Homo. Most archaeologists agree that this was a multi-stage process, beginning with the exploitation of natural fires and ending with the ability to create fire from scratch. Some have argued that in the Middle Palaeolithic (MP) hominin fire use was limited by the availability of fire in the landscape. Here, we present a record of the abundance of polycyclic aromatic hydrocarbons (PAHs), organic compounds that are produced during the combustion of organic material, from Lusakert Cave, a MP site in Armenia. We find no correlation between the abundance of light PAHs (3–4 rings), which are a major component of wildfire PAH emissions and are shown to disperse widely during fire events, and heavy PAHs (5–6 rings), which are a major component of particulate emissions of burned wood. Instead, we find heavy PAHs correlate with MP artifact density at the site. Given that hPAH abundance correlates with occupation intensity rather than lPAH abundance, we argue that MP hominins were able to control fire and utilize it regardless of the variability of fires in the environment. Together with other studies on MP fire use, these results suggest that the ability of hominins to manipulate fire independent of exploitation of wildfires was spatially variable in the MP and may have developed multiple times in the genus Homo.The use of fire played an important role in the social and technological development of the genus  Homo1, allowing for warmth, cooking, birch tar production, protection from predators, a venue for social interactions and access to high latitudes and dark caves2. Evidence of hominin fire use is present in the archaeological record beginning around 1.5 million years ago5, and while it has long been assumed that a variety of hominin species could use fire7, the degree to which hominins were able to intentionally create and control fire (pyrotechnology) is debated17. Recently, some have argued that this ability was exclusive to modern humans16, with other hominins, such as Neanderthals, limited to exploiting wildfires. Neanderthals went extinct during the late Pleistocene. The reasons for that extinction remain unclear, and recent genetic data indicate that Neanderthal DNA persists among certain modern populations of Homo sapiens18. Neanderthal extinction has been linked specifically or in combination to fire use, foraging behaviors, a lack of clothing, demography, climate change, and interactions with expanding populations of Upper Palaeolithic Homo sapiens20. Evidence for the use of fire among MP hominins includes burning found in archaeological sites, and the construction of hearths. Manganese dioxide blocks, which are useful for fire-starting, have also been excavated at MP sites and are interpreted by some as evidence of Neanderthal fire production21. However, research at MP sites in France claims that fire frequency, measured by thermally altered flint and burnt bone, is positively correlated with warmer periods, when wildfire frequency is assumed to be highest, rather than with colder, glacial periods when fire use would have provided greater benefit; this correlation is derived by associating chronometrically dated stratigraphic layers at these sites with particular phases of Pleistocene global temperature records. These data are collectively interpreted as evidence that Neanderthals had not mastered pyrotechnology, and instead harvested natural fires caused by lightning strikes16, though this interpretation is not accepted by all15.The use of fire played a key role in the evolution of the genus 24. Organic molecular markers of fire, including PAHs, can provide a quantitative record of fire over geological time scales26, though lighter PAHs may be more susceptible to the effects of degradation due to their higher solubility27. During biomass combustion PAHs with variable structure are formed typically containing two  to six  rings  with four or fewer rings concentrate in the gaseous phase, whereas high molecular weight PAHs (hPAHs), with five or more rings tend to concentrate in the particulate phase36. Hearths in archaeological contexts can reach maximum temperatures of 1000 °C with mean temperatures of 600 °C, which provides sufficient energy for the production of hPAHs38.In order to test the hypothesis that MP hominin fire use was correlated with natural fire frequency, we developed a record of polycyclic aromatic hydrocarbons (PAHs) for the MP site of Lusakert Cave 1 (LKT1) in the Armenian Highlands from eighteen sedimentary units associated with MP lithic technology39. Following forest fires in South Korea, lPAH concentrations were found to be 3 to 28 times higher than hPAH concentrations40. Soil O-horizons in Russia analyzed two, ten and 16 years after a wildfire found that hPAH concentrations were not higher than background soils, despite 9-fold increases in total PAH concentrations41. hPAHs comprised 35% of the total PAH load in unburnt samples from fire prone regions in Spain, but less than 10% in burnt soils after wildfires due to the addition of lPAHs42. Finally, in savannah fires in Australia the frequency of emitted lPAHs was 13 to 30 times greater than hPAHs43. These data attest to the widespread production and dispersal of lPAHs associated with wildfire events.Unlike wood combustion in hearths, studies document the low production of hPAHs in wild fires of varying intensities, which produce mostly 3- and 4-ring PAHs. For example, soils measured in a recently burned area in southern France did not measure hPAH abundances higher than control samples45. Different explanations have been given for the lack of hPAHs relative to lPAHs, including changes in temperature or burn intensity44, as higher molecular weight PAHs require higher activation energies for synthesis. Another potential explanation is the proximity to the source of the fire45, as hPAHs are less likely to travel from the source of the fire. Therefore, in the context of a spatially confined archaeological site such as LKT1, we expect that the primary mode of deposition of lPAHs will be through long-range dispersals of wildfire, whereas the primary mode of deposition of hPAHs will be local fire use within the cave by hominins. Given the low abundance of hPAHs produced during wildfires relative to lPAHs, and their concentration in the particulate emissions from wood burned in hearths, it is the most parsimonious explanation that accumulation of hPAHs in the sediments of archaeological sites like Lusakert Cave will be due to increased particulate deposition of residues from wood combustion. These particulate emissions of hearths will concentrate locally, on the scale of 10 s of meters.In sedimentary records from fire-prone regions, hPAHs are typically lower in abundance than lPAHswax) and carbon (δ13Cwax) isotope composition of long-chain n-alkanes, the molecular remains of epicuticular waxes of terrestrial plants, to constrain regional climate and hydrology through the period of hominin occupation , demonstrating that they did not undergo significant microbial46 or thermal47 alteration . In C3 plants, δ13C values are influenced by physiological changes as plants balance water loss and CO2 uptake through the regulation of stomata, altering the partial pressures of intracellular (Ci) relative to extracellular (Ca) CO249, causing a positive shift in δ13C values in C3 plants experiencing water stress50. Fractionation during lipid biosynthesis causes n-alkane δ13C values to be lower relative to bulk plant tissues52. Therefore, sedimentary δ13Cwax values of n-alkanes predominantly reflect plant photosynthetic type and factors that affect isotopic discrimination during carbon fixation53. δDwax record the isotope values of ambient water during the period of growth54, reflecting mostly precipitation isotope values in terrestrial systems. δD values of precipitation are influenced by temperature, amount of precipitation, and cloud transport history56. Most of the variability in modern precipitation δD values in the Armenian Highlands is explained by changes in temperature, and there is no significant trend associated with amount of precipitation57. This is also documented in Global Network of Isotopes in Precipitation stations in Georgia and Turkey  and on-site fire frequency (hPAHs). If hominin pyrotechnology drove fire use, we would not expect to see a strict correlation between climate and fire frequency.At LKT1, δion Fig. , which ince Fig. , which m13Cwax) and temperature (δDwax) change documented at LKT1 demonstrates that MP hominins used fire in the cave during periods of low wildfire abundance, as recorded by lPAHs  and is not dependent on sediment type  rather than wild fire frequency, MP hominins at the site were able to habitually control fire, and that these activities were independent of climate .The combined record of local fire (hPAHs), regional fire (lPAHs), vegetation δ3Cwax andAHs Fig. . Layers AHs Fig. . The inv63. Overall, the abundance of hPAHs in these modern wood combustion studies is (in order most abundant to least): B(a)P > B(k)F > B(b)F > IP + DA > BP. This order of abundance is the same as the averaged abundance for the layers at Lusakert, B(a)P > B(k)F > B(b)F > IP + DA > BP . B(a)P does seem to be more abundant relative to other hPAHs than in the modern wood burning study, however, other studies demonstrate similar high abundances of B(a)P relative to other hPAHs in burning of pine wood with green needles and savanna grasses , which may be a reflection of the open environment suggested by the high δ13Cwax values65.At LKT1, the most abundant hPAHs are B(a)P (8 layers), B(k)F (8 layers) and B(b)F (2 layers). In a series of experiments on wood combustion in fireplaces and wood stoves, B(a)P is the most abundant hPAH produced of those measured in this study in 20 of the 29 different species of wood with B(k)F the most abundant in 8 species and B(b)F the most abundant in 1n-alkanes are correlated with their molecular weight, and as such lower molecular weight PAHs and n-alkanes are more susceptible to degradation. Denis et al.27 argue that sedimentary units with lPAH concentrations greater than hPAH concentrations are indicative of favorable conditions for organic preservation27. This is observed in Units 3, 4, 7 and 10 at Lusakert, but not in Units 5 and 6. However, based on the consistently high OEP values, n-alkanes at the site do not show any increase in degradation in Units 5 and 6  and no significant relationship with δDwax values , suggest wild fire frequency (lPAHs) is not determined by temperature, but rather by changes in vegetation. More positive δ13Cwax values, representing more open habitats, are associated with higher frequencies of wild fire, whereas more negative δ13Cwax values, associated with more closed habitats, are associated with lower frequencies of wildfire. Other studies have observed this correlation between δ13Cwax values and lPAH concentration66. This is consistent with research on modern wildfires, the frequency of which is not only determined by the frequency of ignition agents (e.g. lightning strikes), but also by a number of variables including precipitation and net primary productivity67. Though global wildfire activity in the Holocene may correlate with temperature68, records of Holocene fire frequency in the Armenian Highlands co-varied with vegetation and aridity changes69.This coupled record of fire and climate also shows the drivers of fire frequency in the past in the Armenian Highlands. There is a significant positive correlation between lPAHs and δAt LKT1, the most robust evidence for fire (hPAHs) correlates with periods during which wildfires (lPAHs) were at their lowest frequency and occupation intensity was at its highest. Therefore, we can reject the hypothesis that fire use among MP hominins was predicated on its natural occurrence in the regional environment. We interpret the evidence as indicating the habitual use of fire by MP hominins during periods of low wildfire frequency. While these data do not preclude the interpretation that MP hominins were harvesting and maintaining wildfires, given that wildfire was still present on the landscape during all periods of occupation at the site, we conclude that the combined evidence demonstrate that MP hominins exhibited significant control over fire, and likely pyrotechnology.13 and evidence for its control dates to earlier than 300 ka14. If the contradictory evidence from MP sites in France is correct, these data suggest that MP fire use was regionally differentiated. This would suggest that the ability of hominins to control and perhaps create fire was either (1) a primitive behavioral trait that was lost in certain MP populations, or (2) a behavioral trait derived independently among MP populations. In either case, it follows that pyrotechnology was not limited to Upper Palaeolithic Homo sapiens and therefore unlikely to have played any role in the demise of MP hominins such as the Neanderthals. By applying the methods outlined here to Palaeolithic sites elsewhere in Eurasia and Stone Age sites in Africa, we will be able to determine what, if any, spatio-temporal patterns exist in pyrotechnology, and how such patterns might be equated with particular hominin species, environments, or climates.These results suggest that pyrotechnology existed among MP hominins. However, its true antiquity has yet to be determined, and evidence for fire creation exists at 50 kan-alkanes and PAHs were separated from total liquid extract by passing samples through a column of activated silica gel (1.25 g) in baked Pasteur pipettes with 2 mL hexane (non-polar fraction), 4 mL dichloromethane (slightly polar fraction) and 4 mL methanol (polar fraction). n-alkanes were quantified through the analysis of the hexane fraction and PAHs were quantified through the analysis of the dichloromethane fraction. We did not identify PAHs in the hexane soluble fraction.We collected 18 sediment samples (~150 g) in the summer of 2014 from each sedimentary unit in Profile 4 at LKT1. We stored these samples at −20 °C, and they were lyophilized prior to lipid extraction. Lipids were extracted from the sediment via Soxhlet extraction in 300 mL of 2:1 dichloromethane:methanol for 48 hours. Following lipid extraction, we saponified the total lipid extract by heating the sample for 2 hours at 85 °C with 5 mL of 1 M KOH in methanol. We stopped the reaction by cooling to room temperature and adding 5 mL of 5% sodium chloride in water. We then extracted neutral compounds with a liquid/liquid extraction. n-alkanes and PAHs were measured on a Trace Gas Chromatograph (GC) Ultra (Thermo-Scientific) fitted with a split–splitless (SSL) injector and flame ionization detector (FID). We quantified n-alkanes using a BP-5 column  with He as the carrier (1.5 ml/min). Oven temperature was set at 50 °C for 1 min, ramped to 180 °C at 12 °C/min, then ramped to 320 °C at 6 °C/min and held for 4 min. We measured PAHs using a DB-5 column  with He as the carrier (1.5 ml/min). Oven temperature was set at 70 °C and held for 4 minutes, ramped to 180 °C at a rate of 15 °C/min, ramped to 290 °C at 4 °C/min, then ramped to 310 °C at 5 °C/min and held for 19 minutes. Samples were analyzed in concert with a 16 component PAH standard  at known concentrations ranging from 0.25 to 400 ng for calibration and quantification. Calibration mixes were also analyzed via Gas Chromatography-Mass Spectrometry (GC-MS) on an Agilent 6890 at the University of Connecticut with a relative standard error of 4%.13C values of individual n-alkanes were measured with a GC-Isolink coupled to a MAT 253 Isotope Ratio Mass Spectrometer (IRMS) (Thermo Scientific) with a BP-5 column  at the University of Connecticut. Oven temperature and ramp was identical to the method used for n-alkane measurement. Isotopic standards (Mix A from A. Schimmelman) were analyzed every four samples across a range of concentrations to correct for size and scale effects. Standard deviations were 0.5‰ for δ13C (n = 7) and 4‰ for δD (n = 9). Stable isotopic composition is expressed in standard delta notation relative to VPDB and VSMOW.The δD and δSupplementary Information


Whenever computers are used to represent genetic data, the computational encoding must be efficient to allow the representation of processes driving the inheritance and variability. This is especially important across simulations in view of the increasing complexity and dimensions brought by genomics. This paper introduces a new binary representation of genetic information. Algorithms as bitwise operations that mimic the inheritance of a wide range of polymorphisms are also presented. Different kinds and mixtures of polymorphisms are discussed and exemplified. Proposed algorithms and data structures were implemented in C++ programming language and is available to end users in the R package “isqg” which is available at the R repository (cran). Supplementary data are available online.The  Generalizations are provided to support a wide range of known polymorphisms. The algorithms to simulate the generation of variability are presented in terms of bitwise operations. The algorithms and data structures are available to users as an R package “isqg” which is available at the official R repository (l), each one indexed by a genome position (x), which may be associated with a physical or map coordinates. Thus, a binary representation of a dna strands consists of a set of bits (n bits expands to An oligonucleotide is a linear sequence of loci (α and β) as both dna strands, the mosaic recombination (δ) can be obtained by bitwise operations. To do this, another set (γ) must be introduced, which works as a bit mask. This mask controls the flow of the content between both chromatids. It works as a logical conditional statement such as an if-else block and thus, allow the composition of the mosaic between both chromatids i.e., an 1 entry in γ means the bit is taken from α, while a 0 implies it is taken from β. As α, β, δ and γ are binary sets, a single bitwise statement must be performed combining the operators and (&), or 1: mask2: if then3: 4: forchiasmainchiasmatado5: 6: 7: 8: if then9: 10: 11: γ) beforehand, i.e., ones turn zeros and vice-versa, which is carried out by the not operator  with complex quantitative traits driven by multi-allelic factors.In addition, as recombination depends only to the loci’s positions, the linkage among a collection of bits assigned to the same position will never break, working as a single loci. This framework also supports a mixture of different types of polymorphism coexisting together, such as the one at the sequence-level or those with multiple alleles, which may unveil other understanding about the association of i from parent to offspring can also be performed by binary operations. Given the index of the loci to be changed (edited or mutated), a right shift on an empty bitset results in a bit mask for this event. Then, an exclusive or (xor/Mutations and/or gene editing changing the state of locus gpl-2); source codes and binary version of the software are available at the official public repository of R packages (cran): https://cran.r-project.org/package=isqg. The authors affirm that all data necessary for confirming the conclusions of the article are present within the article, figures, and tables. Supplemental material available at FigShare: https://doi.org/10.25387/g3.8162051.Software are released under a public license  is loaded and an specie is started. Then, two completely contrasting individuals are initialized and the Example 1: Simple simulation of crosses and double-haploids with “isqg”library(isqg)> data(ToyMap)> ## start specie given the map> spp <- set_specie(ToyMap)> ## start some ”founders” individuals from specie> AA <- spp$founder(code = ”AA”)> aa <- spp$founder(code = ”aa”)> ## making some crosses > ## are accessible by standalone function and R6 methods> F1 <-cross # the hybrid> F2 <- F1$selfcross(n = 1E3) # segregating population> RILs <- F1$dh(n = 1E3) # inbreeds> ## retrieving the genotypic data> M <- genotype(RILs) # as numeric> N <- F1$genotype(phase = TRUE) # as character (phased)> M) and the meiosis recombination is performed through a count location process > data(ToyMap)> ## standard specie as in Example 1> standard <- set_specie(ToyMap)> ## sample 100 gametes from standard process> standard$gamete(100)> ## require Rcpp package> library(Rcpp)> ## compile and link Example 2 at file Independent.cpp> sourceCpp> ## specie with custom recombination> custom <- set_specie> ## sample 100 gametes from custom process> custom$gamete(100)> i.e., the coefficient of identity by descent. To do so, a specie is initialized with a genetic map having fives chromosomes each one with 1000 equally spaced biallelic loci and a quantitative trait is defined with zero mean and additive effect of one for all loci. From this settings, two contrasting individuals and the Considering a breeding scenario where Example 3: Initializing structures and data for a simulation with “isqg”## generating map: 5 chromosomes and 1000 ”genes” per chromosome> map <- expand.grid)> map$snp <- paste0)> ## initializing ”specie” and ”infinitesimal” quantitative trait> spp <- set_specie(map)> trait <- set_infty> ## initializing two contrasting parents and the F1> P1 <- spp$founder(code = ”AA”)> P2 <- P$mirror> F1 <- cross> After that, one may define an R function to simulated the above mentioned breeding scenario:i.e., N individuals as well as P individuals each;generating inbreeding generations getting the true breeding values regarding the defined quantitative trait and simulating phenotypes adding random deviates accoring to the a desired heritability ; andcollecting and delivering the estimated additive and residual variances.Replications of the simulated scenario are then obtained in which the collected statistics can be further analyzed (Example 4). Consider to load the package rrBLUP beforehand as its functions are used over the simulation .Example 4: Defining and simulating breeding schemes with “isqg”simulation <- function(zzz) {> ## (i) inbreeding generations:+ F2 <- F1$selfcross(n = N)+ F2_3 <- unlist x$selfcross(n = P)),+ recursive = TRUE)+ ## (ii) true breeding values **standardized** and phenotypes:+ alphas <- scale x$alpha(trait)))+ phens <- rnorm / h2))+ ## (iii) VanRaden realized relationship matrix:+ G <- A.mat(t(genotype(F2_3)))+ ## (iv) fitting the mixed models with A and G:+ X <- rep+ Z <- diag(N * P)+ mP <- mixed.solve+ mG <- mixed.solve+ ## (v) collecting and delivering statistics:+ # additive variancevgs <- c + # residuals varianceves <- c + return)+ }+ ## repeating 100 times the simulation> output <- sapply> i.e., 2000 simulated individuals, occupy less than one mega byte of memory and 100 replicates of the simulations using only one cpu in a standard laptop spent around 10 min which includes the times to fit the models.This is a simple but realistic simulation that can be performed with “isqg”, trying to highlight how the simulation can be integrated with third part packages to analyze the simulated data such as rrBlup . To reine.g., wheat and bovine can be fully represented (ascii characters (1 byte) or integer numbers (4 bytes) as found in alternative softwares, bits are at least 4 times smaller in modern computing. Moreover, bitwise operations act on chunks of 64 bits that are faster than loops over arrays.Bitsets are implemented with a limit in size of resented . Taking gpl-2). Its low- and high-level interfaces provide great flexibility for users. A sample script showing a basic pipeline with other features is shared as a complementing material simulating a comparison of a few different recombination models.The presented framework was implemented in C++ with an R interface. Access to bitsets is provided by the Boost library, and bit manipulations are carried out by functions in the standard template library. The package is available under a public license (


We demonstrate that the threaded macrocycles in interlocked porphyrin–corrole conjugates provide a mechanical “picket fence” without affecting their electronic properties. Building on recent progress in the synthesis of functional porphyrins for a range of applications using the Cu-mediated azide–alkyne cycloaddition (CuAAC) reaction, we describe the active template CuAAC synthesis of interlocked triazole functionalised porphyrinoids in excellent yield. By synthesising interlocked analogues of previously studied porphyrin–corrole conjugates, we demonstrate that this approach gives access to rotaxanes in which the detailed electronic properties of the axle component are unchanged but whose steric properties are transformed by the mechanical “picket fence” provided by the threaded rings. Our results suggest that interlocked functionalised porphyrins, readily available using the AT-CuAAC approach, are sterically hindered scaffolds for the development of new catalysts and materials. The copper-mediated alkyne–azide cycloaddition (CuAAC)The CuAAC reaction has also been widely applied in the synthesis of interlocked molecules,The active template (AT) approach to interlocked molecules,Here we demonstrate the utility of the AT-CuAAC reaction in the synthesis of mechanically interlocked analogues of previously studied triazole-linked porphyrin–corrole conjugates,4⊂3 was synthesised in excellent yield (98% after size exclusion chromatography) by reaction of azide 1, alkyne 2 and macrocycle 3 in the presence of a CuI salt .Porphyrin–corrole diad CuI salt . The ext4⊂3 shows a molecular ion at m/z = 1123.5 consistent with [M + H]2+. Comparison of the 1H NMR spectra , which is in keeping with their location away from the threaded region of the axle, triazole proton Hk is shifted considerably to lower field (Δδ ∼ 1.8 ppm). This is consistent with previous observations of C–H·N hydrogen bonding between the polarised triazole-Hk and the Lewis basic pyridine nitrogen donorsj appear at higher field in the interlocked structure (Δδ ∼ 1.2 ppm) due to the close proximity of the induced magnetic field of the electron rich aromatic units of the macrocycle.The mass spectrum (MS) of [2]rotaxane dyad  spectra  of [2]roA, HB, HC, shielding of protons HF, and HG of the flanking aromatic units and the splitting of HD and HE into diastereotopic pairs due to the non-centrosymmetric axle desymmetrising the faces of the macrocycle on mechanical bond formation. Analysis of triad [3]rotaxane 5⊂23 and pentad [5]rotaxane 6⊂43 by MS also confirmed the presence of the corresponding molecular ions . Their 1H NMR spectra .21Femtosecond transient absorption spectroscopy of These results clearly demonstrate that, as proposed, threading of the macrocycles around the arms of the porphyrin core does not significantly affect the electronic properties of the system.4⊂3, 5⊂23 and 6⊂43 clearly have very different steric properties; encircling the triazole moieties with macrocycle 3 significantly increases the steric demand of the linker units. This difference is particularly striking in the case of pentad 6⊂43 which lacks sterically bulky aryl groups on the central porphyrin unit; the space filling model of 6⊂43 (II-porphyrin unit is significantly encumbered by the threaded macrocycles. This steric hindrance was quantified by determining the % buried volume (% Vbur) of spheres centered on the central ZnII ion (r < 3 Å), the values of % Vbur for 6 and 6⊂43 are identical suggesting that the ZnII center is still accessible to small molecules, as required for catalysis or ligand binding. However, as r increases, the values diverge as the threaded macrocycles lead to a higher excluded volume. The comparison between the variation in % Vbur of 6⊂43 and 6 suggests that although the interlocked macrocycles do not affect the accessible volume immediately around the ZnII center, they provide a steric wall at higher radii similar to covalent picket fence porphyrinoids that have been developed for catalytic and photochemical applications.Although the electronic properties of the interlocked products are unchanged compared with the axle moiety, rotaxanes  of 6⊂34  shows thZnII ion .24 At lo6⊂43 compared with 6 can be found in their 1H NMR spectra; non-interlocked axle 6 displays resonances that are broadened considerably compared with that of [5]rotaxane 6⊂43 under the same experimental conditions  to direct the formation of [(ZnII-porphyrin)2L] dimers.1H NMR spectra  at L : 6 ratios up to 0.5 : 1 a signal was observed at –4.92 ppm in the 1H NMR spectrum, along with a new signal corresponding to Hn′ which is consistent with the formation of a [62L] dimer in slow exchange on the NMR timescale; (ii) once the ratio of L : 6 exceeded 0.5 : 1 the signal at –4.92 ppm disappeared and Hn′ moved to progressively lower field as further ligand was added, stabilising once L : 6 = 1 : 1, consistent with fast ligand exchange once excess L is present and the formation of monomeric complex [6L] in competition with [62L]. This was further confirmed by cooling the equimolar solution of 6 and L; at 273 K a broad signal was observed at –2.96 ppm alongside the reappearance of the signal at –4.92 ppm, consistent with the monomeric species [6L] in equilibrium with [62L] at low temperature. Thus, the speciation of 6 2L] but instead the resonances corresponding to triazole proton Hk and porphyrin β-protons Hn underwent monotonic changes that continued until 1 equivalent of L had been added. Alongside these changes, a new broad signal appeared at –2.94 ppm (L had been added at which point it disappeared. These changes are consistent with the formation of [(6⊂43)L] that undergoes slow exchange with free porphyrin 6⊂43 on the 1H NMR timescale, progressing to fast exchange once excess L is present. Consistent with this, cooling an equimolar mixture of 6⊂43 and L to 223 K (6⊂43)L]. Thus, the speciation diagram of 6⊂43 with respect to equivalents of L L]. However, surprisingly, a small signal was also observed at –4.62 ppm. Reducing the temperature further to 248 K led to a number of significant changes consistent with the formation of [(6⊂43)2L] alongside [(6⊂43)L] and unbound 6⊂43, including the appearance of resonances at –4.62 and 8.6 ppm attributed respectively to L and the porphyrin β protons Hn of the dimeric complex. Reducing the temperature further to 223 K (6⊂43)2L] at the expense of [(6⊂43)L]. Thus, as the temperature is decreased, the mixture of [(6⊂43)L] and 6⊂43 present at 298 K is converted to a mixture of [(6⊂43)2L], [(6⊂43)L] and 6⊂43 until an approximately equimolar mixture of [(6⊂43)2L] and [(6⊂43)L] is produced at 223 K 2L] complex forms at all is remarkable given the sterically hindered environment provided by the macrocycles. The effect of temperature suggests that the balance between negative steric interactions and the positive binding interaction between the ZnII center and the N donors of the DABCO ligand are finely balanced. Ultimately, the entropic cost of forming the ternary complex, along with the restrictions to conformational freedom associated with forming such a crowded structure, appear finely balanced against the enthalpic benefit of maximising Zn–N interactions, leading to a strongly temperature dependent self-assembly process.That the  (Fig. S38L, 6 and 6⊂43 as the temperature is varied; 6 is selectively consumed in formation of [62L] in keeping with the higher stability constant for dimerisation of the non-interlocked axle and we did not observe any evidence of hetero-complex formation. Thus, the mechanical picket fence provided by the macrocycles leads to self-sorting in a mixture of ZnII-porphyrin hosts.Finally, we examined the speciation of mixtures of  Fig. S38. FurtherIn conclusion, we have demonstrated that the ease and utility of the CuAAC reaction, which has led to its widespread use in the design of functional porphyrinoids for various applications,Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file.


Accumulating evidence suggests that the gut microbiota shapes developmental processes within the immune system. Early life antibiotic use is one factor which may contribute to immune dysfunction and the recent surge in allergies by virtue of its effects on gut microbiota.As a first step towards determining whether a relationship exists between perinatal antibiotic induced changes in the gut microbiota and the later development of a peanut allergy, we exposed newborn mice to either the broad-spectrum antibiotic vancomycin or to a vehicle for 6 weeks and then used a novel murine model of peanut allergy.Early-life treatment with vancomycin resulted in a significant shift in the gut microbiota community characterized by a reduction in the abundance of firmicutes and preponderance of inflammatory proteobacteria. Mice with an antibiotic-altered microbiota, showed a localized allergic-like response characterized by ear swelling and scratching following intra-dermal peanut antigen challenge. Likewise, circulating IgE levels were increased in antibiotic-treated mice, but no evidence of a systemic allergic or anaphylactic-like response was observed. Importantly, we utilized the naturally occurring pro-inflammatory cytokine, tumor necrosis factor-α (TNF-α), rather than the more commonly used cholera toxin, as an adjuvant together with the peanut antigen.Our data suggest that early antibiotic exposure promotes a shift in the gut microbiota community that may in turn, influence how mice later respond to a TNF-α + antigen challenge. However, further studies verifying the capacity of microbiota restoration to protect against allergic responses will be needed to confirm a causal role of antibiotic-induced microbiota variations in promoting allergic disease phenotypes. Over the past few decades, the prevalence of allergic diseases such as asthma, eczema, rhinitis, and food allergy has increased at such an alarming rate that these diseases now represent some of the most common chronic childhood health conditions in affluent countries , 2. RisiDysbiosis-induced immunopathology and dysregulation of the infant immune system is a central tenet of the “microbiota hypothesis” –11, a moClostridium difficile, which can result in intestinal inflammation and changes in gut microbiota [Staphylococcus aureus (MRSA), coagulase negative Staphylococcus, and Enterococcus, as well as infections seeded from a central line, dialysis/other shunt, or prosthetic heart valve.We experimentally tested the hypothesis that early life  exposure to the antibiotic vancomycin would provoke a shift in the gut microbiota community that would in turn be associated with increased sensitivity to the development of an allergic response to peanut antigen. Vancomycin has previously been shown to induce strong shifts in the gut microbiota linked to asthma exacerbation  and is ocrobiota . VancomyOur secondary hypothesis was that the pro-inflammatory cytokine, tumor necrosis factor-α (TNF-α), would act as an adjuvant to increase sensitivity to the peanut antigen when administered concurrently. While most murine allergy models involved the use of cholera toxin (CT) as an adjuvant , 22, we Female and male C57BL/6 mice were purchased from Charles River Laboratories at 6–8 weeks of age. At the time of breeding, female breeders were 17–19 week-old second-time breeders, and males were first-time breeders 6–8 weeks old. Male mice were bred in-house and only male offspring were used in all experiments. Mice were housed in a standard housing environment on a 12-h light–dark cycle and received water and peanut-free Harlan Laboratories 2014 Rodent Chow food ad libitum for the duration of the experiment. All experiments were approved by the Carleton University Animal Care Committee.g for 25 min to obtain the supernatant. The supernatant was collected and the pH was adjusted to 4.0 before being centrifuged at 4 °C for 50 min at 1000×g. Finally, the protein isolate was obtained by washing the precipitate with water adjusted to pH 4.0. The CPE used for the intradermal challenges was made up as a 10 µg/µL solution in distilled water.Crude peanut extract (CPE) for the intradermal challenge was isolated from partially defatted peanut flour  generously donated by the Golden Peanut Company . Briefly, a 1:8 (w:v) solution of peanut flour and 1 M NaCl adjusted to pH 9.5 using NaOH was mixed at room temperature for half an hour and then centrifuged at 4 °C and 5000×Beginning on the day of parturition, six dams and their pups received a clinically relevant dose , 25 of v® cookies). In a pilot study (unpublished data), we found that 2 µg of TNF-α acts as an effective adjuvant to elicit an allergic reaction (ear swelling) upon subsequent exposure to the peanut protein antigen through an ear punch coated in CPE or peanut butter oil. Hence, TNF-α was given together with the peanut protein as an adjuvant to boost the immune response to the peanut antigen. Another one-third of the mice in each antibiotic/control condition were exposed to 2 µg of TNF-α alone (TNF-α group). The remaining third of the mice (control group)_ received 100 µL of distilled water alone on a digestive cookie.At 6 weeks of age and following the antibiotic treatment (Day 43), mice were weaned and placed into individual housing. One-third of the pups in each antibiotic/control treatment condition (i.e. 7–9 mice/group) were orally exposed to peanut antigen and TNF-α (TNF-α + PB-sensitized group). Specifically, these mice received 10 mg of peanut butter  in 100 µL distilled water spread onto 0.5 g of a digestive cookie (Mr. Christie’s ArrowrootTwo weeks after the sensitization treatment (Day 57), all mice were challenged intra-dermally with an ear punch. The intra-dermal ear punch challenge consisted of punching the left ear pinna of each mouse with an ear punch coated in a solution of 10 µg/mL CPE in distilled water. The right ear pinna was punched with a separate ear punch coated in distilled water as a control. Each ear punch was washed with ethanol between animals. One hour following the ear punch, all mice received an intraperitoneal (IP) injection of 5 mg of peanut butter dissolved in 100 µL of saline. We thus assessed both locally (ear) and systemically induced (IP) allergic reactions. It should be noted that IP administration is a non-physiological route of antigen delivery. Yet, this route of systemic challenge was chosen to avoid the stresses of oral gavage, as stress responses can significantly impact immune responses. One hour following the IP injection all animals were sacrificed via rapid decapitation with surgical scissors. Figure At 15-min intervals for 30 min following the ear punch challenge, both the left and the right ears were scored on a scale of 0–3 for signs of swelling and irritation indicative of allergy. On this scale, 0 indicated no swelling, redness, or signs of irritation or scratching; 1 indicated an enlarged ear punch hole; 2 indicated mild swelling of the ear, and swelling at the base of the ear; and 3 indicated an enlarged ear, swelling at the base of the ear causing the ear to protrude from the head, mild redness, and/or bleeding from the ear area ; and 2 indicating severe or frequent scratching  ; and a rating of 2 used to indicate severe or frequent scratching . Mice were also rated on sickness behaviour at these same time points using our well validated scale , with a g for 15 min to obtain plasma and stored at − 80 °C. Total circulating IgE levels were determined by sandwich ELISA using a commercial mouse IgE ELISA kit after being sent to ITR Laboratories Canada Inc . Briefly, a 96-well plate was coated with a rat anti-mouse IgE monoclonal antibody. Standards and samples were then added to the wells, followed by a biotinylated rat monoclonal anti-mouse IgE detection antibody cocktail. These antibodies were subsequently detected by adding avidin-horseradish peroxidase followed by TMB Substrate Solution and absorbance was read at 450 nm with a microplate reader. The data was acquired using the Biotek Powerwave XS microplate reader coupled to Biotek Gen5 Secure software for data analysis and curve fit purposes.Immediately following the 1-h observation period after the IP challenge, mice were sacrificed by rapid decapitation with surgical scissors and trunk blood was collected into chilled EDTA filled (10 µg) tubes for further analysis. Whole blood was centrifuged at 3000×mothur v.1.34.4 software to determine the diversity and composition of the microbial contents of the samples.Fecal samples were taken from each of 12 cages of animals on Days 21, 28, 35, and 42 and stored at − 80 °C in a 1:1 glycerol: water solution until needed. Samples were sent on dry ice to Microbiome Insights Inc., Vancouver to be subsequently analyzed for their microbial content. Briefly, DNA was extracted from fecal samples using the Qiagen QIAamp DNA Stool Mini Kit and prepared for sequencing on a MiSeq machine using primers targeting the V3 region of the 16S rRNA gene and according to the Schloss lab protocol “16S Sequencing with the Illumina MiSeq Personal Sequencer” Standard Operating Procedure v3.1  by Kozich et al. , with miAll statistics were done using SPSS Version 20. Results were analysed using a 2 (early life treatment: antibiotic vs vehicle) × 3  factorial ANOVA followed by Fischer’s least significant difference (LSD) post hoc tests. For significant interactions, LSD tests were used to compare means of the effects comprising these interactions. When litter was included as a covariate in the analysis (ANCOVA) on the basis of finding significant differences in microbiome composition among litters, this did not alter findings that were significant and thus only results from the simple ANOVA are reported.1,38 = 13.356; p < 0.05; Fig. 2,38 = 5.062, p < 0.05), and the interaction term between antibiotic treatments and sensitization treatment was significant , with post hoc LSD tests showing antibiotic-treated mice scratching more than control mice. There was also a significant interaction between antibiotic and sensitization treatments . Pairwise comparisons of individual means showed that scratching scores were significantly higher among vancomycin-treated mice sensitized with TNF-α + PB compared to those that received vehicle sensitization or that did not receive the antibiotic pre-treatment  than control animals . Sickness ratings were generally quite low in all mice, with no animal scoring above a 2 (modest sickness symptoms) at any time point. Thus, there was no indication of an anaphylactic-like response following antigen challenge using our present experimental allergy model.Paralleling the allergy scores, circulating IgE levels varied among treatment conditions but were quite variable. Indeed, although the initial ANOVA comparing treatment groups was not significant (p = 0.20), we conducted follow up comparisons based on our a priori hypothesis. These comparisons revealed that antibiotic pre-treated mice later exposed to the TNF-α + peanut antigen treatment had significantly elevated IgE levels compared to non-antibiotic vehicle treated control animals p < 0.05). However, these antibiotic-treated mice did not differ from their non-antibiotic treated counterparts that were also sensitized with the PB + TNF-α treatment  allergic phenotypes observed in the present study may have been due to increased baseline inflammatory tone (owing to an elevation of Proteobacteria) or alternatively to a loss of Clostridia bacteria in the gut of the antibiotic-treated mice. Indeed, Clostridiales are among the bacterial orders within the Firmicutes phylum often most affected by vancomycin treatment , and theStaphylococcus aureus, which can lead to intestinal inflammation. Other broader spectrum antibiotics would be expected to differentially affect the gut microbiota and hence, potentially impact different allergic outcomes. Moreover, having now shown that broad-spectrum antibiotic exposure induces shifts in the microbiota and affects markers of allergy, future work should also focus on using more narrow-spectrum antibiotics to determine the relative importance of specific populations of bacteria in mediating these immunological effects. To further show a causal role for antibiotic-induced microbial shifts in mediating peanut allergy, fecal transplant experiments aimed at determining the ability of microbial restoration to rescue allergic phenotypes could be useful [Although we raise the possibility of a link between antibiotic-induced early life disruption to the gut microbiota and later sensitivity to certain immunogenic effects, further studies are required to be able to verify such a link and provide mechanistic details as to the nature of any such relationship. Furthermore, it should be underscored that using different antibiotics might yield different outcomes. Vancomycin is often reserved for serious infections and targets gram positive bacteria such as e useful , 55.The lack of obvious sickness symptoms observed following the peanut protein challenge as would be expected with a systemic or anaphylactic reaction may be related to findings of only elevated circulating IgE levels in sensitized and antibiotic-treated mice. Of course, an important caveat of our study is that we assessed total and not peanut-specific IgE. At the time of this study, we did not have access to this assay and we presently have no biological samples remaining. Thus, we may have missed peanut-specific antibody changes.Taken together, our findings provide evidence of the utility of TNF-α as an adjuvant in a murine model of peanut allergy and are consistent with the hypothesis that early life exposure to antibiotics alters microbial colonization of the intestinal tract  and this


The high-resolution structure of authentic infectious prions and the structural basis of prion strain diversity remain unknown. Here we use cryo-electron microscopy and atomic force microscopy to examine the structure of highly infectious PrP rods isolated from mouse brain in comparison to non-infectious recombinant PrP fibrils generated in vitro. Non-infectious recombinant PrP fibrils are 10 nm wide single fibres, with a double helical repeating substructure displaying small variations in adhesive force interactions across their width. In contrast, infectious PrP rods are 20 nm wide and contain two fibres, each with a double helical repeating substructure, separated by a central gap of 8–10 nm in width. This gap contains an irregularly structured material whose adhesive force properties are strikingly different to that of the fibres, suggestive of a distinct composition. The structure of the infectious PrP rods, which cause lethal neurodegeneration, readily differentiates them from all other protein assemblies so far characterised in other neurodegenerative diseases.Seeded polymerisation of proteins forming amyloid fibres and their spread in tissues has been implicated in the pathogenesis of multiple neurodegenerative diseases: so called “prion-like” mechanisms. While  Sc 3. Prions propagate by means of seeded protein polymerization, a process that involves the recruitment of PrP monomers to fibrillar assemblies followed by fission of the polymer to produce more “seeds”. Different prion strains can propagate in the same inbred host to produce different disease phenotypes and appear to be encoded by distinct PrP conformations and assembly states3. It is now clear that elucidating the molecular processes involved in mammalian prion propagation and strain diversity will be of major relevance to understanding other human neurodegenerative diseases (including Alzheimer’s and Parkinson’s disease) where seeded polymerisation or “prion-like” propagation of other protein assemblies is now a major research focus7. To date, despite enormous international effort, the high resolution structure of an infectious mammalian prion remains unknown. Consequently, protein assemblies in other diseases cannot yet be classified as “prion-like” according to defined structural criteria. In this context, it is important that animal models of other neurodegenerative diseases involving propagation and spread of assemblies of different proteins including tau, amyloid-β and α-synuclein do not generally result in lethal neurodegeneration, implying that the basic architecture of mammalian prions may be unique and critical to their ability to act as lethal pathogens3.Mammalian prions are infectious agents composed principally or entirely of multichain assemblies of misfolded, host-encoded prion protein (a glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein), some of which acquire protease-resistance and are classically designated as PrP8. Subsequently, using sensitive cell culture infectivity assays we established that the PrP rods are intrinsically infectious and that changing the number of aggregates into which the rods are distributed proportionally changes the number of infectious units available to cells at inoculation9. Electron tomography of negatively-stained prion rods from multiple prion strains revealed a common three-dimensional architecture comprising a pair of short, intertwined fibres, each with a double helical repeating substructure, separated by a distinct gap of 8–10 nm in width9. Hierarchical assembly of PrP into a paired fibre structure (20 nm in width) appears to be a key feature of infectious prions and contrasts markedly with non-infectious amyloid PrP fibrils generated in vitro from bacterially-expressed recombinant PrP which consist of only a single fibre (10 nm wide) composed of a double helical arrangement of two protofilaments10.Progress in understanding prion structure has been severely hindered by the difficulty of isolating relatively homogeneous prion particles from infected tissue and unequivocally correlating infectivity with composition and structure. However, we recently developed new methods for isolating exceptionally pure, high-titre infectious prion preparations from mouse brain and showed that pathogenic PrP in these preparations is assembled into rod-like assemblies (PrP rods) that faithfully transmit prion strain-specific phenotypes when inoculated into miceex vivo PrP rods but not in recombinant PrP fibrils) may be involved in linking the paired fibres of the PrP rods and might reside predominantly in the 8–10 nm central gap of the rod9. However, the use of negative stain in our previous studies precluded the ability to visualise material in this central region of the rod. Accordingly, in this study we have now examined infectious ex vivo PrP rods and non-infectious recombinant PrP fibrils by cryo-electron microscopy (cryo-EM) and atomic force microscopy (AFM) using unstained samples. Images collected show that negative stain makes little difference to the overall dimensions of these assemblies and confirms their key structural differences. AFM imaging and concurrent quantitative measurement of the adhesion interaction with a silicon tip shows unequivocal evidence for biological material in the central 8–10 nm gap of the infectious PrP rods, which now formally excludes the possibility that the paired fibre assembly of the PrP rods is an artefact of staining methods. This central material has an irregular topography and adhesive properties that are significantly different to that of the outer surface of the paired fibres of the PrP rod, consistent with the idea that N-linked glycans may be fulfilling a key structural role9. The first cryo-EM images of unstained, high titre, ex vivo infectious PrP rods formed from wild-type PrP shown here, allied with significant new AFM findings, now defines the configuration of the authentic infectious prion assembly state that should be targeted in future high resolution structural studies of mammalian prions.Previously we hypothesised that some of the PrP N-linked glycans  have a more complex structure9. Each PrP rod is composed of a pair of short, intertwined fibres, each with a double helical repeating substructure, separated by a distinct gap 8–10 nm in width9  by negative stain EM  was 20.5 ± 2 nm  compared to a width of 18.5 ± 2 nm  measured by cryo-EM, indicating that dehydration and staining have little effect on the dimensions of the rods. By AFM the paired fibres of the ME7 rods appeared smooth with no apparent protrusion of PrP N-linked glycans from their surface. Importantly, analogous to findings with RML prion rods, AFM revealed the presence of material in the central 8–10 nm gap of ME7 PrP rods with an adhesivity that was greater than that of the rod fibres  separated by a gap of 8–10 nm. AFM of unstained samples reveals that this central gap contains irregularly-structured material that appears to be compositionally distinct from the surface of the individual fibres. This finding is consistent with a proposed involvement of PrP N-linked glycans in linking the paired fibres of the rod9 as the marked protease-resistance of the rods9 is difficult to reconcile with irregularly structured material if this were comprised solely of protein. In contrast to the structural complexity of the PrP rods, non-infectious recombinant PrP fibrils are single fibres ~10 nm wide with a double helical arrangement of two protofilaments.In this study, we have examined the architecture of unstained preparations of 9 and this study, we have defined PrP rods as the target for future high resolution studies of authentic prion structure. Recently, Wille and colleagues also sought to investigate mammalian prion structure using cryo-EM11, however to our surprise, the structure they described comprised a single PrP fibre ~10 nm wide composed of two protofilaments11 with an overall architecture closely similar to that of our non-infectious recombinant PrP fibrils. We consider that this finding is directly attributable to their use of prions isolated from the brains of transgenic mice expressing GPI-anchorless PrP (tg44 mice) infected with RML prions11. RML prion-infected tg44 mice replicate prions (that can be subsequently titrated in wild-type mice) but they also develop intense PrP amyloid deposits throughout their brain which are not seen in RML prion-inoculated wild-type mice13. Notably, the GPI-anchor-deficient PrP expressed in these mice is mainly non-glycosylated13, so that these mice are in effect producing a high level of what is essentially recombinant PrP along with a minor population of glycosylated PrP species. Caughey and colleagues have described two morphologically distinct PrP fibril assemblies in prion-infected tg44 mouse brain16 and inspection of their images shows clear evidence for the presence of paired fibre PrP rods (~20 nm in width) together with other single PrP fibres of ~10 nm in width. The PrP fibrils isolated from the brain of RML prion-infected tg44 mice by Wille and colleagues showed tight uniformity in width 11 indicating that their purification protocol had resulted in preferential enrichment of these single PrP fibres. Importantly, the specific prion infectivity of their purified PrP fibre preparation (with respect to protease-resistant PrP concentration) was dramatically reduced compared to the RML prion-infected tg44 mouse brain homogenate used for purification. Using the original prion titration data for RML prion-infected tg44 mouse brain reported by Chesebro and colleagues12, the mean prion incubation periods of 153 days for RML prion-infected tg44 mouse brain homogenate and 203 days for purified PrP fibrils observed in C57Bl/6 mice by Wille and colleagues11 would correspond to a 102–103 fold reduction in infectious prion titre12. Collectively, based upon the published data, we conclude that RML prion-infected tg44 mouse brain propagates paired fibre PrP rods (20 nm wide) which account for the transmissible prion infectivity and structurally distinct single PrP fibres (10 nm wide) which account for the abundant PrP amyloid plaques that characterise these mice. It appears therefore that Wille and colleagues have structurally characterised the far more abundant, single fibre PrP fibrils rather than the infectious PrP rods.Based upon our previous findings18. These include PRNP stop mutations that result in C-terminally truncated GPI-anchorless PrP isoforms and a range of PRNP missense point mutations some of which are associated with Gerstmann-Sträussler-Scheinker (GSS) disease phenotypes22. A common feature of some of these missense point mutations is that the expressed full-length mutant PrP forms two distinct disease-associated assemblies of misfolded PrP. One assembly forms N-terminally truncated protease-resistant fragments that correspond to those generated from classical PrPSc  which is enriched in brain areas showing synaptic PrP deposition, spongiform vacuolation and neurodegeneration. The other disease-associated assembly leads to smaller N- and C-terminally truncated protease-resistant fragments  which is associated with PrP amyloid plaques31. These distinct disease-associated PrP assembly states from GSS patients with the P102L PrP mutation transmit different phenotypes to experimental reporter mice resulting in either a lethal transmissible spongiform encephalopathy  or a clinically silent PrP amyloidosis 33. Based upon the evidence that RML-infected tg44 mice are propagating PrP rods and structurally distinct single PrP fibres, it seems probable that both PrP rods and distinct single fibre PrP assemblies might also be propagating in some inherited prion diseases. Variation in the substructure of PrP rods or the single PrP fibrils (governed by the specific PrP missense mutation) may dictate their strain-specific transmission properties and host range via conformational selection35, while temporal and spatial differences in their accumulation (either predominantly PrP rods or single PrP fibrils within particular brain regions) might underlie the diverse clinicopathological phenotypes that are seen in family members with the same PRNP mutation36. Although the work of Wille and colleagues11 does not report the structure of infectious PrP rods, the architecture of an ex vivo single PrP fibril is nevertheless of major interest as the propagation of single fibrils may significantly influence clinical and pathological disease phenotypes in humans.Generation of GPI-anchorless PrP mice has provided significant insight into the pathogenesis of inherited forms of human prion disease in which prominent amyloid PrP plaque deposition is a major feature7 and from numerous other PrP aggregates proposed by others as infectious prion assembly states (for reviews see refs 38). Our finding that the characteristic 8–10 nm gap between the paired fibres of infectious PrP rods contains material with an irregular topography that is likely to be compositionally distinct to the protein surface of the outer fibres is consistent with our proposal that some of the PrP N-linked glycans are located in the gap and are contributing to the stability of the assembly, and as a consequence its infectivity9.In summary, the structure of infectious PrP rods that we describe here readily distinguishes them from the available characterised structures of tau, amyloid-β and α-synuclein that propagate in other neurodegenerative diseases8 in which work with animals was performed in accordance with licences approved and granted by the UK Home Office (Project Licences 70/6454 and 70/7274) and conformed to University College London institutional and ARRIVE guidelines. All experimental protocols were approved by the Local Research Ethics Committee of UCL Institute of Neurology/National Hospital for Neurology and Neurosurgery. Prion purification and cell based prion bioassay was conducted at UCL in microbiological containment level 3 or level 2 facilities with strict adherence to safety protocols. Work with infectious prion samples at Bristol University and Birkbeck College London was performed using dedicated sample holders and equipment with strict adherence to safety protocols. Prion samples were transported between laboratories in packaging conforming to UN 3373 Biological Substance, Category B specifications.Frozen brains from mice with clinical prion disease were used to generate purified prion samples. These brain samples were generated by us as part of a previous study9. Brain homogenates (10% (w/v)) from uninfected CD1 mice (isolates I10340 or I14040) were used to generate control samples. Full details of the purification method have been described and we used purified P4 fractions prepared without proteinase K digestion for all experiments8. The method produces a recovery of ~10% of the prions present in the starting 10% (w/v) brain homogenate so that resuspension of the purified P4 pellet fraction in buffer at one tenth of the volume of the 10% (w/v) brain homogenate from which it was derived produces prion preparations whose infectivity titre matches that of the starting 10% (w/v) brain homogenate8. RML isolate I6200 has a prion titre of 107.3 ± 0.5  intracerebral LD50 units/ml when end-point titrated in Tg20 mice8. Concentration of purified prions or buffer exchange was achieved by centrifugation at 16,100 g for 30 min and resuspension of the pellet fraction into the desired volume and buffer of choice. Routine SDS-PAGE, silver staining and PrP immunoblotting to characterise purified prion preparations was performed using published procedures39.Prions were purified from 10% (w/v) brain homogenates from terminally affected CD1 mice propagating the RML prion strain (isolate I6200) and terminally affected C57Bl/6 mice propagating the ME7 prion strain (isolate I14050) as described previouslyPrnp allele a; amino acid residues 23–231) was purified from E. coli BL21(DE3) and folded in to a β-sheet rich conformation (β-PrP) in 10 mM sodium acetate/10 mM tris-acetate buffer pH 4 containing 1 mM DTT as previously described40. Samples were subsequently adjusted with 1 M HCl to pH 3 and a final protein concentration of 0.5 mg/ml in 10 mM sodium acetate/10 mM tris-acetate buffer, after which 100 µl aliquots were incubated for three to five months without agitation at 25 °C in sealed 1.5 ml tubes. Recombinant PrP fibrils were harvested by centrifugation at 13,000 g for 45 min after which pellets were resuspended in 100 µl of 10 mM sodium acetate/10 mM tris-acetate buffer pH 3.0. These stocks were stored at 25 °C.Recombinant mouse PrP (9. Briefly samples were loaded onto carbon-coated 300 mesh copper grids (Electron microscopy Sciences) that were glow discharged for 40 seconds using an PELCO easiGLOW™ glow discharge unit . Samples were left to bind for 2 minutes, blotted, washed briefly in one water drop, blotted, and then stained with either NanoW stain (Nanoprobes) 2 × 1 min or 2% (w/v) uranyl acetate in water for 45 sec, then blotted and air-dried. Grids were inserted into the microscope using a dedicated sample holder for mouse prions with strict adherence to risk assessment and microbiological containment level 2 safe working practice. The sample holder was decontaminated directly after use by exposing to plasma using a Fischione 1020 plasma cleaner. Images were acquired on an FEI Tecnai T10 electron microscope  operating at 100 kV and recorded on a 1 k × 1 k charged couple device (CCD) camera (Gatan) at a nominal magnification of 44,000 with a pixel size of 3.96 Å. Fibril dimensions were measured in ImageJ41 and IMOD42. Because of their helical twist, prion rods and recombinant PrP fibrils viewed on a surface alternate between wider, face-on and narrower, edge-on views of the structure. The dimensions reported here were measured on the widest parts of the fibrils.Full methods used have been published previously41 and IMOD42. Fibril and rod dimensions were measured on the widest regions of the fibrils, as for the negative stain EM.We examined purified prion rods  brain homogenate from which they were derived) and recombinant PrP fibrils (stock preparations diluted 5-fold in 10 mM sodium acetate/10 mM tris-acetate buffer pH 3.0). Protein samples were sonicated for 20 sec in a Sonicator 3000 (Misonix) at 40 W at 4 °C and then mixed with Protein A-10 nm gold solution (Electron Microscopy Sciences) (pre-sonicated for 5 sec as above) at a ratio of 1:25 (v/v). 3 µl of sample was loaded onto carbon-coated 300 mesh copper or gold grids (Electron Microscopy Sciences) that had been glow discharged for 1 min using a PELCO easiGLOW™ glow discharge unit  and allowed to bind for 1.5 min. Grids were then blotted for 4–7 sec prior to freezing in liquid ethane using a manual plunging device after which the grids were stored in liquid nitrogen. Samples were imaged using a dedicated Gatan cryo-transfer holder (model 626) on an FEI Tecnai T12 electron microscope  operating at 120 kV and recorded using a US4000 CCD camera (Gatan) typically using 1 to 3 µm defocus and a nominal magnification of 42,000 with a pixel size of 4.1 Å. After use, the holder was decontaminated using a Fischione 1020 plasma cleaner. Fibril dimensions were measured in ImageJ−1 protein) were washed once with water to reduce the concentration of contaminating salts and detergents. This was done by resuspending 40 µl of each sample in 500 µl of ultrapure water and centrifugation at 16,100 g for 30 min after which the supernatant was discarded and the bottom of the tube containing the sample resuspended in 40 µl ultrapure water. Samples were then sonicated briefly for 10 sec using a Sonicator 3000 (Misonix) at 40 W at 4 °C after which 1 µl of sample was dispensed onto a freshly cleaved mica surface and air-dried. Samples were investigated at room temperature using a Multi-mode VIII microscope with Nanoscope V controller and utilising a non-resonant, PeakForce feedback mechanism. In addition, a fast scanning head unit was employed with SCANASYST-AIR-HR cantilevers (Bruker) with nominal spring constant, k of 0.4 N/m and nominal tip radius 2 nm for imaging large areas (up to 50 µm × 50 µm) in order to identify candidates for high resolution investigation. SCANASYST-FLUID+ cantilevers (Bruker) with a nominal spring constant, k of 0.7 N/m and nominal tip radius <2 nm were used for high resolution imaging of individual PrP rods or recombinant PrP fibrils and also for mapping of adhesion interactions across the surface of each. Adhesion interaction forces were extracted in real-time from the force curves collected as part of the PeakForce control mechanism. Whilst the cantilever type was kept constant between samples, fresh cantilevers were used for each to ensure the highest possible topographic resolution was obtained. When comparing adhesion interaction forces for different samples it was therefore necessary to negate possible influences of small differences in tip radius through normalising to that of the mica background consistent across all samples44. Data was analysed using Nanoscope Analysis software (Bruker). Dimensions reported by AFM in Table Prior to loading onto mica, purified prions  brain homogenate from which they were derived) and recombinant PrP fibril samples  brain homogenate from which they were derived and 10-fold serial dilutions thereof), were applied to the mica surface and air-dried. Mica sections were then placed in the bottom of the wells of tissue culture plates  followed by the addition of 2.5 ml of PK1/11 cell suspension (80 cells µl−1) in OFCS  to give a total of 200,000 cells per well. In parallel, replicate tissue culture plates were prepared in which 1 µl of each purified prion or recombinant PrP sample was applied as solution to wells containing PK1/11 cells (as above). For both conditions cells were then incubated for 3 days. Cells growing on mica were harvested by transferring each mica section to a well of a new tissue culture plate followed by washing with 2 × 500 µl OFCS medium to remove cells from the mica surface, after which the wash fractions were pooled. Cells from the replica plates in which samples had been applied as solution were thoroughly resuspended in 1 ml OFCS medium. All recovered cell suspensions were then adjusted to a density of 16 cells µl−1 with OFCS media and 250 µl aliquots (4000 cells) transferred to 6 wells of a new 96 well tissue culture plate  and grown to confluence for 7 days. Subsequently cells were split at a 1:8 dilution into fresh OFCS media and grown to confluence for three days and then passaged in a standard Scrapie Cell Assay format (three successive cycles of 1:8 splits with three days incubation between splits) before transferring aliquots of the cells to ELISPOT plates for analysis of the number of cells containing proteinase K-resistant PrP “spot count”46. As a negative control, purified P4 samples from non-infected CD1 mouse brain were analysed in an identical fashion. We analysed three different purified P4 preparations from RML-infected CD1 mouse brain using these methods and in each case the prion titre of the P4 samples (as determined using the standard Scrapie Cell Assay46) was entirely consistent with our published findings8. Preliminary experiments in which freshly cleaved blank mica sections were incubated with PK1/11 cells showed no deleterious effects on cell health or viability.To measure RML prion infectivity bound to mica, samples of mica (Agar Scientific) were cut into 0.5 cm


Stereotaxic surgery to implant guide cannulas into the rodent brain is a frequently used technique to deliver drugs to targeted brain regions in awake, freely moving animals. There are limited reports, however, of central injections in young animals, and no information on cannula implantation for drug administration into the dorsomedial hypothalamus (DMH) in young rats. Our protocol describes a simple and successful method for implanting guide cannulas in the brains of young, male Sprague-Dawley rats and outlines newly developed stereotaxic coordinates to accurately target the dorsomedial hypothalamus.•Stereotaxic surgical procedure for guide cannula implantation in the DMH in young rats.•Development of stereotaxic coordinates of the DMH in young rats.•Microinjection of drugs into the young rat brain. Specifications TableThe effects of drugs in specific brain regions can be assessed in awake rodents by surgically implanting guide cannulas that can be later used to microinject drugs into the brain. Although this technique has been used extensively in adult rodents, there are fewer reports in young animals that are more susceptible to surgery-induced mortality and for which detailed stereotaxic coordinates are lacking. Young rats are also extensively used in the neuroscience field for elecrophysiological and other studies that may require stereotaxic injections; thus this technique may be applicable for a wide range of scientists. Here we provide newly developed stereotaxic coordinates for cannula implantation into the dorsomedial hypothalamus (DMH), an important appetite-regulatory center, in young, male Sprague-Dawley rats. We also outline the surgical procedure that yields a less than 2% mortality rate and an 81% success rate of proper implantation in the DMH [-1 mm screws-2 μL Hamilton microsyringes (×2)-24-well plates-40 cm connector tubing-Anesthetic machine with induction chamber and nose cone-Baxedin Pre-Op (0.5%)-Clear frozen section compound-Clippers-Cryostat-Dental cement (fast curing custom tray acrylic resin powder and liquid)-Dental drill with a foot petal; 1 mm and 2.38 mm drill bits-Dummy cannulas -Electric heating pad-Ethanol (100%)-Gauze (sterile)-Guide cannulas -Guide cannula holder (made on site to fit guide cannulas)-Hot bead sterilizer-Internal cannulas -Lamp-Lubricant -Needles (26 gauge)-Optixcare tear gel-Oxygen supply for the anesthetic machine-Paraformaldehyde-Phosphate buffered saline-Q-tips-Rat brain matrix-Razor blades-Rodent stereotaxic frame-Scavenger system and fume hood-Sodium azide-Stereo microscope-Sterile air laminar flow cabinet-•35 mm serrefines (×4)•Forceps•Needle drivers•Scalpel•Scissors•Screwdriver for 1 mm screwsSurgical tools-Syringes-Thermometer -Ultra fine point permanent marker-Underpads-Xylocaine (2%)-Turn on heating pad and lamp-Sterilize all surgical instruments and equipment using Baxedin Pre-Op (0.5%) and ethanol (100%) and place surgical instruments in a hot bead sterilizer for 15 s-Set up the stereotaxic frame to ensure rat is positioned in the center of the frame-Add isoflurane to the anesthetic machine-Insert dummy cannula into guide cannula and screw the guide cannula into a holder and secure this to the arm of the stereotaxic frame  and warm to ˜37 °C-Prepare syringes containing 0.1 ml of 0.1 mg/kg buprenorphineStereotaxic surgical procedures are carried out in young , male rats weighing approximately 100–120 g between 8 h00–12 h00. In preparation for surgery, the following steps are taken (beginning approximately 40 min prior to planned anesthesia time):Following preparation, turn on the isoflurane machine and ensure that the flow rate of oxygen is ˜0.8 L/min. Fill the induction chamber with 0.5% isoflurane in oxygen for two minutes. Place the animal in the induction chamber and then increase the percentage of isoflurane in 0.5% increments every 20 s until it reaches 3.5%. Keep the animal in the chamber at 3.5% for an additional 2–3 min until a deep level of anesthesia is achieved. Transfer the animal to a nose cone attached to the bite plate of the stereotaxic frame and carefully move the tongue to the side while ensuring the top incisors are over the bite plate. The animal should be placed on an underpad on a towel on top of an electric heating pad. Confirm a lack of sensation with a toe pinch and then center and secure the animal with the ear bars of the stereotaxic frame. Once the ear bars are securely in place, decrease the percentage of isoflurane and maintain an isoflurane concentration of 2.5–3.0% for the remainder of the procedure. Insert a lubricated rectal thermometer and carefully monitor body temperature throughout the procedure. Keep the heating pad warm enough so that the animal’s core body temperature does not drop below 37 °C. If the temperature rises above 37.5 °C, turn off the heating pad. Using clippers, clear a section of the head from the neck to just posterior to the eyes and apply an antiseptic  and a topical anesthetic  to the shaved region to sterilize and anesthetize the region. Apply tear gel (Optixcare) to the eyes to prevent them from drying out. Administer 2 ml of warmed sterile physiological saline  and 0.1 ml of buprenorphine (0.1 mg/kg) in separate subcutaneous injections in the subscapular area.Use a scalpel to make a small incision (2 cm) medial to the eyes and beginning at the posterior region of the eyes. Clip open the incision to expose the skull using 4 × 35 mm serrefines. Clear away the blood and other connective tissue from the surface of the skull using Q-tips until the skull is dry and bregma, a landmark on the skull where the coronal and sagittal sutures converge, is clearly visible. Position the bilateral guide cannula above the most posterior part of bregma and then move the cannula to the following coordinates that we have confirmed correspond with the DMH in young rats: 2.0 mm posterior and ±0.5 mm lateral . Note: we have also successfully implanted cannulas in the DMH with the cannula positioned 2.25 mm posterior to bregma; with this coordinate, the cannulas are typically positioned from mid to posterior DMH. When the cannula has been positioned in place using the anterior-posterior and medial-lateral coordinates, draw a box on the skull immediately around the cannula tips using an ultra fine point permanent marker to indicate the target insertion site. Record the exact position of the stereotaxic arm holding the guide cannula and then move it aside to allow room for drilling. Using an electric drill with a 1 mm bit, drill a small hole in the skull anterolateral to the target insertion site and insert a 1 mm screw (1 mm diameter) into the skull to serve as an anchor for dental cement. Next, drill a hole at the target insertion site (indicated by the box) using a 2.38 mm drill bit. Return the guide cannula to the previously recorded position and from the surface of the skull, lower the cannula 6.0 mm into the brain. Note: we have also successfully reached the DMH using a coordinate of 6.75 mm ventral to the skull surface. This coordinate will position the guide cannula approximately 2 mm above the DMH. This prevents the guide cannula from potentially damaging the target area, and allows for the internal cannula (which extends 2 mm below the guide cannula) to reach the top of the DMH during drug injection. The final stereotaxic coordinates for the implanted guide cannula should be (relative to bregma): 2.0 mm posterior, ±0.5 mm lateral, and 6.0 mm ventral (the latter is from the surface of the skull). For comparison, the coordinates often used in adult animals are 3.1–3.3 mm posterior, ± 0.5 mm lateral, and 6.8–9.0 mm ventral .Ensure the skull is completely dry and then apply dental cement with a liquid consistency completely around the screw and cannula to secure both in place. Allow the cement to completely harden, and then remove the stereotaxic arm, leaving just the guide cannula in place. Use a 4-0 silk suture kit with a 19 mm 3/8c reverse cutting needle to stitch up the incision site with a total of four stitches . Fill the remaining ˜1.3 μL with the desired drug in preparation to inject 0.5 μL of drug per side to the DMH. This volume has been used for microinjections into the DMH and other hypothalamic nuclei . Remove This procedure yields an 81% success rate. To verify whether cannulas are successfully implanted for drug injection into the DMH, fix brains via transcardial perfusion, and store them in 4% paraformaldehyde in phosphate buffered saline (PBS) for 24 h, followed by incubation in 30% sucrose in PBS for 24 h, both at 4 °C. Remove brains from the sucrose solution and place them in a rat brain matrix designed to hold brains from rats weighing between 35–175 g. Beginning 1 mm anterior to the optic chiasm, cut a 6 mm section containing the hypothalamus using razor blades and mount it with the posterior side facing down on a freezing block at −50 °C. Coat the brain with Clear Frozen Section Compound and wait for it to freeze. Using a cryostat, cut 30 μm slices and place them in 24-well plates containing 0.02% sodium azide in PBS and store them at 4 °C. To determine whether cannulas are successfully implanted in the DMH, view brain slices showing clear cannula tracks in a petri dish containing PBS under a stereo microscope . Using t


As an important category of CRE, enhancers play crucial roles in gene transcriptional regulations in a distant manner. Further, the disruption of an enhancer can cause abnormal transcription and, thus, trigger human diseases, which means that its accurate identification is currently of broad interest. Here, we introduce an innovative concept, i.e., abelian complexity function (ACF), which is a more complex extension of the classic subword complexity function, for a new coding of DNA sequences. After feature selection by an upper bound estimation and integration with DNA composition features, we developed an enhancer prediction model with hybrid abelian complexity features (HACF). Compared with existing methods, HACF shows consistently superior performance on three sources of enhancer datasets. We tested the generalization ability of HACF by scanning human chromosome 22 to validate previously reported super-enhancers. Meanwhile, we identified novel candidate enhancers which have supports from enhancer-related ENCODE ChIP-seq signals. In summary, HACF improves current enhancer prediction and may be beneficial for further prioritization of functional noncoding variants.Deciphering the code of  Enhancets (CRE) ,3,4. On ts (CRE) ,6 and thts (CRE) . On the ts (CRE)  and somets (CRE) . TherefoHowever, this is a challenging problem because an enhancer acts in its regulating role with both a distal and bidirectional manner. In contrast to promoter, distal enhancers are typically located more than 10 kb away from the gene it regulates , and somin vivo. ChromHMM [In the past two decades, researchers developed several distinct experimental strategies from different viewpoints for inferring the locations of active enhancers, such as Vista enhancer browser using transgenic mouse assay , EP300  enhancer sequence contains combinations of short sequence motifs known as transcriptional factor binding sites (TFBSs) and disrupting their core motifs substantially reduced the activities of the enhancers , suggestcis-regulatory code (CRC) [k-mer features of varying length (3–10 bp)  are predictive sequence features for discriminative prediction of enhancer when analyzing ChIP-seq data of EP300 [1–4 mers and other 11 combinatoric features [k-tuple nucleotide composition features for identifying enhancers and their strength [The computational strategy aims to learn de (CRC)  hidden ide (CRC) ,32,33,34of EP300 . Furtherfeatures . iEnhanck-mer to be predictive sequence features. Notably, DRMs like “GCGCGC” and “GAGAGA” were proven to be necessarily required for active enhancers and a high occurrence number of DRM was found to be a main feature of broadly active enhancers [k-mers (termed as “subwords” in mathematics) appearing in the DNA sequence. This enables SCF or TE to be the most appropriate tools for characterizing repeat sequence [4-mer motif features, hybrid abelian complexity features (HACF) are proven to be predictive for enhancer prediction.Although different tools adopted distinct technologies, a consensus is that one should try to learn combinatoric properties of DNA motifs or nhancers . In mathsequence  because topH(intron) > topH(exon) > topH(promoter) [topH(intron) > topH(exon) > topH(promoter)still holds. And, as expected, topH(enhancer) is significantly larger than that of exon, intron or promoter  ,37, leadentropy) , we chosentropy)  and the promoter A, suggesIt is noteworthy that the difference of TE between promoter and enhancer is large B, leadin−20, light blue points in Next, we proposed a new concept of sequence complexity—abelian complexity to construct a prediction model of enhancer based on the dataset of Enhancer4589. We first applied SACF484 (Methods 2.4) to run a SVM classifier and found a predicting accuracy of AUC of 0.877 . We furtmer features by counting frequencies of four continuous bases) were proven as predictive features for identifying enhancers in DEEP [DNA composition features are fundamental features for representing DNA sequences and tetra-nucleotide frequency features  (access on 5 May 2018), we ran the HACF model and listed a detailed comparison based on a leave-one-out test in iEnhancer-2L  construcIn summary, comprehensive comparisons with three state-of-the-art methods might lead us to conclude that our HACF approach is a more accurate and robust model for enhancer prediction.Finally, we applied the HACF model to scan the whole DNA sequence of human chromosome 22 for validating its generalization ability. We adopted a scanning strategy by sliding a 1 kb-window with step size 10 bp across human chromosome 22 , by which 3,488,530 DNA fragments were obtained and then were put into the HACF model for prediction. To control the false positive, we adopted a similar analysis strategy with ChIP-seq for the quality-control task: (1) each 1 kb DNA fragment that had a predicted value larger than 0.5 was regarded as a candidate read; (2) all candidate reads along with their predicted values were put into MACS2  to perfoMeanwhile, we tested the generalization ability of the HACF model on two known super-enhancers (SE) located within chromosome 22 which were reported to be related with MYC oncogene in multiple myeloma . Lovén ehttps://www.ncbi.nlm.nih.gov/geo/roadmap/epigenomics/) access on June 2, 2018. To comprehensively evaluate validities of novel candidate enhancers, we made comparisons between 7045 candidate enhancer regions (±2Kbcentered at the predicted enhancers) and the same number of random intergenic regions (background) on their average signal intensities of H3K4me1 and H3K27ac in two cell lines or tissues (iPS-20b and kidney). It was found that both H3K4me1 (red curves) and H3K27ac (purple curves) signals on candidate enhancer regions generated obvious peaks around the locations of predicted enhancers which are significantly larger than the signals on the random regions (blue and green curves)  E,F, supphttp://fantom.gsc.riken.jp/5/datafiles/latest/extra/Enhancers/ . The original data was presented as a matrix E65423×1829 with 65,423 rows and 1829 columns, where each row represents a specific enhancer, each column represents a specific tissue or cell line in human and element in ith row and jth column eij epresents the relative log expression (RLE)-normalized expression value  of CAGE of ith enhancer in jth tissue or cell line.In this study, we constructed a large-scale enhancer dataset based on FANTOM5 enhancer data , which cTPMrowmin ≥ α’  to select most active enhancers. When α was determined as 0.08 (we showed that the prediction performance was not sensitively dependent on the selection of parameter α in the supplementary information and α = 0.08 is an appropriate choice), only 5386 enhancers passed this criterion. Next, although some existing works used 2 kb as the length of enhancer sequence [http://www.bioinformatics.org/cd-hit/) . with a cutoff threshold of 0.8 to remove the redundancy between enhancer sequences, meaning 4653 enhancers remained. In the last step, for a fair evaluation of scanning chromosome 22, we discarded those enhancers located within chromosome 22 and obtained a total of 4589 enhancers as the final positive sample dataset for training  studies combinatorial features of sequence by counting the number of different subwords appearing in the sequence and topological entropy (TE) is a mathematical index for quantitatively measuring the asymptotic exponential increasing speed of SCF . They weDefinition 1.ω: N→N is given by Fwhere Card denotes the number of elements of a set and μ < ω represents that μ is a subword (or a factor) of ω.ωp(n) ≤ 4n holds for an arbitrary DNA sequence, and ωp(n) = 1 represents a complete repeat sequence, whereas ωp(n) = 4n represents the sequence containing all subwords with full complexity. In early works, Kirillova [ω over Ω is defined as:Mathematically, the nature of 1 ≤ irillova  and Coloirillova  studied irillova . To grabirillova  noted thDefinition 2.to is defined by For an ω was computed at the maximal value of n such that 4n + n – 1 ≤ |ω| based on the following theorem and corollary [The direct computing of TE is difficult because the classic definition of TE is based on infinite sequences equipped with the limit operation, which requires us to give an asymptotic algorithm to estimate the value of TE. A rational estimation of TE of orollary :Theorem 1.n + n – 1 such that(Existence of sequence with full complexity ) For allCorollary 1.0 be the unique integer such that4n0 + n0 – 1 < |ω| ≤ 4n0+ 1 + (n0 + 1) − 1, then Let ω bn0 instead of the entire SCF for measuring the complexity of a DNA sequence. By applying this to the intron and exon elements of the human genome, he found that the complexities of introns are larger than those of exons, suggesting that intron is more random and is under less selective pressure. After that, Jin et al. [n0 should be integrated together to give a more accurate estimation of TE. By applying it to three categories of genomic elements, they found that intron > exon > promoter of their complexities. Recently, Wu et al. [n0 are good predictors for predicting DNA methylation. These related works altogether demonstrate that specific positions of SCF where it may reach full complexities are intrinsic features as well as good predictors for discriminating distinct functional genomic elements.Using the above algorithm, Koslicki gave a unique value of TE at n et al.  found thu et al.  found thRecently, Richomme et al.  compreheDefinition 3.|ω|→N4, given by For a fA denotes the number of occurrences of the letter A ϵ Ω in the finite sequence ω.where |ω|Definition 4.ab: N→N is defined as: For a fwhere ψ(ω) is the Parikh vector of subword u of the finite sequence ω.ω, if n > |ω|, then n < |ω| when computing n ≥ 1. Another marked difference between ACF and SCF was that ACF usually showed a fluctuation while SCF strictly increased in the first part. For example, for the given DNA fragment ω=AAGCAGTCGG used in Note that for the finite sequence We next investigated the resource consumption of computation of ACF by running our script on a test computer with Ubuntu 18.04 on Intel Xeon 2.10 GHz processors and 256 GB RAM. When running on 500 enhancer sequences with a length of 1 kb, a total of 1319.767 s and 3656.48 Mb were needed for such computations, implying that the average computation time and memory consumption of each DNA sequence was about 2.64 s and 7.313 Mb, respectively.For a long DNA sequence, we now face the same problem of high-dimensionality as with the subword complexity situation. The main experience learnt from previous studies ,37,49 isTheorem 2.(Upper bound of ACF) For every finite word ω and every positive integer satisfying 1 ≤ n ≤ |ω|, the absolute upper bound of ACF is given bywhere C means the binomial coefficient. Proof. We gave the proof of theorem 2 in the supplementary information.n0 such that 4n0 + n – 1 ≤ |ω| because the minimal length required for reaching full subword complexity 4n0 is 4n0 + n – 1 (theorem 1). Here, we determine such a length for full abelian complexity by the following theorem:Under the guidance of the main idea of computing TE, we employed the upper bound of ACF in theorem 2 to estimate the minimal length required for reaching full abelian complexity. When determining the most appropriate position for computing TE, one chose the largest integer Theorem 3. For all m ≥ 0, if there exists a word ω such that, thenProof. ω with full abelian complexity, which means ω such that it has at least Suppose there exists a word ω| = 1000, we determined the integer m0 = 16 such that ω|=1000 , specificity (Spec), precision, accuracy (ACC), Matthew’s correlation coefficient (MCC) value and GM value, which are calculated as:An enhancer is one class of the most important CRE and plays significant roles in the regulation of gene expressions. Identification of the locations of enhancers is currently of broad interest because it is a prerequisite for a better understanding of complex mechanisms of transcriptional regulations and for evaluating non-coding DNA variants. Subword complexity function and topological entropy were proven useful tools for discriminating distinct classes of CREs, such as intron, exon and promoter. However, it is no longer valid for the identification of enhancers, which is why we developed a new mathematical tool termed “abelian complexity function” to try achieve this task. Under an estimation of the upper bound, a feature selection approach finally determines 209 abelian complexity features that outperformed three existing methods in fair comparisons. Moreover, in addition, to be consistent with existing enhancers of FANTOM enhancers and known disease-related super-enhancers, scanning human chromosome 22 with our model finds some novel candidate enhancers with supports of histone modification markers of H3K4me1 and H3K27ac.(1)New contribution to prediction methodology: for the first time, we proposed an innovative method, i.e., selected abelian complexity features together with DNA composition for accurate enhancer identification. In comparison with three state-of-the-art enhancer prediction tools, our method shows greater accuracy and more robust performances on imbalanced datasets.(2)New contribution to the prediction of novel candidate enhancers: when we applied our trained model to scan human chromosome 22, 7045 genomic regions were identified as enhancers. Some of them are consistent with known FANTOM5 enhancer, and the majority of them are considered as candidate enhancers by the evidences of surrounding histone modification markers including H3K4me1 and H3K27ac. This demonstrates that the predicted enhancers by our scanning approach have the potential to be true enhancers.On the basis of current efforts, we conclude that our method is an accurate and robust model for enhancer identification and we summarize that our work brings some new contributions into the area of enhancer biology:TPMmin ≥ 0.08“, implying that the enhancers we chose for training are actually ubiquitously active across all tissues and cell lines  was constructed by the criteria of “hancers” ). A rati


Letter to the Editor,n = 17) or prospectively (n = 11) had undergone whole exome sequencing (WES) [We read with interest the article by Pausepp et al. about 28 patients with suspected mitochondrial disorder (MID) who retrospectively n = 7 or prosATP6 variant was detected and in another patient mtDNA depletion was detected on muscle biopsy [PDHA1 carrier should be regarded as a MID [The authors mention that only in 4/28 patients a MID was genetically confirmed . Howevere biopsy . FurtherATP6 mutation, no heteroplasmy rates are provided. In which tissue were heteroplasmy rates determined and was the degree of heteroplasmy compatible with the phenotype? Did the mother of this patient also carry the mutation?Only in 5 of 28 patients did the authors detect a mutated gene associated with a MID and in 1 patient a possible mitochondrial depletion syndrome (MDS) was diagnosed? Were any of the genes associated with MDS screened for a mutation ? In how SMN1 gene?Spinal muscular atrophy (SMA) should not be diagnosed upon the muscle biopsy findings. Did the patient with “typical” histological features of SMA also carry a deletion/duplication of the Muscle hypotonia was reported in 11 patients [ATP6 variant, and from stressing that clinical assessment with or without scores may falsely suggest MID in a significant number (n = 21) of patients.Overall, the study may profit from inclusion of the family history, from provision of heteroplasmy rates for the There are no conflicts of interest.No funding was received.JF: design, literature search, discussion, first draft, SZM:literature search, critical review.