Diamond formation has typically been attributed to redox reactions during precipitation from fluids or magmas. Either the oxidation of methane or the reduction of carbon dioxide has been suggested, based on simplistic models of deep fluids consisting of mixtures of dissolved neutral gas molecules without consideration of aqueous ions. The role of pH changes associated with water–silicate rock interactions during diamond formation is unknown. Here we show that diamonds could form due to a drop in pH during water–rock interactions. We use a recent theoretical model of deep fluids that includes ions, to show that fluid can react irreversibly with eclogite at 900 °C and 5.0 GPa, generating diamond and secondary minerals due to a decrease in pH at almost constant oxygen fugacity. Overall, our results constitute a new quantitative theory of diamond formation as a consequence of the reaction of deep fluids with the rock types that they encounter during migration. Diamond can form in the deep Earth during water–rock interactions without changes in oxidation state. The cause of diamond precipitation has previously been attributed to poorly understood redox changes at depth. Here, the authors propose that a drop in pH during water–rock interactions leads to diamond formation as a consequence of the migration of reactive fluids at elevated temperatures and pressures. Diamonds record fluid migration events over billions of years in the deep carbon cycle in the Earth's interior124567891011191112131442, CH4, H2 and H2O  without consideration of aqueous ions or species derived from silicate rock components4Traditional models of the fluids involved in diamond formation have long treated the fluids as mixtures of neutral gas molecules such as CORecent advances in theoretical and experimental aqueous geochemistry resulting in the Deep Earth Water (DEW) model enable calculation of equilibrium constants involving minerals and aqueous ions, metal complexes and organics to 6.0 GPa and 1,200 °C refs , 20, 21.22ξ values of −2.0 to about −0.30; ξ values greater than about −0.30, An initial fluid chemistry  was set 25ξ<−0.30), corresponding to the smallest extents of fluid–rock interaction, could correspond to the formation of diamond in a Mg-rich carbonate-metasomatized eclogite. It might be expected that the carbonate phase could disaggregate during transport in a kimberlite magmaξ>−0.30), corresponding to much larger extents of fluid–rock interaction, could correspond to the formation of diamond, clinopyroxene and garnet-metasomatized eclogite. Under these circumstances, diamonds may well be preserved in their eclogitic host–rock during the kimberlitic eruption process. If such large extents of fluid–rock interaction are uncommon, the overall model in It is interesting to note that the first part of the reaction path shown in f2O is almost unchanged (<0.01 log units) during the entire reaction path. However, in It can be seen in The univariance of the equilibrium in ξ<−0.30) causes the precipitation of diamond and a secondary Mg-rich carbonate mineral according to the reactionsIt can be seen in 2+ ion and add the (AlO2)SiO2− complex and H+ ions to the fluid, causing the ratio of These reactions remove the MgIn the later stages of reaction progress (−0.30<logξ<+0.1), the fluid is directly altering primary eclogitic clinopyroxene to secondary garnet with an increasing pyrope component  accordin2+ ion and add H+ ions to the fluid, again causing the ratio of These reactions remove the Mg2,aq increases by 623 mmolal, which is mainly balanced by decreases in the concentrations of the Ca(HCO3)+ complex, HCO3− and CO32−  plus a decrease of about −160 mmolal of C from organic carbon species +). Although each of the reactions in 2,aq concentration and therefore of the f2O. Consequently, the overall reaction precipitating diamond is independent of f2O and the f2O barely changes during the calculated reaction path in  because of small fluctuations in solution chemistry associated with the disappearance of the Mg-rich carbonate solid solution. This predicted behaviour may mirror the often noted precipitation and dissolution features in natural diamondsDiamond precipitation in 2 or CH4 previously pictured as the only possible sources of carbon for diamond formation. The involvement of aqueous organic species in our model arises, because it is predicted that at pressures above ∼3.0 GPa it is possible to have a complete equilibrium between all oxidation states of aqueous carbon species293031323334It is also interesting that the precipitation reactions for diamond in The predicted model evolution of the fluid chemistry during the precipitation of diamond is shown in 2 is much higher in the model fluid (47%) than has been derived for the natural fluid inclusions (11%). A possible cause of this discrepancy might be the simplistic assumption of a unit activity coefficient for the the aqueous CO2 species in the model. Finally, it should be emphasized that the calculations plotted in Significant differences, however, are apparent when a more detailed comparison of individual elemental concentrations is made. For example, in f2O increases could result in multiple cycles of diamond precipitation and dissolution, which might explain some observed diamond resorption featuresOur theoretical calculations provide a model clearly demonstrating that diamond can form by pH decreases during water–rock interaction under constant redox conditions in subcratonic lithospheric mantle environments. The theory implies that diamonds and their solid and fluid inclusions could be the natural, and perhaps even the common result of changes in water chemistry rather than redox changes alone. We wish to emphasize that our proposed pH-change mechanism for diamond formation represents a mechanism in addition to potential changes in redox, temperature and pressureThe theoretical model developed here could also be used to investigate subcratonic lithospheric diamond formation in peridotites. Furthermore, the model could be expanded to include more sophisticated models of solid solution in the minerals, the partitioning of trace elements between the minerals and fluids, a much wider range of aqueous organic species, and liquid and crystalline hydrocarbons. It would then be possible to integrate quantitative theoretical models of evolving fluid chemistry with detailed studies of natural diamonds including their solid and fluid inclusions, their trends with time during diamond growth and their relationships to host eclogites and peridotites78927415Diamond formation occurs particularly in the roots of continents known as mantle keels that extend from about 40 to 250 km depth. In this subcratonic mantle lithosphere environment, diamonds have formed at temperatures in the range of 900–1,400 °C and pressures of about 4.0–8.0 GPa ref. . DiamondThe initial fluid was derived by reaction of fluids assumed to be low in chloride with a carbonated peridotitic mineral assemblage envisioned as passing through the carbonated mafic part of a subducting slabThe calculations referred to in The two codes use a database of equilibrium constants calculated in advance using the DEW model43193)+, because of the predicted abundance of the bisilicate anion HSiO3− at high pressuresRemarkably, little experimental data exist for mineral and silicate-rock solubilities in aqueous fluids at elevated temperatures and at pressures >3.0 GPa, in particular as the melting point of the rocks is approached454647484950514953How to cite this article: Sverjensky, D. A. & Huang, F. Diamond formation due to a pH drop during fluid–rock interactions. Nat. Commun. 6:8702 doi: 10.1038/ncomms9702 (2015).Supplementary Figure 1, Supplementary Tables 1-2, Supplementary Note 1 and Supplementary Reference.


The highly programmable positioning of molecules  on surfaces has potential applications in the fields of biosensors, biomolecular electronics, and nanodevices. However, the conventional techniques including self-assembled monolayers fail to position the molecules on the nanometer scale to produce highly organized monolayers on the surface. The present article elaborates different techniques for the immobilization of the biomolecules on the surface to produce microarrays and their diagnostic applications. The advantages and the drawbacks of various methods are compared. This article also sheds light on the applications of the different technologies for the detection and discrimination of viral/bacterial genotypes and the detection of the biomarkers. A brief survey with 115 references covering the last 10 years on the biological applications of microarrays in various fields is also provided. The u methods . The in rawbacks ,8. The drawbacks –12. The rawbacks –15. Hencrawbacks –18.The performance of DNA chips was under shadow due to the several issues including the probe design, the reaction conditions during spotting, the hybridization and washing conditions. Furthermore, the suppression of nonspecific binding, the distance between the oligonucleotides and the surface also add to the factors responsible for DNA chip problems. The lateral spacing between the immobilized oligonucleotides also determines the performance of the DNA chips . Many re2.DNA probes are short oligonucleotides which can hybridize with specific target sequences. The immobilization step for the DNA probes is essential to develop a whole range of microarrays. Immobilization can be defined as the attachment of molecules to a surface resulting in reduction or loss of mobility. The way in which DNA's are immobilized determines the property of a microarray. Generally, the choice of a suitable immobilization strategy is determined by the physicochemical properties of both surface and DNA probes. DNA microarrays developed by different strategies have a common critical step of immobilization of the DNA probes on the surface. To develop microarrays, the probes can be made base-by base on the support or pre-synthesized and then spotted on the surface.As shown in the 2.1.Physical adsorption is the simplest immobilization method because it does not require any nucleic acid modification. Immobilization has been reported based on ionic interactions occurring between the negatively charged groups present on the DNA probe and positive charges covering the surface as depicted in The limitation of the adsorption mechanism is random orientation and weak attachment of DNAs to the surface. Due to the weak attachment the DNA probes can be removed by some buffers or detergents when performing the assays. Moreover, the problems related to the mass transport effect and high background signals emanating from nonspecific interactions can result not only in the false calculation of kinetic rate constants, but also false detection of pathogenic infections.2.2.In the immobilization of DNA on solid-state surfaces using the electrostatic interaction as a driving force, environmental changes such as ionic strength, pH, and temperature can cause desorption of the adsorbed DNA probes. Therefore, a covalent coupling route for the immobilization of DNA probes to achieve good stability and high binding strength is more facile ,42. ChemAs shown in The technique of immobilization of thiol-DNA probes by self-assembly on gold electrodes is widely used in the fabrication of electrochemical and DNA biosensors. The technique uses the advantage of the strong interaction (chemisorption) between thiolated DNA and gold surfaces. Self-assembled monolayers obtained by using thiol-tethered oligonucleotides mixed with alkanethiols such as mercaptohexanol represent a simple yet effective means to control the density and availability of the capture probe . The secThe covalent immobilization method requires chemical modification of the surface when fabricating microarrays. The SiOH groups on the glass surface are modified to either nucleophilic or electrophilic functionalities that then react with the amine or carboxylated oligonucleotide probes. Therefore, the surface chemistries for the attachment of oligonucleotides on the surfaces have been extensively investigated . HoweverDifferent covalent attachment chemistries involving various functional groups on the surface are summarized in the The Schiff-base reaction between aldehyde groups on the substrate surface and amine groups of DNA probes results in the covalent attachment of DNA probes on solid surfaces . The intHowever, the vast majority of the immobilization chemistries described in It was generally considered that chips with high immobilization density perform well upon hybridization with the target DNA probes. However, due to the compactness of the immobilized probes, the incoming target probes do not have enough space to get into the immobilized probes and to bind with them. Thus, many methods use a high temperature hybridization step to allow the target probe to bind with immobilized probes. The use of high temperature hybridization and the crowding effect of the immobilized probes are the main causes of low signal to background ratios, non-specific hybridization, and low sensitivity. Thus the crowding effect as depicted in the 2.3.aK = 1015 M−1). Moreover, avidin and streptavidin are tetrameric proteins that have four identical binding sites for biotin. Streptavidin with an isoelectric point (pI) equal to 5 is thus preferably used over avidin, which has a pI of 10.5, to avoid nonspecific interactions [Various methods for the immobilization of streptavidin on the solid surfaces have been reported. The formation of the streptavidin and biotin complex is useful in a wide variety of applications ,77. The ractions .The avidin (or streptavidin)-biotin interaction is often used to develop DNA microarrays. DNA probes were succesfuly bound to a SAM of 2-mercaptoethanol and 11-mercaptoundecanoic acid through streptavidin-biotin interactions. Avidin can be adsorbed on the silica surface before immobilizing a biotinylated molecular beacon (MB). Three out of four sites in avidin remain free to interact with the biotinylated DNA probes .Though streptavidin is easily immobilized on various surfaces the binding capacity decreases over time. Moreover, the the synthesis of streptavidin-immobilized surfaces involves multiple steps, including synthetic modification of the surface, immobilization of streptavidin, and blocking. Each step increases the production time and cost. Moreover, the immobilization of streptavidin or biotin on the suface suffers from drawbacks like the instability of the immobilized proteins and non-specific interactions, thus resulting in the low sensitivity amd specificity .2.4.mesospacing between the apexes of the dendron molecules. The dendron-modified substrate provided lateral spacing between the immobilized probes [The problems due to the high compactness of immobilized prones as depicted in the d probes . The pro2.5.The preparation of the 9G DNAChip  and the 3 in the para position of an aromatic ring or the presence of a trimethylammonium group) [Earlier, it was reported that the cavities of AMCA derivatives have a preference for structurally flat guests  –85. TherTo gain insight into the molecular recognition properties of the AMCA monolayer on the AMCA slide, further investigation was done by immobilization of the probes appended with nine adenine (9A), nine thymine (9T), nine guanine (9G), and nine cytosine (9C) subunits, respectively, on the AMCA slide. The 9T and 9C probes showed the lowest fluorescence intensity. The 9A probe also showed significant fluorescence intensity, but not as good as the 9G probe. Hence, oligonucleotides with nine consecutive guanine bases were used to generate 9G DNAChips. These findings pioneered the basis of the 9G technology.The phenomenon of molecular recognition to immobilize oligonucleotides for the production of the DNA chips was first introduced based on the 9G technology. The 9G probes are immobilized by the multiple interactions of the nine consecutive guanines on the AMCA monolayer. The lateral spacing between the immobilized probes provides high accessibility leading to the more than 80% hybridization efficiency in 5 min at 25 °C. Moreover, the nine consecutive guanines can be easily added to the oligonucleotide probes during their synthesis. The excellent properties shown by the 9G DNAChip enables it to be a powerful and promising tool for nanotechnology, biotechnology and allied sciences.3.Microarray technology has altered the scope of life science research and boosted the biomarker diagnostics industry ,87. MicrThe regeneration of a surface-immobilized probes allows the reuse of DNA microarrays without the loss of hybridization activity. Thus, it can help in the reduction of the cost per test. However, for the reuse of the DNA microarrays three requirements should be taken into account with probe immobilization. Firstly, the immobilization chemistry needs to be stable, second the probes have to remain functional after attachment and lastly the DNAs have to be immobilized with an appropriate orientation and configuration .The detection of protein biomarkers whose change in concentration with the progression of a disease is becoming increasingly important to monitor the right direction of treatment. However, use of the protein microarrays biomarker detection has been limited by issues such as the surface immobilization of proteins without loss in bioactivity ,91. To s3.1.In the last few years the development of new methods for the detection of infectious diseases such as human papillomavirus (HPV), human influenza virus (H1N1), pneumonia and tuberculosis has increased significantly . Such inAs shown in The limitation of the PCR technique is that it cannot discriminate the SNP efficiently and has a very low clinical sensitivity and specificity. Multiplex PCR, a much more powerful technique than PCR itself, also suffers from the problem of detection of multiple infections in a single sample and has a low sensitivity and specificity . AlthougThe 9G DNAChips obtained by the 9G DNAChip technology successfully solved these problems . The 9G 3.2.Enzyme-linked Immunosorbent Assays (ELISAs) are one of the earliest techniques employed for the detection of the biomarkers. Even though ELISA has several advantages for the detection of the biomarkers, it suffered from limitations as shown in Protein microarrays had been developed for the detection of the biomarkers . The proDNA-Directed Immobilization (DDI) was employed to improve the stability of the proteins by immobilizing them on the surface shortly before the detection of antigens . In the ® technology of Luminex Corp. , allows multiplex analysis of samples. The beads used are color coded by different ratios of two fluorescent dyes [One of the drawbacks of the biomarker detection methods is that, their limit of detection is in the nanomolar range . The othent dyes ,115.According to the recently reported DNA-Guided Detection (DAGON) method  based on4.etc.) nanopatterns of the various molecules can been created on a surface.The 9G technology uses nine consecutive guanines in the immobilized probe which provides nanometer scale lateral spacing in the immobilized probes. However, by altering the number of guanine subunits in the immobilized probes, the lateral spacing between the immobilized probes can be controlled. Nanometer-sized patterns of biomolecules can be produced by using a known number of guanine residues in the immobilized probes. Thus, by hybridization of the immobilized probes with the conjugated target probes  on the surface. Unfortunately, conventional techniques such as self-assembled monolayers fail to position the molecules on a nanometer scale, which is a key to produce highly organized monolayers on the surfaces. The conventional techniques used for the immobilization of the probe DNA's and the proteins to produce molecular and biomarker diagnostic platforms suffer from the serious drawbacks. The drawbacks, such as crowding effect in immobilized probe DNA's, distortion of the three dimentional structure of the immobilized proteins limits the clinical applicability of micorarrays and the protein chips. Recently reported 9G DNAChips based on the 9G technology allow 80% hybridization in 5 min at 25 °C with 100% specificity. Therefore, the advantages and the drawbacks of conventional methods are compared with those of the 9G technology. It was noticed that the linear spacing between the immobilized probe DNAs plays a crucial role for the sensitivity and specificity of the microarray. We believe that the recent development in the chemical synthesis and modification of DNA will further allow to use the DNA as molecular building blocks for the highly functional microarrays.


This study evaluated the morphology and immunohistochemistry of 85 canine cutaneous histiocytic tumours. The tumours were classified morphologically as either canine cutaneous histiocytomas (71 tumours) or canine cutaneous histiocytic sarcomas (14 tumours). The immunohistochemical analysis was conducted on paraffin sections using an antibody panel . Histochemical staining with toluidine blue and Gomori silver impregnation was also performed. A follow-up was conducted via surveys. The histiocytic origin of the tumour cells was confirmed in 65 of the canine cutaneous histiocytomas and in 4 of the canine cutaneous histiocytic sarcomas. The tumours that had been misdiagnosed as canine cutaneous histiocytomas included plasmacytomas, epitheliotropic T-cell lymphomas and undetermined entities. The tumours misdiagnosed as canine cutaneous histiocytic sarcomas included plasmacytomas and non-epitheliotropic T-cell lymphomas, but the majority of them remained undetermined. The canine cutaneous histiocytomas showed MHCII, CD18 and E-cadherin expression, but in several of the tumours, the expression of CD18 or E-cadherin was confirmed in only a small percentage of the tumour cells. The regressing canine cutaneous histiocytomas showed increased T- and B-lymphocyte infiltration, a decreased mitotic index, transport of the MHCII molecules from the cytoplasm to the cell membrane and loss of E-cadherin expression in the tumour cells. The canine cutaneous histiocytic sarcomas showed both high morphological diversity and expression of MHCII and CD18. Two of the evaluated histiocytic sarcomas also showed expression of E-cadherin. In conclusion, immunohistochemistry, including analysis of MHCII, CD18 and the lymphocytic markers CD3 and CD79, should be performed for the diagnosis of canine cutaneous histiocytic tumours. The expression of E-cadherin in canine cutaneous histiocytic sarcomas suggests an origin of the tumour cells among Langerhans cells. Canine cutaneous histiocytoma (CCH) is a common benign skin tumour of the dog, characterised by clonal proliferation of Langerhans cells and showing expression of CD1 and specific β2 integrins . The tumours included 71 CCHs and 14 cutaneous histiocytic sarcomas. The tumours were diagnosed on the basis of the histopathological examination according to widely used criteria  and Pannoramic Viewer software . The histiocytic origin of the tumour cells was confirmed by immunohistochemistry  according to Fernandez et al. . A detaiA follow-up was conducted by surveying the veterinarians or owners. The survey included assessment of the incidence of local recurrence and the survival time up to 2 years after the diagnosis.The results were analysed statistically. A Spearman rank correlation analysis was used to detect the correlations between the evaluated morphological and immunohistochemical features (r: Spearman rank correlation coefficient) of CCH. The number of histiocytic sarcomas was too low to perform a reliable statistical analysis. The correlations were considered statistically significant when P < 0.05. The statistical analysis was performed using Statistica software .The histiocytic origin of the tumour cells  was confirmed in 65 CCHs and 4 cutaneous histiocytic sarcomas. Two tumours diagnosed histologically as CCH showed expression of CD3; therefore, their final diagnosis was epitheliotropic T-cell lymphoma Fig. . Two othr = −0.259; P = 0.037). Prominent necrotic foci were found in 38 % of the cases, but necrosis or apoptosis of single tumour cells was common and associated with the lymphocytic infiltration. The epidermal changes included ulceration (92 % of the cases), parakeratosis (21 % of the cases), intra-epidermal aggregates of tumour cells . Neutrophils were mostly observed as small or large foci and were commonly associated with epidermal ulceration and necrotic foci within the tumour. Eosinophils were noted in 21 % of the cases, as either single cells or small foci. Mast cells were confirmed in 28 % of the cases, mostly as small foci, and were localised within the tumour or at the tumour periphery. The mast cells within the tumour and at the tumour periphery were positively correlated . The number of mast cells within the tumour was negatively correlated with the intensity of the lymphocytic infiltration .The T-lymphocyte infiltrate was predominantly focal and diffuse  and negatively correlated with the number of mast cells within the tumour  and at the tumour periphery . The CD18 expression pattern was either strictly membranous or membranous with slight cytoplasmic accentuation. Strong variations between the labelling intensities were noted. Strong expression was confirmed in 11 % of the CCHs; moderate, in 67 % of the CCHs; and weak, in 22 % of the CCHs. The number of tumour cells expressing CD18 was positively correlated with the intensity of the CD18 expression , the intensity of the lymphocytic infiltration , the number of tumour cells expressing MHCII  and the MHCII expression pattern . The E-cadherin expression pattern was membranous. The positive cells were usually subepidermally situated, gradually losing E-cadherin expression in the deeper parts of the tumour .A quantitative analysis of MHCII, CD18 and E-cadherin expression is summarised in Table our Fig. . There wFollow-up was available in 44 of the CCH cases. Recurrence was noted in two cases, but a subsequent histopathological examination was not performed. Two dogs (a 10-year-old Boxer and a 10-year-old American Staffordshire Terrier) died within one year after the surgery, but the reason for death was not associated with CCH. Necropsies were not performed.The first case of histiocytic sarcoma occurred as a solitary cutaneous nodule situated in the area of the elbow in an 11-month-old mongrel male. The tumour was poorly circumscribed, nonencapsulated, localised under the epidermis, and extending to the deep dermis. The neoplastic cells formed a solid mass, obliterating the adnexa, with a dense cellular arrangement and abundant reticulin fibres. The neoplastic cells were round to polygonal and oval to fusiform, with abundant, slightly eosinophilic cytoplasm and indistinct cell borders. The nuclei were oval, round or reniform, with marginated chromatin and indistinct nucleoli. The mitotic index was 16. The second case of histiocytic sarcoma occurred as a solitary cutaneous nodule situated in the thigh area in an 8-year-old Cane Corso male. The tumour was poorly circumscribed and nonencapsulated, localised in the dermis, and extending deeply into the subcutis and focally to the epidermis. The neoplastic cells formed sheets and cords, with a loose cellular arrangement and a moderate amount of reticulin fibres. The neoplastic cells were polygonal and stellate, with short cytoplasmic projections. The neoplastic cells had a tendency to coalesce Fig. . The abuIn all cases, the tumour cells were accompanied by numerous T-lymphocytes  and less numerous B-lymphocytes . Neutrophils formed large foci, mostly related to the necrotic areas. Mast cells and eosinophils were observed sporadically. The MHCII expression pattern was membranous or cytoplasmic and confirmed in more than 80 % of the tumour cells (three cases) or in 51-80 % of the tumour cells (one case). The CD18 expression pattern was membranous and confirmed in more than 80 % of the tumour cells (two cases), in 51-80 % of the tumour cells (one case) or in 20-50 % of the tumour cells (one case). The tumour cells were E-cadherin positive in two cases. The expression of E-cadherin was membranous . In the third case, the dog was euthanised due to a poor prognosis following the diagnosis. In the fourth case, the dog died within two years of the diagnosis. According to the survey, the cause of death was related to the tumour, but a necropsy was not performed.The present study revealed that a routine histopathological examination based solely on morphology is insufficient for diagnosing CCH. Six of the 71 tumours (8 %) that were diagnosed as CCH had to be reclassified after an immunohistochemical examination. The tumours misdiagnosed as CCH included epitheliotropic lymphoma and plasmacytoma. Lee Gross et al. revealed that CCH and cutaneous T-cell lymphoma can mimic each other perfectly; thus, immunohistochemistry is often required for differentiation 2005). Plasmacytoma (or B-cell lymphoma) has also previously been misdiagnosed as CCH  diagnosed as histiocytic sarcomas had to be reclassified after the immunohistochemical examination, and most of them remained undetermined, as they were negative for all or nearly all of the evaluated markers. The reason for the high rate of misdiagnoses may be either the poorly specified diagnostic criteria or strong morphological variations in this group of cutaneous tumours. Canine histiocytic sarcomas can mimic other spindle cell or round cell sarcomas; therefore, immunophenotypic evaluation is required (Affolter and Moore The small number of cutaneous histiocytic sarcomas that were evaluated does not allow any generalisations or conclusions to be made about their incidence, their breed or sex prevalence, or their preferred locations. However, the difficulty in collecting more tumour samples indicates that these tumours are rare, especially compared with their benign counterpart, or are frequently misdiagnosed and therefore underestimated. Furthermore, the histiocytic sarcomas described in this report showed marked morphological diversity. The cellular shape varied from round or polygonal to fusiform. None of the described tumours was characterised by the presence of all three cellular populations of round, spindle and multinucleated giant cells, as described previously (Affolter and Moore In conclusion, a routine histopathological examination is insufficient for diagnosing CCHs and canine cutaneous histiocytic sarcomas. Immunohistochemistry, including analysis of MHCII, CD18 and the lymphocytic markers CD3 and CD79, should be performed in questionable cases of CCH and in any case of canine cutaneous histiocytic sarcoma. E-cadherin expression is of limited diagnostic value in CCH. Certain canine cutaneous histiocytic sarcomas are E-cadherin positive and are therefore possibly derived from Langerhans cells. The spontaneous regression of CCH, determined by T- and B-lymphocyte infiltration, is connected with a decrease in the mitotic index, an increase in the transport of MHCII molecules from the cytoplasm to the cell membrane, and a loss of E-cadherin expression in the tumour cells. The present study highlights the practical aspects of diagnosing canine cutaneous histiocytic tumours.


The fiducial localization error distribution (FLE) and fiducial configuration govern the application accuracy of point-based registration and drive target registration error (TRE) prediction models. The error of physically localizing patient fiducials (We built a ground-truth (gt)-based unbiased estimator (We conclude that  Knowing the accuracy of the navigation system is crucial in image-guided surgery. The target registration error (TRE)  is the dIn simplest cases of TRE prediction models, the FLE is assumed to be zero-mean isotropic normal distributed  but therThis paper studies the simplest case of skull-mounted fiducial screws, as this is known to be the most precise case for point-based registration. In patient-space, In image-space, however, the cost of manual localization is high and samples are not guaranteed to be bias-free localizations. Moreover, without knowing the ground-truth fiducial locations the or (FRE) , measurior (FRE)  approximThis study presents techniques to directly estimate FLE distributions of the human fiducial localization process in image-space without the use of FRE. In order to directly measure FLE, the exact locations of the fiducials have to be known in a reference dataset. When using a physical CT device to create the dataset, complex phantoms have to be manufactured and positioned inside the CT machine with high precision . Recent Such a controlled environment is required for ground-truth measurements to evaluate human and algorithmic performance in image fiducial localization. The acquired ground-truth-based measurements are the best estimates that one can ever get for Section “Ground-truth FLE” defines a probabilistic viewpoint on measurement processes and ground-truth-based FLE. Sampling strategies for crowd and single-person-based experimental FLE with and without fiducial orientation dependence are defined. Section “FLE estimation without ground-truth data” defines the “difference-to-mean” (dtm) estimator which does not use the ground-truth data. Section “Testing equality of the gt and dtm estimators” utilizes a distribution-free kernel-based two-sample hypothesis test  to checkIn this section, we define various alternative interpretations of the FLE distribution, when ground-truth fiducial locations are available. These estimators are assumed to be the best possible estimators of the underlying fiducial localization error distribution. Several parameters  determine the final information content of the dataset in which the localization is made. Other parameters  are specific to the procedure with which the data collection is executed. All constant parameters of the imaging process and the measurement methodology are assumed to be implicitly encapsulated in a measurement process p on a randomly chosen fiducial s at repetition r. The values of p and s are running over all possible persons and fiducials, respectively: In the generic case, a sample f from s and p is assumed to follow the probability density function f for fiducial f to the error vector pointing from the true position of fiducial k to the acquired sample f. The probability distribution  scene. The orientations were randomly chosen but similarly to earlier plastic skull  participated in data collection. The experiment consisted of ten repetitions. At each repetition, the participants were asked to localize nine fiducials in the image set. In order to minimize the effect of short-term memory (repeating the same pixel measurement instead of fully reprocessing the dataset), the persons were encouraged to schedule a few hours break between repeated sessions. The participating persons applied the same methodology used on real CT datasets and had no access to the ground-truth locations. Each individual localized each of the 9 screws 10 times, giving 90 samples; in total, 900 samples were obtained.The definitions of ground-truth and dtm FLE were evaluated, resulting in a set of 3D vectors for each different FLE estimator. Table x, y, z axes and the best fit Gaussian. The collected data are not normally distributed  version of the dtm estimator seems to be the most reliable estimation technique. It seems to describe the data spread; for the crowd case, the zero-mean assumption is also viable. For the majority of the test cases, the resulting estimation shows no statistically significant difference to the ground-truth-based reference distribution.On the other hand, in the conditioned versions the tests have shown a much higher chance for a person and/or fiducial specific dtm estimate to significantly deviate from the ground-truth reference distribution. After inspection, the primary reason for the difference seems to be systematic bias introduced by the individuals in the markup process. The presence of this bias indicates that in practice more advanced TRE prediction methods are required that are capable of handling the presence of bias in the FLE distributions .Although the advantage of crowd-based data collection is clear, it is questionable if it can become a common practice to determine The ground-truth data in the crafted virtual datasets can aid the optimization or evaluation of the performance of persons/algorithms in image fiducial localization by allowing direct numerical validation. Apart from evaluating the performance of other estimators, the ground-truth-based estimators of the presented approach could also provide a sound


Introduction: Study goals attempt to identify the variables most commonly associated with successful tweeted messages and determine which variables have the most influence in promoting exponential dissemination of information  and trending (becoming popular) in the given disaster affected region.Methods: Part II describes the detailed extraction and triangulation filtration methodological approach to acquiring twitter data for the 2013 Hattiesburg Tornado. The data was then divided into two 48 hour windows before and after the tornado impact with a 2 hour pre-tornado buffer to capture tweets just prior to impact. Criteria-based analysis was completed for Tweets and users. The top 100 pre-Tornado and post-Tornado retweeted users were compared to establish the variability among the top retweeted users during the 4 day span. Results: Pre-Tornado variables that were correlated to higher retweeted rates include total user tweets (0.324), and total times message retweeted (0.530).  Post-Tornado variables that were correlated to higher retweeted rates include total hashtags in a retweet (0.538) and hashtags #Tornado (0.378) and #Hattiesburg (0.254). Overall hashtags usage significantly increased during the storm. Pre-storm there were 5,763 tweets with a hashtag and post-storm there was 13,598 using hashtags.Conclusions: Twitter’s unique features allow it to be considered a unique social media tool applicable for emergency managers and public health officials for rapid and accurate two way communication.  Additionally, understanding how variables can be properly manipulated plays a key role in understanding how to use this social media platform for effective, accurate, and rapid mass information communication.  The goal of Part III is (1) to identify the variables most associated with successful tweeted messages and (2) determine which variables have the most influence in promoting exponential dissemination of information  and trending (becoming popular) in the given region. By understanding the variables associated with Twitter messaging, disaster communication managers can purposefully and properly manipulate these factors to promote dissemination to at-risk populations and better understand and react to on the ground changes and events as they occur. This includes identifying those individuals that become ‘superspreaders’ who have a real-time exponential outreach within a local community. Understanding these variables of Twitter offer a way to create and monitor effective communication and information dissemination in real-time during a disaster.The study utilized a two-fold approach. First, the potential of message virality by variables associated by the top 100 regionally retweeted Twitter users was analyzed and second, the discovery of the overall use of hashtags in the region, a known tactic to stimulate viral Twitter message dissemination.DATA EXTRACTION AND FILTRATIONPart II describes the detailed extraction and filtration methodological approach .h Figure .1 The daRETWEETED USER VARIABLESTable 2 comprehensively lists the variables that were analyzed and collected through the 48 hours pre- and post- windows. The variables were publicly accessible from any Twitter profile or message. Of note, a Klout score, a commercially accepted (proprietary) rank is used to indicate the influence of a Twitter user as a proprietary variable based mainly on retweets and user-mentions.MULTIVARIANT METHODOLOGY,User data from the top 100 retweeted users was inputted into XLSTAT Version 2013.6.04, a plug-in for Microsoft Excel 2010, and a multivariate analysis was completed by ANOVA using the post-storm retweeted count as the independent variable.The study received an IRB exemption for human subject research from the William Carey University IRB Committee.RETWEETED USER VARIABLESTwenty-five of the top 100 post-storm retweeted users are listed in Table 1. Only 9 out of the top 25 (36%) post-Tornado retweeted users were within the top 25 of the pre-Tornado retweets (indicated with a † sign in Table 1). Those 9 retweeted users are the most identifiable superspreaders in the affected region.MULTIVARIANT RESULTSThe multivariant analysis (Table 2) revealed several criteria that correlated to higher retweeted user rates. The significant ones, p-values <0.05, are indicated with the † symbol. Variables that demonstrated correlation both pre- and post- Tornado included User follower counts, Retweets that contained a hashtag, Klout Score, retweet included hashtag MSWX, and total user mentions. These variables demonstrate the strongest correlation for possible modification.Pre-Tornado variables that were correlated to higher retweeted rates include total user tweets (0.324), and total times message retweeted (0.530). Post-Tornado variables that were correlated to higher retweeted rates include total hashtags in a retweet (0.538), and hashtags #Tornado (0.378) and #Hattiesburg (0.254) (Table 2).HASHTAG RESULTSOverall hashtag usage significantly increased during the storm. Pre-storm there were 5,763 tweets with a hashtag and post-storm there was 13,598 using hashtags. Twenty-five of the top 100 hashtags are listed in Table 3. Values with a † represent p-values <0.05. There was a 0.630 correlation between post-Tornado hashtags and a pre-Tornado hashtags correlation of 0.256 among top retweeted users. Top retweeters that used more hashtags post-Tornado in their retweet also had a correlation of 0.538 with their retweet rate.–Traditional methods of communications have transformed with the development of smart phones and the Internet. Television is slowly being replaced by personal video services such as Netflix, and the radio is being replaced by customizable systems such as Pandora or Spotify.Twitter provides an instantaneous publically accessible cost effective communication network. The cost is being able to afford a smart phone with access to the Internet—which is now readily available in the United States through state funded programs to socio-economically challenged communities. These research efforts have also demonstrated that Twitter can be purposefully targeted towards the geographically at-risk population if the right messenger is used or if the message is modified appropriately, particularly with the use of hashtags. These findings provide a framework in which Twitter can be utilized in the mitigation and preparedness planning and response phases of the disaster management.For the disaster plan phase the disaster management team should initially focus on identifying and recruiting the individuals that have large followers in the local region and in building individual disaster response follower bases. User followers along with the Klout score were shown to be the strongest pre-storm significant indicators associated with exponential message distribution. The average Twitter follower has 208 followers but our local superspreaders (out of the top 100 retweeted users) were found to have an average of 1,000 Twitter followers (5x the average follower). Increasing followers for those individuals and agencies involved in disaster response is something that should be expedited as part of the planning process and before large scale events. It should be noted that total number of tweets sent during the event did not correlate with virality so other variables play a stronger role.In this study it was found that local populations are more likely to follow individuals rather than organizations for crisis news. For example, while the broader local news station WDAM did not generate much activity during the storm, the weather news reporter from WDAM garnered a substantial viral outreach level. It should also be noted that just because one has a popular following does not guarantee viral messaging at the time of the disaster. While these users understand their population’s characteristics, they should be notified of the effective modifiable techniques, particularly the use of the correct hashtag, in order to bolster information relay during a disaster event.Also observed was an underutilization of individuals who possessed sufficient Twitter communication skills. While these individuals were among the Top 100 retweeted users prior to the storm they did not effectively communicate post-storm. It is speculated that perhaps they were personally and adversely affected by the storm or did not effectively modify their tweets. These individuals need to be targeted for Twitter education and training in the disaster planning phase. Importantly, many were sports reporters who had extensive Twitter experience and a previous history of using hashtags. Religious/spiritual professionals also proved to have large followers but did not necessarily effectively tweet post-storm. Part of the educational process should revolve around encouraging the use of the regional and event driven hashtag. The challenge however is that hashtag usage can evolve rapidly and unexpectedly requiring one to remain nimble with the flow of information.Figure 2). One might conclude that multiple hashtags would be effective for distribution, but this study did not demonstrate that. Purposeful employing of multiple hashtags may prove to bridge evolving hashtag trends and may have statistical significance in twitter message virality in the future.Retrospectively examining the Hattiesburg and Tornado hashtags showed that the initial hashtag utilized (about 4 hours prior to impact) for the storm was #MSWX (standing for Mississippi), followed by the specific event that occurred #tornado, and later focused on the area it struck (#hattiesburg) #911 and turn GPS on to allow search and rescue teams to rapidly locate them in real-time. The power of Twitter is an effective bimodal communication in real time with the affected at-risk populations.This study validates the value of Twitter in disaster planning, mitigation, preparedness and response and possibly in assisting the prioritization of recovery and rehabilitation efforts. It is hypothesized that Twitter can be effectively used in the disaster recovery phase, however our study solely focused on the initial disaster response phase. Future studies should explore Twitter’s potential across the entire disaster cycle.The findings are limited by the scope of time (96 hrs), and nature of event that was monitored in the region. Regardless the pre-tornado activity was typical of most normal days and activities in the region. The team was still able to extract and triangulate a total of 81,441 tweets and 10,646 twitter users, 27,309 retweets, and 2,637 tweets with GPS coordinates (pre and post).Search terms only utilized the English language and may miss minor misspellings of those terms not caught in the small user sampling. City names were not used outside of Birmingham and Hattiesburg due to corresponding cities in other states. Social media sites often provide anonymity that many users wish to preserve making defining their region impossible. Some users may be on business accounts and some view tweets without creating accounts.Twitter’s unique features allow it to be considered a unique social media tool applicable for emergency managers and public health officials for rapid and accurate two-way communication. Additionally, understanding how and if a variable can be manipulated plays a key role in understanding how to use this social media platform for effective, accurate, and rapid mass information communication. This knowledge will create a better framework for understanding how to create and alter messages that will be effectively received by the at-risk population and potentially mitigate adverse outcomes. This platform can be accessed via a smart phone so is relatively ubiquitous for all populations regardless of social and economic status.The authors have declared that no competing interests exist.


Infinium HumanMethylation 450 BeadChip Arrays by Illumina (Illumina HM450K) are among the most popular CpG microarray platforms widely used in biological and medical research. Several recent studies highlighted the potentially confounding impact of the genomic variation on the results of methylation studies performed using Illumina’s Infinium methylation probes. However, the complexity of SNPs impact on the methylation level measurements  has not been comprehensively described.In our comparative study of European and Asian populations performed using Illumina HM450K, we found that the majority of Infinium probes, which differentiated two examined groups, had SNPs in their target sequence. Characteristic tri-modal or bi-modal patterns of β values distribution among individual samples were observed for CpGs with SNPs in the first and second position, respectively. To better understand how SNPs affect methylation readouts, we investigated their impact in the context of SNP position and type, and of the Illumina probe type (Infinium I or II).Our study clearly demonstrates that SNP variation existing in the genome, if not accounted for, may lead to false interpretation of the methylation signal differences suggested by some of the Illumina Infinium probes. In addition, it provides important practical clues for discriminating between differences due to the methylation status and to the genomic polymorphisms, based on the inspection of methylation readouts in individual samples. This approach is of special importance when Illumina Infinium assay is used for any comparative population studies, whether related to cancer, disease, ethnicity where SNP frequencies differentiate the studied groups. Human DNA methylation studies have become an important part of many biological and medical research areas , 2. A nuThe number of methylation studies performed with the use of Illumina HM450K array have been published in various research fields, including studies on cancer, disease, ethnicity, aging, and gender, and it still grows exponentially , 10–15. Recently, some technical limitations of the Infinium platform have been recognized, leading to the recommendation for a more careful interpretation of DNA methylation results from the HM450K array , 16–19. The presence of SNPs at the CpG loci targeted by Illumina Infinium probes is one of the most important confounding variables, especially when methylation levels are compared among individuals from genetically non-homogeneous populations. Our interest in comparative population genetics prompted us to characterize the theoretical and practical impact of SNPs on the methylation readouts and on the interpretation of population data. Using exemplary results obtained with the use of Infinium probes in male B-lymphocyte lines from two human continental populations: Caucasian (European) and East Asian (Chinese), we illustrate how SNPs at the interrogated CpG loci affect the resulting methylation calls. Our study provides important practical clues for using HM450K array in comparative population studies.p < 0.05. Of those, 120 CpG sites were characterized by q < 0.05, and arbitrarily set |Mav_diff| > 0.3. These sites included 33 CpGs targeted by Infinium probes type I and 87 by probes type II, and were used to demonstrate the impact of genomic SNPs on the interpretation of methylation readout from HM450K array. A subset of 96 CpG sites , representing the highest inter-population differences  was provisionally termed “population-differentiating” (pop-diff) CpGs. The cut-off at |Mav_diff| > 1 roughly corresponded to that of an absolute difference in β value > 0.2 [Methylation microarray analysis of 36 European and East Asian cell lines revealed a total of 34,024 sites differentiating two populations) at the significance level p = 0.67).There was no global methylation difference between interrogated populations  for the presence of neighboring probes, located 500 bp up- or downstream from the investigated CpG. 53 of the 96 pop-diff probes had no other probes in the vicinity; the remaining 43 had from one to six neighbors. Altogether, there were 106 additional probes, 33 of which (31 %) differentiated two populations (Mdiff 0.2–0.49); however, the majority of these were not included in the list of 96 pop-diff because they did not fulfill the requirement of q < 0.05.We examined the 96 pop-diff probes ; cg26367031 and cg00862290 were 226 bp apart on chromosome 3 . The remaining pop-diff probes, when localized on the same chromosome, were separated from one another by much larger distances .Among 96 pop-diff probes, only two pairs (with no SNP in the interrogated CpG) mapped to the same chromosome region each. cg04287289 and cg26513180 were 8 bp apart on chromosome 16 , we frequently observed a high proportion of probes with a tri-modal or bi-modal distribution of the individual β values. This distribution resembled clustering expected for heterozygous and homozygous forms of SNP genotypes, also reported in earlier studies [Among 96 pop-diff CpGs representing the highest inter-population differences between Europeans and Asians  of the pop-diff CpG sites contained common genomic SNPs  with the allele frequencies strongly differentiating two analysed populations (MAF_diff > 0.1). Fst values calculated for each of these SNPs were in the range of 0.51–0.013.). The average genetic distance between European and East Asian populations based on these 66 SNPs was 0.26 (+ − 0.11), which is significantly more than the average Fst of 0.10–0.15 or less, reported for inter-continental comparisons of human populations , only 25 % had a SNP in the interrogated CpG. These observations suggested that the large proportion of population differences detected by the HM450K probes reflected genomic rather than epigenetic differences. None of these CpGs corresponded to the 65 control Illumina probes (rs) that assay highly-polymorphic single nucleotide polymorphisms (SNPs) and are intentionally included in the HM450K array to allow sample quality control and indicate the degree of technical variance between samples. All the probes targeting pop-diff CpGs with SNPs in the interrogated sequence belonged to the class flagged to be discarded from methylation analyses by Naeem et al. [In contrast, among 24 weakly differentiating CpG sites  than for type I Infinium probes . When the position of a SNP within the interrogated CpG was considered, seven type I pop-diff probes had SNP in the first, and five—in the second CpG position; for type II pop-diff probes, 46 had SNP in the first, and eight—in the second CpG position. No significant differences between the number of probes with SNPs in the first and second CpG position were observed in a set of 24 weakly differentiating probes. This comparison clearly indicated that the population-differentiating capacity was mainly restricted to type II probes, with a frequent SNP in the first position of the interrogated CpG. This effect was seen both when a SNP in CpG was the only confounding factor, and when it was combined with other factors listed by Naeem et al. , like unThe excess of Infinium type II among the pop-diff probes with SNPs can be related to the fact that most of the CpGs interrogated by these probes are located in the genomic sea, outside of the presumably more evolutionary conserved CpG islands and shores  interrogated by Infinium I probes.In our study, the majority of pop-diff Infinium probes targeting CpG sites with common SNPs, yielded characteristic patterns of β values distribution among the examined cell lines: tri-modal, bi-modal, and cloud-like. In order to facilitate interpretation of such distributions, we examined potential factors influencing these patters. For individual probes, the pattern depended on: the SNP position in the interrogated CpG site; the probe type (Infinium I or II); and the methylation status of the wild type CpG allele at the examined locus. It has to be emphasized that in over 40 % of the pop-diff probes with a SNP in the targeted dinucleotide, other potentially confounding features were present; however, for most of them the presence of a SNP in the interrogated CpG appeared to exert the strongest effect upon methylation readouts. For the sake of clarity, examples presented below represent probes with SNPs in the interrogated CpG sites and with no other confounding factors. The examples are not intended to cover the whole range of variability in β values distribution. Instead, they are chosen to illustrate some of the mechanisms that influence the readouts, and possibly to help in the interpretation of some ambiguous results that may be encountered when using Illumina Infinium probes.The most conspicuous and very frequent pattern observed among pop-diff CpGs in our study, was that of a tri-modal distribution of β values  fluorescence signal from the probes.A similar tri-modal distribution was also observed when C in the first position of the interrogated CpG sites was substituted by A or G. In most CpG sites with a SNP in the first position, the concurrent presence of another confounding factor, like SNPs, indels or repeats under a probe, had only minor effect compared to the influence of SNP in the interrogated site and did not disturb the tri-modal distribution of β values (not shown).Another striking pattern, that of a bi-modal β distribution, was typically seen in CpGs with a G/A SNP at the second position  heterozygotes therefore, the only significant contribution to the fluorescence readout was from the probes indicating methylation , and β values did not differ from those obtained for methylated CpG homozygotes , as described above, affected both type I and type II probes interrogating CpG site with a G/A SNP in the second position. A similar effect was observed when Infinium type I probes were used to interrogate CpG sites with a C/T SNP in the first position. Here, the mechanism of low fluorescence signals reflected the nature of Infinium type I probes, which assume concordant methylation of the neighbouring CpGs. In the example illustrated in Fig. For a number of pop-diff probes, the impact of substitutions in the interrogated CpGs was ambiguous, with no clear correlation between the β values and the SNP genotypes. A typical characteristics of these patterns was a cloud-like pattern, with varying level of β values for CpG homozygotes, which indicated non-uniform level of CpG methylation. In these cases, β values for heterozygotes were variable, and homozygotes for non-CpG alleles usually had low β values Fig. .Fig. 5Exav values could be ascribed to real differences in the level of methylation between the studied groups . In the example presented in Fig. It has to be emphasized that similar cloud-like patterns of individual β values distribution were also observed when probes targeted “clean” CpGs (with no SNPs or other confounding factors). In these cases, inter-population differences in βThe impact of SNPs on methylation readouts  in the Illumina HM450K array, while predicted, has not been well characterized so far. Here we analyzed the theoretical, expected outcome in the context of different SNPs and probes, and compared it to the observed distribution of β values in the examined cell lines.Among the factors that influence the impact of SNPs on the β values, the polymorphism’s location and type  are the most obvious. Furthermore, because of the differences in the chemistry underlying signal detection when using Infinium I and II probes, SNP’s effect strongly depends on the type of Infinium probes . Any otThe effect of under-a-probe SNPs is not uniform and cannot be easily predicted from the probe sequence and sample genotype. This is reflected by the inconsistencies in the literature: some authors had suggested that SNPs within 10 bp of the interrogated CpG can affect probes , while oSNPs located in the interrogated CpGs have a potential to affect the Illumina HM450K array readouts in a more direct manner. Tables For type I probes, the genomic C>T substitution in the interrogated CpG site renders it undistinguishable from the bisulfite-converted unmethylated dinucleotide (TpG). However, it has to be remembered that if the interrogated CpG was methylated, the C>T substitution would change the presumed concordance in the methylation status between the tested dinucleotide and under-a-probe CpGs. This would lead to an inefficient attachment of not only one but of both probes. The resulting low fluorescence readouts from both probes, compared to relatively high readouts from the same probes efficiently hybridizing with CpG alleles, would affect β values and return false estimates of the methylation level . The influence of the C>A or C>G substitutions in the interrogated CpG sites is difficult to predict; these substitutions would affect hybridization of the probe at its 3′ end, thus lowering the extension efficiency of both M and U probes.For type II probes, the genomic C>T in the interrogated CpG site also mimics the unmethylated status of the examined dinucleotide in the bisulfite-converted DNA, resulting in the red fluorescence signal (incorporation of a complementary A). The genomic C>A substitution would give a similar signal ; the genomic C>G, in turn, would mimic the methylated status of the dinucleotide . Unlike in type I probes, the readouts of degenerated type II probes do not depend on the concordance between the methylation status of the interrogated dinucleotide and adjacent CpG.Substitutions in the second position of the interrogated CpG site  have a strong impact on methylation measurements using Infinium probes. They all act in a way similar to SNPs complementary to the 3′-most end of the probe, by affecting the hybridization and thus strongly lowering or completely blocking the efficiency of the probe’s extension. This results in very low fluorescence readouts for any type of the Illumina probes , affecting the calculation of β value and returning false estimates of the methylation level (see below). It has to be emphasized that any substitution in the second position of the CpG affects also the first position of the interrogated dinucleotide in the bisulfite-converted DNA; e.g. genomic CpA becomes TpA.av or Mav) is calculated, and used to assess the population/group difference of the methylation status for each probe [av value depends on its methylation status. However, in a population with the high frequency of a non-CpG allele, the βav value does not reflect the real (un)methylation status. The analysis of the distribution of individual β values, as presented above, provides important clues for discriminating between βav differences due to the methylation status and to the genomic polymorphisms affecting the analyzed CpG.In the routine analysis of the data from Illumina HM450 array, an average methylation level in each studied group . In these cases it is advisable to inspect individual values of normalized fluorescence signals in search for cases where the sum of intensities of both fluorescence signals (green and red) are abnormally low compared to other samples. When Infinium probes efficiently hybridize to the interrogated locus, the increase of the green signal indicating methylation occurs at the cost of the red signal indicating lack of methylation, and vice-versa. In case of an inefficient hybridization of the probes , individual beta values are artificially inflated, leading to a high βSome observations regarding the impact of SNPs on the results from Illumina HM450K array have been reported in earlier studies on methylation in tissues and phenotypes , 16, 19,The recently published list of Infinium probes affected by factors that confound methylation readouts in Illumina HM450K array , is a usdif in the range of 1.1–2.9 (manuscript in preparation). Our data suggest that the best candidates for comparative methylation studies could be found among probes with the “cloudy” distribution of individual β values. Some probes with such a pattern were classified as “clean” (free of SNP and other confounding effects); others had SNPs in the interrogated CpGs. The observed cloudy pattern in the latter could be due to overlapping effects, with the methylation differences “masked” by the presence of a SNP. Discarding such probes  may lead to missing some possibly important methylation differences. Inspection of individual probes in search for a “cloudy” appearance of the β values distribution may help to find out, which probes should be retained for the analysis, even if a SNP is present.It has been estimated that only a small fraction of CpGs across the genome vary in their methylation across human populations , 22. In Accounting for the presence of SNPs potentially confounding the readout of methylation signals should be mandatory in any research involving comparison of methylation level in populations. This may be crucial not only in comparative population studies, but also in a medical research during control group selection, when a natural SNP variation existing in a population could be incorrectly interpreted as revealing methylation state associated with the disease.n = 18), further referred to as Europeans and Han Chinese from Bejing, further referred to as East Asians (n = 18) populations were used in the study. All lines were derived from unrelated healthy adults males . The cells were cultured under the same standard conditions and harvested at 1 million cell lines per 1 ml. Shortly, immortalized B lymphocytes were purchased from Coriell Cell Repositories. Cells were grown in RPMI medium supplemented with 10 % FBS, 2 % glutamine and 1 % HEPES as well as 1 % penicillin/streptomycin solution . Cell lines were maintained in suspension in T flasks in a humidified 5 % CO2 atmosphere at 37 °C.Human B-lymphocyte cell lines representing Caucasian from Georgia (DNA samples isolated from the cell lines were bisulfite converted (according to Zymo Research protocol) and analysed using Illumina Infinium HumanMethylation450 BeadChip array . Samples were evenly distributed between three slides: on each slide 6 European and 6 Asian samples were analysed. The quality control was performed in Genome Studio Methylation Module (Illumina), according to the Illumina recommendations. All the samples passed the quality criteria.Genotyping of genomic SNPs in CpG loci was based on the 1000 Genomes database resources and/or direct sequencing of PCR products (primers and PCR conditions are available from the authors upon request).The study was approved by the Bioethical Committee at the Central Clinical Hospital of the Ministry of Interior in Warsaw (No 67/2100).The data were preprocessed using the quantile normalization method SWAN  , implemeTo evaluate the batch effect, we performed the unsupervised analysis . The main source of variation in the dataset represented the difference between European and East Asian population; no batch effect related to the difference between arrays was observed (data not shown).To check, whether the pre-processing filtering of probes has the significant impact on the results of our analysis, we calculated the detection P for all the probes. There were 2844 failed probes with a detection p-value greater than 0.01 in at least one sample. We filtered out all the 2844 failed probes from the normalized data and applied the Student’s t-test followed by FDR. We applied the criteria described in the paper  > 1) to select differentially methylated CpGs and obtained 76 such probes (instead of 96). However, the four probes that are described in details in the paper passed the criteria of differentially expressed genes. It shows that adding the filtering step does not have an influence on the paper’s conclusions.For each site and each individual, β values  were calculated using the following formula:β values, ranging from 0 (100 % unmethylated) to 1 (100 % methylated), have a more intuitive biological interpretation and were used here for visualization of signals from individual measurements. M values, which are more statistically valid for differential analysis , 26, werav_diff (the differences between average methylation levels in the two populations) were calculated for each interrogated position. After correction for multiple testing  [To identify differentially methylated CpGs, the Student’s t-test was performed and absolute values of Mte, FDR) , 28, onlIllumina HM450K array consists of two assays, Infinium I and II .The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus  and are


As an oncology advanced practitioner, you may find yourself called upon to write or partner with a nurse scientist to write a grant to support or improve educational, patient care, or professional activities. There are different types of grants that can be used to support a variety of activities, such as research grants, educational program development grants, and quality improvement/clinical practice grants, to name a few.Writing your grant application requires quite a bit of preparation. Before you begin, it is important to review the grant application and its specific instructions—they can vary widely in their parameters. Adherence to the grant instructions is a critical aspect of grant preparation. Grant proposals that do not adhere to page length, font type, and other specifications may not even be reviewed.Pay careful attention to the grant personnel who will carry out the proposal. Some grant mechanisms require a doctoral degree as the minimum preparation for the primary investigator or for educational grants, the program director. You must consider what additional expertise may be required to carry out the project or research successfully. You may need a nurse clinician or other provider to grant access to a patient population, a statistician, or another specialty  that would provide expertise and services to complete the project or research.Data analysis for the research or project outcomes should be considered at the very start of the proposal. Secure the advice of a statistician in determining the study sample size, measures to be used, and the plan for data analysis.As protection of human subjects and informed consent are of paramount importance, even in simple survey or educational research methods, a thorough understanding of Internal Review Board (IRB) requirements and submission for IRB approval requires planning; it can take several months to gain approval. Lastly, consideration of budget costs and constraints is necessary to be sure to be able to cover the costs of completing the proposed project or research.Clarity and attention to detail are aspects of grant writing that make for a successful application. Be clear, concise, and precise. Define what you are proposing, for whom it is intended, and exactly how it will be accomplished. For those new to grant writing, seek out an experienced mentor to assist you in the development of your ideas and the review of the drafts of the proposal prior to submission. The National Institutes of Health peer review criteria for scoring grants is helpful when considering the areas a well-written grant addresses.Your proposal should speak to the significance of the issue or problem to be addressed and how, if the aims are achieved, the findings may change the field to improve clinical practice or the care of patients. The review of the investigative team should explicate the appropriateness of the team members for the project or research and show that they have a track record of accomplishments that make them ideal partners for your work. Innovation in clinical practice and approaches or methods that are novel or result in a paradigm shift are quite appealing. The approach should be well specified, detailed, and well reasoned, with consideration of burden to the subject or population. Lastly, be sure to demonstrate that the environment for the conduct of the project or research is supportive and that resources are available to support the successful completion of the grant.There are many reliable online resources available for both grant-writing support and available funding. You will find a good number of such resources listed in the Table to the left. But don’t neglect your own institution for funding information as well. Many institutions have foundations for grant support of approved projects.Grant writing can be a daunting process. But if you approach it step by step and stay positive and focused, it will not be nearly as difficult as you might imagine. Remember, the more preparation and attention to detail you put in, the more likely that you will have success in securing your desired funding.


Chem. Chem. Phys., 2015, DOI: ; 10.1039/c4cp05278a.Correction for ‘Orbital entanglement and CASSCF analysis of the Ru–NO bond in a Ruthenium nitrosyl complex’ by Leon Freitag  The authors would like to explicitly acknowledge the exchange funding of a European Short Term Scientific Mission (STSM) between Austria and Switzerland, and therefore the amended Acknowledgments should read as follows:Financial support from the University of Vienna, the COST Action CM1305 ‘‘Explicit Control Over Spin-states in Technology and Biochemistry’’ (STSM 21153), the Research Council of Norway through a Centre of Excellence Grant (Grant No. 179568/V30), the Italian Ministry of Education and Research (MIUR)-Grant No. RBFR1248UI, the Uppsala University, eSSENCE, and the Swedish Research Council (VR) is gratefully acknowledged. Furthermore, we thank Yingjin Ma for the help with the MAQUIS-MOLCAS interface, as well as Vladimir Arion and Gabriel Büchel for fruitful discussions.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.