.The spin conservation rules predict that the change in spin multiplicity, that is, a singlet-to-triplet transition, is of low probability. As such, electronically excited neration , 28. As − occupying each of its two π∗ SOMOs; it is bifermionic. Ground state O2 can be in either the Ψ  = 1) or the −Ψ  = −1) state; thus, the multiplicity is |2(1) + 1| = 3, that is, triplet.As per the maximum multiplicity rule, ground state molecular oxygen is a triplet multiplicity, paramagnetic diradical with one eS = 0—and, as such, present bosonic frontier orbitals. For such molecules, chemistry is confined to bosonic HOMO-LUMO exchange of a composite orbital electron-couple. The fermionic orbital character of radicals is atypical and favors radicals-radical reaction. Antisymmetric fermionic frontier orbital overlap is constructive resulting in covalent bonding of the bosonic singlet product. As described in The vast majority of biological molecules are singlet multiplicity—that is, 2 with singlet multiplicity organic molecules violates the spin conservation rules, and, as such, combustion is not spontaneous. Overlap of the bifermionic π∗ (the 2 SOMO) frontier orbitals of triplet multiplicity O2 with the empty LUMO or bosonic HOMO of singlet multiplicity organic molecules is not constructive. Such reactions are symmetry-restricted, and the only product possible would also be a bifermionic triplet. O2 with its triplet multiplicity ground state is relatively exotic. The triplet multiplicity states of most molecules are electronically excited. The relaxation of such excited triplet multiplicity molecules to their singlet ground state, that is, triplet-to-singlet transition, violates the conservation rules and is of low probability. The delayed relaxation of an excited triplet to its singlet ground state by photon emission is responsible for the phenomenon of phosphorescence [Direct reaction of ground state triplet multiplicity Orescence .Considering the complexity and variety of potentially pathogenic organisms, neutrophil microbicidal action must be simple, potent, and focused so as to maximize microbicidal action and minimize collateral damage. Microbicidal combustion is realized by changing the frontier orbitals of oxygen from bifermionic to bosonic. Neutrophil combustive action is expected to produce light. Oxygenation reactions yield electronically excited products that relax to ground state by emission of light in the visible spectral range. Bioluminescence and chemiluminescence reactions are essentially limited to such oxygenation reactions. Neutrophil luminescence is energetic proof of such combustive activity.2. One equivalent  reduction is unusual in cytoplasmic metabolism. When it does occur, the semiquinone of a riboflavin prosthetic group is typically involved. Such flavoenzymes mark the departure from two equivalent cytoplasmic transfers to one equivalent cytochrome transfer. One equivalent reduction of O2 changes its multiplicity from triplet to doublet, thus reducing its overall fermionic orbital character. The flavocytochrome enzyme NADPH oxidase catalyzes the reduction of bifermionic triplet multiplicity O2 to fermionic doublet multiplicity HO2 [2 yields its conjugate base, doublet multiplicity superoxide anion (O2−) [2 with O2− produces singlet multiplicity ground state H2O2 plus 1O2∗ [Respiratory burst metabolism mobilizes the reducing equivalents required for changing the frontier orbitals of Ocity HO2 , 10. Acin (O2−) . As per us 1O2∗ .2O2 produced serves as substrate for myeloperoxidase (MPO) oxidation of chloride producing hypochlorous acid; that is, H2O2 + Cl−→ H2O + HOCl. Note that all reactants and products are exclusively singlet multiplicity (bosonic). The hypochlorous acid (HOCl) produced reacts directly with additional H2O2. This reaction involves the bosonic HOMO of H2O2 and the bosonic LUMO of HOCl producing the intermediate singlet multiplicity chloroperoxy acid (HOOCl) and singlet multiplicity H2O [2O2 + HOCl →1O2∗ + H2O + Cl− + H+. The bosonic orbital symmetry of 1O2∗ allows direct constructive spin-allowed overlap with the bosonic frontier orbitals of biological substrates producing singlet multiplicity dioxygenated products of bosonic symmetry.The Hcity H2O . Disinte2. As described in In conventional combustive burning, sufficient energy must be applied to a nonradical (singlet) substrate to produce homolytic bond cleavage yielding two radical (doublet) products. The fermionic SOMO of these doublet multiplicity radical products can now constructively overlap with one of the fermionic SOMOs of triplet multiplicity ground state O2, the bifermionic orbitals of ground state triplet O2 are converted to the bosonic orbitals of 1O2∗. In its electronically excited singlet multiplicity state, the bosonic frontier orbitals of oxygen can participate in spin-allowed electrophilic oxygenation reactions with a broad spectrum of singlet multiplicity biological molecules. As per spin conservation, the products of these reactions are singlet multiplicity. Such products are nonradical, diamagnetic, and unable to participate in the radical propagation reactions that are characteristic of burning.Neutrophil microbicidal action is combustion by a different pathway. Instead of radicalizing a substrate to facilitate reaction with diradical O1O2∗ is restricted to its metastable lifetime. The microsecond lifetime range of 1O2∗ confines reactions to within about a 0.2 μm radius from its point of origin [1O2∗ in close proximity to the target microbe guarantees that combustive action is focused on the microbe with minimal collateral damage to the host. By changing the spin quantum number of oxygen, the neutrophil not only realizes its potent electrophilic microbicidal action, but also limits such combustive action to the target microbe.The potent electrophilic reactivity of f origin , 28. Thuπ∗ singlet multiplicity state. As depicted in π∗ description indicates that an electron from the ground state nonbonding (n) orbital of the atomic oxygen (O) component occupies the π∗ orbital of the electronically excited carbonyl function. When the spin of the excited π∗ electron is antisymmetric to that of the ground state n electron of O, the electronically excited carbonyl is singlet multiplicity. Thus, π∗-to-n relaxation to ground state is spin-allowed and yields a photon with energy proportional to the difference between the two orbital states.The endoperoxide and dioxetane products of dioxygenation are of relatively high energy. These dioxygenated products are typically unstable and of singlet multiplicity. Disintegration of such products yields two carbonyl functions. One carbonyl is in the ground singlet multiplicity state, and the other carbonyl is in the electronically excited nπ∗ electronically excited carbonyl. This electronically excited state is chemically generated. In fluorescence, electronic excitation to the nπ∗ state can occur when a photon of proper energy is captured by an electron in the nonbonding (n) orbital of the O component of the carbonyl.In bioluminescence and chemiluminescence processes, dioxygenated products disintegrate by oxygen-oxygen bond cleavage producing the singlet multiplicity nπ∗ of the singlet excited carbonyl back to the n orbital of O results in photon (hν) emission and returns the carbonyl function to its ground singlet state. The frequency of the emitted photon is the energy difference that separates the n orbital from the π∗ orbital. Photons are symmetric bosons; their absorbance or emission does not affect the electron spin.As depicted in π∗ electronically excited singlet multiplicity carbonyl to its singlet ground state by fluorescence or chemiluminescence is spin-allowed, and, as such, the lifetime of the excited state is very short-lived, typically in the nanosecond range. Consequently the excited carbonyl is unable to participate in reaction chemistry.In fluorescence, photon capture promotes an electron of an orbital to an appropriately higher energy orbital. Such photon excitation results in transient antibonding and fermionic character, but the overall spin symmetry is retained; that is, singlet character is retained. Relaxation of an nπ∗ electronically excited carbonyl products. Such substrates increase the sensitivity for detecting dioxygenation activity by several orders of magnitude [The chemiluminescence produced by neutrophils is an energy byproduct of microbicidal combustion and varies with the molecular composition of the microbe. Different microbes present substrates that vary with regard to dioxygenation susceptibility and combustion quantum efficiencies, that is, the photon yield per dioxygenation reaction. Native luminescence is easily measured using a luminometer, but the photon yield is insufficient for measurement of less than a hundred thousand neutrophils. In order to increase photon yield and impose reaction specificity, chemiluminigenic substrates can be introduced to the milieu. Chemiluminigenic substrates are susceptible to dioxygenation producing endoperoxides or dioxetanes ultimately yielding nagnitude . In addiagnitude .The state of the circulating neutrophil reflects the state of inflammatory immune defense . The sen2O2 production. Fusion of the lysosomal azurophilic granules with the microbe-containing phagosome produces a phagolysosome with a relatively high MPO concentration in an acidic milieu replete with H2O2. The phagolysosomal milieu is optimal for combustive microbicidal action.Neutrophil leukocytes respond to cytokines and other agents by fusing specific granules with the cytoplasmic membrane, that is, specific degranulation. Such fusion effectively increases the surface-to-volume ratio of the neutrophil, as is required for locomotion and phagocytosis, and for upregulated membrane expression of cytokine and opsonin receptors, as is required for effective recognition and phagocytosis. Microbicidal effectiveness is optimized by coordinating phagocytosis with activation of NADPH oxidase, resulting in dynamic acidification and H2O2 as metabolic products [2O2 necessary for effective MPO-mediated microbicidal combustion and chemiluminescence [The neutrophils of patients with chronic granulomatous disease (CGD) have defective NADPH oxidase function and consequently have defective microbicidal combustion and do not chemiluminesce . These Cproducts , 39. Phanescence .Human neutrophils contain a relatively high concentration of MPO (about 5% of the dry weight of the neutrophil) in their azurophilic granules . Followi2O2 as metabolic products. The transit of neutrophils into such spaces delivers MPO into milieux that are rich in LAB.When neutrophils transit from the blood to tissue and body spaces, they carry MPO into these spaces. Each neutrophil carries about four femtograms of MPO. As such, neutrophils deliver relatively large quantities of MPO into body spaces on a routine basis. The oral and vaginal cavities have acidic pH. The normal florae of these spaces are cytochrome-negative LAB that generate lactic acid and H Staphylococcus aureus, and Gram-negative, for example, Pseudomonas aeruginosa and Escherichia coli, microbes [MPO selectively binds to Gram-negative and many Gram-positive bacteria. However, LAB show relatively poor MPO binding . When smmicrobes . Damage microbes .1O2∗ is generated in close proximity to the target microbe, resulting in selective, focused combustive microbicidal action. The 1O2∗ lifetime of a few microseconds limits damage to within about a 0.2 μm radius of its point of generation [Selective binding of MPO to a microbe guarantees that neration , 28. Thuneration .There are no restrictions on biologic evolution other than those imposed by physical reality. The spin conservation rules describe fundamental symmetry restrictions that define reactive possibilities. These rules forbid the direct reaction of triplet oxygen with singlet organic molecules, and, as such, combustion is not spontaneous. Chemical reactions involve frontier orbitals. Changing the multiplicity of oxygen changes its frontier orbitals, eliminating the symmetry barrier to its electrophilic reactive potential.2 to singlet electronically excited  1O2∗. In addition, other singlet multiplicity  microbicidal reactants, for example, H2O2 and HOCl, are generated. These agents are uniformly singlet multiplicity  reactants with bosonic frontier orbitals. Their reactions with microbes are consistent with the spin conservation rules. 1O2∗ has one empty (LUMO) π∗ orbital and one full (HOMO) π∗ orbital with bosonic character. This highly electrophilic LUMO can participate in spin-allowed bosonic LUMO-HOMO reactions with biological molecules. The dioxygenated products of such combustions yield the electronically excited carbonyl functions that relax by photon emission, that is, luminescence or chemiluminescence.Neutrophils kill microbes by a unique, focused combustive action. The redox reactions of neutrophil respiratory burst metabolism change triplet ground state  OConsidering frontier orbital reactivity in the bosonic-fermionic terms of particle physics broadens the perspective for explaining oxygen chemistry in particular and for appreciating reaction chemistry in general. Biological reactions typically involve constructive frontier HOMO-LUMO overlap. Radical reactions involving singly occupied atomic or molecular orbitals (SO(A)MO) are uncommon in biology under normal conditions, that is, in the absence of high energy radiation or heat producing homolytic bond cleavage. Biological reactions involving one electron transfer are typically restricted to flavoenzymes and to highly ordered cytochrome electron transport systems such as those found in mitochondria. Cytoplasmic redox reactions typically involve two equivalent transfers and are nonradical. Orbital pairing of antisymmetric fermionic electrons imposes a bosonic  character.+ yielding NADPH; these redox reactions involve balanced two equivalent bosonic transfers. Bosonic orbital pair transfers are the norm in biochemistry. The bosonic nature of the antisymmetric orbital electron-couple may be essential for such biologic redox transfer. Any fermionic electron transfer would open the possibility for reaction with bifermionic oxygen.Chemical reactions can be generalized as constructive bosonic transfers  or constructive fermionic annihilations. Both reactions produce bosonic products. Radical-radical MO-SO(A)MO) orbital overlap is typically constructive; that is, fermionic radicals react with fermionic radicals. Overlap of a bosonic HOMO with a fermionic SO(A)MO is not constructive. As illustrated in S = 0. This has significance with regard to the Heisenberg uncertainty principle which states that the uncertainty of momentum (Δp) multiplied by the uncertainty of position (Δx) is always equal to or greater than (1/2)ħ; that is, ΔpΔx ≥ (1/2)ħ [There are advantages to transferring electrons as a bosonic orbital-couple. As stated by Dirac, “If a state has zero total angular momentum, the dynamical system is equally likely to have any orientation, and hence spherical symmetry occurs” . A boson≥ (1/2)ħ . The spi≥ (1/2)ħ . Such a