The observed loss of ORF2p function is independent of the endonuclease or reverse transcriptase activities of ORF2p and RNA interaction required for reverse transcription. In addition, the loss of function is physically separate from the cysteine-rich motif sequence previously shown to be required for RNP formation. Our data suggest an additional role of the less characterized carboxy-terminus of the L1 ORF2 protein by demonstrating that this domain, in addition to mediating RNP interaction(s), provides an independent and required function for the retroelement amplification process. Our experiments show a functional modularity of most of the LINE sequences. However, divergent evolution of interactions within L1 has led to non-reciprocal incompatibilities between human and mouse ORF2 cys-domain sequences.LINE-1 (L1) retroelements emerged in mammalian genomes over 80 million years ago with a few dominant subfamilies amplifying over discrete time periods that led to distinct human and mouse L1 lineages. We evaluated the functional conservation of L1 sequences by comparing retrotransposition rates of chimeric human-rodent L1 constructs to their parental L1 counterparts. Although amino acid conservation varies from ∼35% to 63% for the L1 ORF1p and ORF2p, most human and mouse L1 sequences can be functionally exchanged. Replacing either ORF1 or ORF2 to create chimeric human-mouse L1 elements did not adversely affect retrotransposition. The mouse ORF2p retains retrotransposition-competency to support both Alu and L1 mobilization when any of the domain sequences we evaluated were substituted with human counterparts. However, the substitution of portions of the mouse cys-domain into the human ORF2p reduces both L1 retrotransposition and Alu  The activity of LINE-1 (L1) elements has contributed both directly and indirectly to almost a third of the human genome mass A full-length human L1 is about 6 kb, consisting of a 5′ untranslated region (UTR), two open reading frames separated by an intergenic sequence, and a 3′ UTR which ends in a poly(A) signal and an A-tail Comparison of the consensus sequences of the currently active young human and mouse L1 elements reveals different lineage-specific areas of amino acid conservation. In this manuscript, we present data from an array of chimeric human-mouse L1 elements and chimeric ORF2p that allowed us to determine species-specific differences that influence retrotransposition. In addition, these data provide insight into the less characterized cys-domain and suggest an important functional role for a less conserved region of the cys-domain that is distinct from the RNA binding function of the highly conserved cysteine-rich motif.ex vivo assays RP  contain multiple premature polyadenylation signals and splice donor and acceptor sites within their sequences The human and rodent L1 lineages have evolved independently for approximately 80 million years. The ORF1p and ORF2p of the currently active human and mouse elements share approximately 35% and 63% amino acid identity, respectively. To test for a species-specific interaction between L1 ORF1–ORF2 proteins, we individually substituted the ORF1 or ORF2 sequences of a tagged mouse (psmL1) and human (L1PA1) L1 construct with the same ORF from the opposite species and used these chimeric L1 elements in the retrotransposition assay. All of the chimeric ORF1–ORF2 L1 constructs maintain retrocompetence ; howeverWe generated 12 human-mouse ORF2 chimeric proteins by swapping homologous sequences between species and selected sequence breakpoints to preserve distinct functional domains . We subdChimera breakpoints were introduced in areas of conserved amino acid sequence ranging from 3–9 identical residues surrounding the breakpoints . The proin trans in the human HeLa cell line. Because ORF2p alone is sufficient to drive Alu retrotransposition in the assay HMM; and human with mouse endo: MHH) and RT domains  were all functional, with only MHM showing more than a 35% drop in retrocompetence (30.9% activity relative to mouse: MMM)  .H and MMmhh, and MMmmh). The same is not true for the human ORF2 protein. The only human chimera with a portion of the mouse cys domain that effectively supported Alu retrotransposition in trans was HHhhm  did not support Alu retrotransposition. Transfections with HHhmm and HHhmh chimeras produced significantly fewer colonies than the negative empty vector control  and HHM transfections showed a similar trend but were not significantly different than empty vector. We therefore considered that the colonies from these three sets of transfections likely represent background colonies derived from endogenous L1 proteins in HeLa cells i.e., ∼1000 fold reduction), a decrease in retrotransposition rate of this magnitude compares to that observed for the endonuclease and reverse transcriptase mutant L1 constructs, which are considered to be essentially “non-functional” i.e., not significantly different from the empty vector control; two sample T-test, p≥0.238) irrespective of the cell line utilized, while HHhhm was not significantly different from the single species ORF2p positive control .The mouse ORF2p is functional when any portion of its cys domain is substituted with human counterparts  for evaluation in the context of our tagged human L1 construct. As with periment , suggestInefficient expression could explain the reduced retrotransposition activity observed for the ORF2 chimeric protein. We assessed protein expression using western blot analysis of extracts from cells transfected with all the chimeric ORF2 constructs. A band with an apparent molecular weight (∼150 kDa) corresponding to ORF2p was detected for human and chimeric ORF2p transfections . These r−RT− ORF2p control  compared to the empty vector and the EN control . We nextin vivo work demonstrated that the L1 5′UTR can be completely removed and replaced by another unrelated promoter, such as the composite CMV enhancer modified chicken β-actin promoter RPRP retrotransposition rate relative to L1.3, an increased amount of the full length L1 transcript, and possibly an increase in translation efficiency due to the codon optimization of the construct.L1 elements have been replicating and evolving in mammals for approximately 100 million years. Because a retroelement is generally parasitic upon its host genome, the potential for parasite-host coevolution may have contributed to some independent evolution of L1 sequences and host factors in different lineages. The basic organizational structure of L1 is the same for all mammalian elements, consisting of the 5′UTR-ORF1–ORF2-3′UTR and poly(A). Previous The modularity of L1 sequences becomes more evident with our observation that chimeric L1 human-mouse ORF1–ORF2 constructs maintain retrotransposition competence. Of particular interest, the ORF1 sequence was subjected to intense positive selection during a brief period of primate evolution, resulting in a high rate of amino acid replacement in the coiled-coil domain, which is currently conserved in the human ORF1p in trans was independent of the species (mouse or human) of the transfected cell line. We previously demonstrated that ORF2p from both mouse and human L1 sources mobilizes tagged Alu transcripts in chicken cells in vitro work demonstrated that this domain maintains endonuclease activity independent of the rest of the ORF2 protein The ability of chimeric human-mouse ORF2 proteins to mobilize Alu The simplest interpretation of our data is that predominantly human ORF2 protein is incompatible with the middle subsection of the mouse cys domain. All three of our chimeras that fit this description were essentially non-functional, while reciprocal mouse ORF2 chimeras maintained retrotransposition competence. Functionality of these chimeras could not be rescued by the use of cells from either species or by the addition of ORF1p in the context of the bicistronic L1 constructs. Given the important role that the cys-domain appears to play in functionality, it is notable that the highly conserved cys-motif is not associated with the observed species-specific difference. Reciprocal chimeras that only affected the third subsection of the cys-domain containing the conserved cys-motif (HHhhm and MMmmh) were both highly functional.i.e., HHM→MHM). However, the MHM ORF2 is only ∼30% active relative to the parental HHH or MMM constructs. We observe a higher recovery effect with the RT domain, as functionality is also restored when the non-functional HHM is compared to the functional HMM construct with ∼66% activity of HHH or MMM. Because activity is not fully restored in either of the two chimeric proteins, it is possible that the decrease in retrocompetence is due to negative sequence interactions and/or inefficient structural folding of the ORF2 protein.The inhibitory effect caused by one or more sequence stretches of the mouse cys-domain in the context of the human protein appears to be independent of the endonuclease or reverse transcriptase activities of ORF2p, as demonstrated by our functionality assays. Previous work demonstrated that substitutions of the cysteines in the cysteine rich motif lowered the amount of ORF2p in the RNP fraction and reduced LEAP activity, suggesting a possible reduction in the ORF2p-RNA interaction Interestingly, the smallest tested sequence with negative impact (165 aa of the middle sub-section of the cys-domain) is also the one with the lowest amino acid identity , which cCHmneo, contains the fully codon optimized ORF1 and ORF2 of the human L1RP cloned in the pBluescriptII vector (Stratagene). A schematic of the synthetic L1 vector is shown in http://www.changbioscience.com/primo/primoo.html), which makes synonymous changes to optimal codons within the nucleotide sequence. A 63 bp inter-ORF with 92% sequence identity to L1RP was utilized for all constructs. Sequence changes to this region were generated to introduce restriction enzyme sites used for plasmid construction. The optimized nucleotide sequence is 74.9% identical to the sequence of L1RP. The “notag” constructs contain an SV40 polyadenylation signal at the 3′ end, and the others contain the mneoI cassette including the SV40 polyadenylation signal from JM101/L1.3 pBS-L1PA1CHrescue, contains the L1RP ColE1-mneo cassette of SynL1neo mneoI cassette. The ColE1-mneo cassette was amplified with the FseI-NeoTag: 5′-TCCTAGCTGGCCGGCCTTTTATTGCCGATCCCCT-3′ and Not-NeoTag: 5′ –TCCTAGCTGCGGCCGCAACGCGCGAGGCAGCCGGATCATA-3′ and cloned into the FseI and NotI sites of pBS-L1PA1CHmneo (pBS-L1PA11CHmneo .spa (a kind gift from Dr. Jef Boeke) RP ORF2 protein sequence and was codon optimized by Blue Heron Biotechnology, Inc . The open reading frames are cloned into the expression vector pBudCE4.1 (Invitrogen), under control of the CMV promoter. The nomenclature and break points of the chimeric ORF2 constructs are described in pBudORF2 human-mouse chimera were generated by a PCR approach using overlapping primers mneo and pBS-L1-1M,2Hmneo were derived from pBS-L1PA1CHmneo and contain either the mouse ORF1 or ORF2 of psmL1 pBS-L1-1H,2MRP) in psmL1 pCEP-L1synM-1H and pCEP-L1synM-2H contain the codon optimized human ORF1 or ORF2  JM101/L1.3, referred to as “wildtype” L1, contains a full-length copy of the L1.3 element and the All plasmid DNA was purified by alkaline lysis and twice purified by cesium chloride buoyant density centrifugation and quality assessed by the visualization of agarose gel electrophoresed aliquots stained with ethidium bromide. Constructs were confirmed by sequencing .5/T75 flask or 2.5×105 cells/T25 flask. Transient transfections were performed the following day with Lipofectamine Plus (Invitrogen) following the manufacturer's protocol using 1 µg of Alu-neoTET vector plus 0.33 µg of the ORF2 construct or empty vector (T25 flask). For L1 retrotransposition assays between 1 or 3 µg of the tagged L1 plasmids was used for transfection (T75 flask). The next day, the cells were treated with the appropriate media containing 400 µg/ml for HeLa and 800 µg/ml for NIH3T3 cells of Geneticin/G418 (Fisher Scientific). After 14 days, cells were fixed and stained for at least 30 minutes with crystal violet . Retrotransposition rate was then determined as the number of visible G418R-resistant colonies/106 cells seeded. Both L1 and Alu G418 resistant colonies were validated as authentic retrotransposition events (Transient L1 or Alu retrotransposition assays were performed as previously described with some minor modifications n events .6 for transfection with 5 µg of the L1 plasmid constructs. Cells were harvested 48 hours post-transfection for RNA extraction. RNA extraction and poly(A) selection was performed as previously described 5′-TAATACGACTCACTATAAGGACGAGGCAGCG-3′ and Neo northern(+): 5″- GAAGAACTCGTCAAGAAGG-3′. The cyclophilin riboprobe was generated from pTRI-cyclophilin template (Ambion) to use as a control for transcript quantitation analyses. Riboprobes were radioactively labeled by incorporating 32P-UTP (Amersham Biosciences) using the MAXIscript T7 kit (Ambion) following the manufacturer's recommended protocol. The radiolabeled probes were purified by filtration through NucAway Spin columns (Ambion). Separate hybridizations were performed overnight with 4–12×106 cpm/ml of each individual probe at 60°C. The membrane was washed twice at high stringency  at 60°C before analysis using a Typhoon Phosphorimager (Amersham Biosciences) and the ImageQuant software.HeLa cells were seeded at a density of 4×10Protein was extracted from trypsinized and PBS-washed transiently transfected NIH3T3 or HeLa cells using standard Tris SDS glycerol buffer plus 2-mercaptoethanol and boiled for 15 min. Extracts were electrophoresed on 4–12% NuPage Bis-Tris gels (Invitrogen). Proteins were transferred to a nitrocellulose membrane using the iBlot gel transfer system (Invitrogen). The goat anti-LINE  and secondary HRP-conjugated antibodies were purchased from Santa Cruz Biotechnology Inc. The membrane was incubated for 1 h at room temperature with the primary or secondary antibody diluted 1∶500 and 1∶5000 in PBS pH 7.4 plus 0.05% Tween 20 and 3% non-fat dry milk (Biorad), respectively. Signals were detected using the SuperSignalWest Pico Chemiluminescent Substrate  and Amersham ECL hyperfilm .5 cells/ml. Gamma-irradiated (5 Gy) cells were used as positive control, untreated cells as background and the empty vector plus an endonuclease and reverse transcriptase ORF2 mutant as negative controls. The alkaline comet assay was performed by using the CometAssay Reagent Kit for Single Cell Gel Electrophoresis  following the manufacturer's instructions. Cells were mixed with low melting agarose  and embedded on a slide . Slides were incubated in cold lysis solution (Trevigen) for 45 minutes at 4°C. Slides were then incubated in Alkaline Solution, pH>13  for 40 minutes at room temperature in the dark. Cells were then electrophoresed in Alkaline Electrophoresis Solution, pH>13  in a horizontal gel apparatus  at 1 V/cm for 30 minutes at 4°C. Slides were rinsed briefly in dH2O and then incubated in 70% ethanol for 5 minutes. Slides were air-dried overnight and DNA stained with SYBR Green I (Trevigen). Cells were analyzed using an epifluorescence microscope . Digital images were acquired with a SensiCam QE digital camera . Tail moments were determined for at least 75 random cells per sample by using Comet Assay IV  comet scoring software. Tail moment is defined as the ratio of DNA in the comet tail to total DNA.HeLa cells were transfected with 1 µg of plasmid expressing the different ORF2 expression plasmids and controls. After 24 hours, cells were harvested and gently resuspended in phosphate buffer saline (PBS pH 7.4) to a concentration of 106/T75 flask) were transfected with 10 µg of the ORF2-expressing constructs. Cells from two transfected flasks were pooled and harvested 48 h post-transfection following the previously described protocol 5′-GAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTTVN-3′ and LEAP NA: 5′-GCGAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTT-3′. No LEAP primer was added to the negative control. The LEAP cDNA product was PCR amplified using the anchor primer (5′- GCGAGCACAGAATTAATACGACT-3′) and a primer specific to a sequence shared by both the mouse and human ORF2p constructs near the 3′end of the gene (ORF2-3′B: 5′-ACGGATCCGAACAAAAACTCATC-3′). A 3 µl aliquot of the LEAP product was PCR amplified for 35 cycles of 20 s at 94°C, 30 s at 62°C and 30 s at 72°C, with a final cycle of 5 min at 72°C and visualized on a 2.5% agarose gel.HeLa cells  to align ORF2 protein sequences from human (L1RP), mouse (L1spa), rat, dog, rabbit, cow, tree shrew, and horse. Sequences other than human and mouse were collected from the Repbase Update database (TIF)Click here for additional data file.Figure S2The human and mouse chimeric HHM, HHhmm, and HHhmh chimeric L1 ORF2ps are unable to support Alu retrotransposition in a rodent cell line. NIH3T3 (mouse) cell line was transiently transfected with the three ORF2p chimeric proteins that were non-functional in HeLa and a tagged Alu vector. All three constructs  generated Alu colonies at a rate that was not significantly different than the empty vector control. The results in rodent cells NIH3T3 emulate the results obtained in the human HeLa cells, the other cys-domain mutant (HHhhm) was functional. The functionality of these ORF2 human-mouse chimera appears to be independent of cell line species (mouse or human). Two sample T-test analysis showed: * Significantly different than HHM, HHM2, HHhmh, and empty vector p<0.01; $ Not significantly different than empty vector p≥0.238; # not significantly different than MMM p = 0.210.(TIF)Click here for additional data file.Figure S3Amino acid alignment between the non-exchangeable region of the L1 ORF2 cys domain of mouse and human shows low and dispersed amino acid identity. The region encompassing 160 amino acids of the mid-section of the cys domain of the L1 ORF2p is shown. The consensus is shown at the top; identical amino acids are shown and non-identical amino are shaded in gray.(TIF)Click here for additional data file.Figure S4The L1 and Alu inserts recovered present the features of bona fide retrotransposed elements. L1 and Alu G418R colonies were selected and grown to confluency. DNA was extracted from the cells using the DNA Easy kit (Qiagen) following the manufacturer's recommended protocol. A. L1 inserts were recovered and analyzed using a previously published method CH tagged with the mneoI/ColE1 cassette B. The extracted DNA from cells transfected with the tagged Alu vector plus the different ORF2 expression constructs was evaluated by performing nested PCR with primers designed to amplify the sequences flanking the self splicing intron, F1: 5′-GGGCGCCTGTAGTCCCAGCTA -3′; F2: 5′-TAGCAGCCAGTCCCTTCCCGCTTCA-3′; R1: 5′-GTCAGCGCAGGGGCGCCCGGTTC-3′ and R2: 5′-ACTGGGCACAACAGACAATCGGC-3′ ). The annealing location of the primers are shown in the schematic of the Alu construct. An open arrowhead indicates the PCR product corresponding to an insert containing the spliced version (open arrowhead) of the Alu expression vector. An open arrowhead indicates the nested 217 bp PCR product corresponding to an insert containing the spliced version (open arrowhead) of the Alu expression vector. Lanes correspond to the following chimeric ORF2 constructs: 1- MMmhh, 2- MMH, 3-HHmhh, 4-HHhhm, 5- MMmmh, 6- HMM, 7- MHH, 8- MHM, and 9- HMH. M denotes the DNA, and the Alu expression plasmid (P lane) was used as the control for 625 bp unspliced product (black arrow).(TIF)Click here for additional data file.


In the majority of countries, surnames represent a ubiquitous cultural attribute inherited from an individual's ancestors and predominantly only altered through marriage. This paper utilises an innovative method, taken from economics, to offer unprecedented insights into the “surname space” of the Czech Republic. We construct this space as a network based on the pairwise probabilities of co-occurrence of surnames and find that the network representation has clear parallels with various ethno-cultural boundaries in the country. Our inductive approach therefore formalizes a simple assumption that the more frequently the bearers of two surnames concentrate in the same locations the higher the probability that these two surnames can be related . Using the Czech Republic as a case study this paper offers a fresh perspective on surnames as a quantitative data source and provides a methodology that can be easily incorporated within wider cultural, ethnic, geographic and population genetics studies already utilizing surnames. The spatial distribution of surnames is far from random. Differences in early naming practices and unique regional, geographic, demographic, or migratory influences have led to considerable specificity with regard to mix of surnames that can be found in a particular place. Such specificity has been shown to capture a great deal of ethno-cultural variation that is often intertwined with the characteristics of an area Such applications of surname research are based on the key assumption that the spatial structure of surnames can, at least to some extent, mirror other aspects of population structure. To extract information from surnames, the challenge is to discern meaningful patterns from complex spatial distributions with little a priori information . To our knowledge there has so far not been any attempt to capture the entire surname structure of a country through the pairwise comparison of geographic distributions of individual names. Previous research has ignored the spatial component altogether This paper seeks to examine the surname structure of the Czech Republic (Czechia) by employing a suitable pairwise measure of relatedness between individual surnames based on their frequency of spatial co-occurrence in terms of their joint spatial concentration. This measure formalizes a simple assumption that the more frequently the bearers of two different surnames concentrate in the same locations the higher is the probability that these two surnames can be “related”. In this context, relatedness corresponds to surnames formed within the same community and those informed by similar cultural, ethno-linguistic or other factors. Using this measure, we depict the aggregate surname structure of Czechia as an undirected network of surnames linked by the degree of their relatedness. This representation can be conceptualised as “Czech Surname Space” and offers a template for similar research in other countries. Our inductive approach focuses on the revealed relatedness; only after the Czech surname space is determined do we map its structure and examine possible coincidences with other aspects of the Czech population differentiation.A focus on the spatial co-occurrence of surnames makes this paper distinct from previous studies. The bulk of the literature typically concerns pairwise comparisons between spatially defined populations based on the (di)similarity of their respective surname compositions The approach adopted here is a departure from previous research in the sense that the spatial distributions of individual surnames are the key input; regional patterns emerge as groupings in the surname space. Such approaches seek to establish the extent to which two or more geographic areas share the same pool of surnames and therefore offer comparisons between spatial units rather than the surnames themselves. With traditional methods, broad surname regions can be reliably produced but at the risk of subsuming some of the smaller groups of surnames with non-contiguous spatial patterning. Migrant surnames may, for example, be well-represented in these smaller groups and therefore more easily isolated than when using a traditional measure to produce more aggregate results. Improved granularity comes at the expense of increased computing overheads and a far more complex result (due to its larger number of comparisons), but we feel that capability to handle and interpret such outputs is increasing all the time and, as such, the methodology will become more widely applicable.LQ). For individual surnames (i) and regions (r), respectively, it can be expressed as:i,rF stands for the absolute number of bearers of the surname i in the region r. The i,rLQ compares the relative share of people with the surname i in the population of the region r relative to the share of this surname in the whole population at a more aggregate level. An i,rLQ >1 indicates that the surname in question is more prevalent in the region r than in the whole population (below we simply say that the surname concentrates in the region r).The first step in defining a surname spatial similarity measure is the selection of an appropriate form of input data for describing the occurrence of individual surnames in particular regions. A simple consideration of the absolute numbers of bearers would be inappropriate in the present context because the size of subpopulations of individual surnames varies immensely. A better metric that accounts for both the spatial concentration and the ubiquity of individual surnames is the location quotient . As stated above, we consider this network as an appealing representation of the Czech surname space. It can be examined both globally in terms of its aggregate patterns, its shape, or the number of communities, and locally through extracting the positions of individual surnames or their groups. Both of these aspects are important with respect to our inductive analysis that is driven by an expectation of detectable clusters or communities and surnames with strong internal and relatively weak external relatedness.Given the large sample of surnames analyzed it was necessary to run a series of computationally intensive calculations to obtain an extensive matrix of surname-surname proximity observations . Such a matrix tends to be very sparse with a large number of zero or negligible observations and very few more significant observations. It is therefore conducive to data mining through network analysis . With this the network can be conceptualised as a physical system where nodes (surnames) influence each other via attracting forces with strengths proportional to their revealed relatedness. The algorithm minimizes the energy of the physical system and assigns the nodes with positions in two-dimensional space accordingly.For the network visualization we used Cytoscape, open source software suitable for handling large complex networks i,jD (denoted as d) determined by, for example, inspecting the frequency distribution of the proximity observations provides a logical criterion. Considering a certain d, a surname space visualisation consists of n surnames and m surname-surname relatedness links, when:For the network visualisation to be interpretable, the majority of negligable links should be removed. A threshold of i is denoted as ik and it corresponds to the number of its revealed relatedness links to other surnames equal or above chosen d:This provides the basis to defining some simple local and global characteristics of the surname network, similarly to basic measures used in the network analysis ik implies that surname in question co-occurs (concentrates in similar regions) with many other surnames within a given surname space or its sub-space. In other words, a high ik indicates that a surname i is highly embedded in the surname space or its sub-space  and that it can be considered an examplar of a local population.This measure is particularly interesting in the present context because it can be considered as a simple measure of the node centrality. A high c) expressed as:ρ): that is the proportion of actual number of links in the surname space relative to the maximum possible number of links:In addition, two basic global parameters of a surname space can be introduced in terms of the mean surname degree . As such, they provide interesting information about the extent of internal population homogeneity.This paper draws on a unique dataset containing the occurrence of individual surnames in each of the 6,253 Czech municipalities derived from the 2009 Central Population Register (produced by the Czech Ministry of the Interior). The data cover all those with permanent residence; that is Czech nationals and foreigners staying on a long-term basis. The 10,705,763 individuals listed share 362,125 unique surnames.It is conventional in Czechia to have male and female variants of the same surname. Both exhibit almost identical spatial distributions negating the need to include both forms and so the female variants were omitted. This dramatically reduced the volume of data. Fortunately, nearly all Czech feminine derivatives are easily distinguishable by the suffix “–á”. The exceptions are comparatively more frequent for certain surnames typical of eastern Moravia and Silesia For the analysis of co-occurrence we decided to work with male surnames with a frequency exceeding 49 bearers in the whole country. With this filter applied the data comprised 15,487 most frequent male surnames and 4,347,283 individuals corresponding to 83% of total male population. The cut-off was chosen in the light of the following: firstly, the size distribution of surnames is heavily right skewed and the inclusion of less frequent names would make our analysis excessively computationally intensive (as described below); secondly, and more importantly, the consideration of less frequent surnames would considerably increase a risk of contamination of results by random co-occurrences of rare surnames; thirdly, we also noted that the spatial distribution of rare surnames in Czechia is quite uneven with significantly higher shares of such surnames in peripheral areas and especially in the region of Silesia . i,j,regD observations attaining a value exceeding 50% of the maximum observation. In other words, while an overwhelming majority from all of analyzed pairs of surnames reveal a negligible relatedness, there is also a tiny proportion of those pairs that are interesting in the present context because of their high mutual proximity.We first examined the co-occurrence of 15,487 unique male surnames over 206 Czech micro-regions. The calculations for all possible pairs of these surnames produced a matrix of 119,915,841 proximity observations , Hrubý , Liška (a fox), Toman (after Thomas the Apostle), Kočí (a coachman), Prokop . The ubiquity of these and similar surnames is determined by a meaning independent of regionally specific naming practices (a common naming practice in many countries). Of the 675 male surnames with the frequency above 1,000 bearers only 17% of them fall into this group of spatially ubiquitous surnames. Importantly, it implies that the majority of the most frequent surnames exhibit some kind of spatial concentration. Arguably, the tendency towards spatial concentration is expected to be even higher for the less popular names.Before examining the most significant links it is worth discussing some of the highly ubiquitous surnames in terms of greater prevalence but low spatial concentration. The following six surnames have more than 4,000 bearers but have no observation exceeding i,j,regD  = 0.525 (or 60% of the maximum) offers a good threshold for distinguishing these important observations as it lies in the area beyond which the rank-size curve rapidly flattens. Using the conditional probability interpretation of the Dice coefficient, we can say that two surnames connected by a link satisfying i,j,regD ≥0.525 have at least 52.5% probability that one of these surnames concentrates in a region where another is concentrated.For this paper, the most interesting information is contained in observations pertaining to the steep left part of the rank-size plot in Unfortunately, despite this cut-off, the surname space still contained too many nodes to be reasonably visualised. We thus further limited the displayed results to surnames with at least 100 bearers. This value is based on insights from a number of preliminary experiments examining the trade-off between the number of surnames displayed (complexity of displayed surname network) and graphical limitations of our network visualisations (readability of the network). As a result, we obtained a set of 8,405 proximity links connecting 2,429 unique male surnames. After applying the weighted force-directed layout algorithm, the aggregate version of the Czech surname space was generated and visualized in The Czech surname space illustrated in The key feature of each network graph is its degree distribution. In a random graph, nodes have a similar probability of being connected and therefore the degree distribution tends to be homogenous as signified by a binomial shape. By contrast, real world networks of various complex phenomena are typically hierarchically organized, with an inhomogeneous, considerably right skewed degree distribution. Here, a highly inhomogeneous degree distribution has been found  suggestii, the region of its prevailing concentration refers to a region with the maximum i,rLQ . The three distinguished groups within the main Bohemian core were indicatively marked as A1, A2, and A3 in The most extensive cluster of surnames in the Bohemian part of the Czech surname network in The group A1 includes typical Bohemian surnames in terms of the most frequent  in addition to other lower frequency, and traditional, Bohemian surnames. Although the more populous of these surnames are widely found across the country, all of the surnames from this group tend to be concentrated in the south and west regions of Bohemia see also . The secIn addition to the main core, there is another dense cluster in the Bohemian part of the Czech surname network. Labelled as A4 in The second Moravian-Silesian part of the Czech surname space has two dense cores in terms of the main Moravian cluster (B1) and Silesian cluster (B2). In addition, there is also a relatively dense area between these main cores consisting of names typical for various more specific regions in the north, central, eastern Moravia. In the case of Moravian surnames, linguistic differentiation of surnames and spatially specific naming practices are clearly recognizable. For example, a majority of names that apparently originated from verbs  are located in the lower left and upper parts of the main Moravian cluster (B1). Some notable examples of these names, with a quite central position in our surname network (see below), are Zapletal , Přikryl (from “to cover”), or Hradil (from “to block”). In In addition, An intriguing exception to these explanations is a typical Czech surname Vlček that can also be found in i,j,munD) among 5,660 surnames selected on the basis of the highest revealed relatedness at more aggregate spatial level. This sample of surnames covers almost a half of the Czech male population. Given the significantly higher number of spatial units considered for this second stage of our analysis, the values of i,j,munD are generally lower than i,j,regD in the first stage which focused on co-occurrence in 206 micro-regions only. At the same time, the size distribution of these second stage results is even more skewed to the right; the maximum i,j,munD  corresponds to 0.687, while only 0.011% of all observations exceed 50% of the maximum value. These differences between the first and second stage results are understandable and go hand in hand with the expectation that the surname network based on the municipality level calculations will be more fragmented.In the second stage of our analysis we examined the co-occurrence of Czech surnames at the finest spatial level of 6,244 municipalities. We began with the calculation of the pairwise indices of revealed relatedness . Regarding Moravian surnames, again the commonality of verb-derived surnames emerges, as they form the majority of names in the B1 area of the network. The Bohemian part of the surname space (A) is structured into three main groups of surnames. The A1 cluster comprises some of the most frequent surnames and those prevalent across most of Bohemian regions, whilst the separation from the secondary cluster (A2) is hardly discernible. By contrast, two other core areas are well recognizable and represent northern and eastern Bohemian names (A3) more specifically and surnames concentrated mainly in municipalities in the north-west and west of Bohemia (A4).i,j,regJ and i,j,munJ were considered instead of the i,j,regD and i,j,munD, respectively). However, the main value of this second stage municipality level exercise should be seen in individual details uncovered with respect to local parts of the surname network. A number of interesting examples of pairs of surnames that have been found as potentially closely related, regionally specific offshoots of the surname network, or specific groups of surnames determined in various ways, could be identified, mapped, and examined in greater depth.The general congruence in macro-structure of the surname networks constructed here and in the first stage of our analysis is an important finding  than those with very different distributions. Through the application of suitable measures of spatial co-occurrence, the extent of revealed relatedness between individual pairs of surnames was quantified. The focus here was not an intensive examination of the proximities between particular surnames; instead, the ultimate goal was to understand the aggregate pattern of the Czech surname space, anticipating that some innovative insights about the Czech population structure can be gathered in this way too.We conceptualized and represented the Czech surname space as an undirected network of surnames linked by their pairwise revealed relatedness. This approach demonstrated the utility of network representations and techniques in the context of surname data that appears to share several properties often attributed to other complex networks. These include a relatively inhomogeneous structure, considerably skewed degree distribution, and multi-layered composition determined by a highly right skewed frequency distribution of surnames. This falls hand in hand with a pronounced hierarchy regarding spatial scales on which the concentrations of these surnames occur.Indeed, the results confirmed a great deal of correspondence between the macro-structure of the Czech surname space and the main cultural and historical macro-divisions of the Czech population. The more detailed analysis has proved useful in offering numerous more nuanced insights about Czech population structure such as the identification of less known secondary divisions or specific clusters of surnames. It has also been shown that the inspection of network parameters such as density or the mean degree between particular parts of the surname space can be used for comparing the extent of homogeneity and stability between different populations or their parts.This work represents an initial foray with a wide range of further applications. Importantly, most of the methods presented here are scalable so that they can be analogously used for analyzing different spatial systems or different parts or regions within one spatial system.Another possible application is related to the identification of the clusters of high degree surnames found in the cores of the Czech surname network. These “hub” surnames can be regarded as the most typical and stable exemplars of their respective parts of the surname space, and together, can be considered a backbone of the Czech surname space. The identification of these most typical and stable surnames (and mapping of the main areas of their concentrations) offers a valuable tool for population geneticists, who for example are seeking to optimise their sampling design. Such names can indicate aspects of population structure, such as rates of population turnover, that may be more or less conducive to genetic sampling. For example, it would be ineffective to target a population group comprising large numbers of migrants if trying to characterise the genetic attributes of the historic population of the specific area in which the migrants reside. In this sense, our study provides another example of promising potential for integration of geography and genetics Although our analysis utilized current cross-sectional data, there exists a potential for insights into long-term population processes. This is most evident in relation to the enduring spatial stability of a majority of Czech surnames in spite of a long history of population movements. Such movements, therefore, appear to have only marginal impacts on regional surname structure. The exceptions are rare but notable as they point to the radical population changes associated with the expulsion of Germans from the post-war Czechoslovakia and subsequent resettlement of the formerly largely German speaking areas. This presents further avenues for research that could, for example, focus on the separate surname network for the former German areas and compare its parameters with the rest of the country. If an appropriate theoretical framework is applied, this one-time population shock can be considered as a kind of “natural experiment” and the persistence and resilience of the affected surname system may be examined using the methodology described above.Another notable feature perturbing the stability of the Czech surname space is the specific spatial behaviour of various minority population groups including international migrants. Although, in quantitative terms, these groups still represent a minor part of the Czech population, this study has shown that they are a well-delineated segment. On this basis, our analysis may be considered a tool for the classification of surnames into ethnic groups based solely on their spatial characteristics. It can be thus considered as an alternative to existing approaches to name-based ethnicity classifications that harness pre-existing ethnic categories of surnames In summary, this study sought to demonstrate the applicability of a new approach to surname research as a means of revealing the underlying surname structure of a country, in this case the Czech Republic. It is our hope that the perspective and methodology adopted here can serve as a template for similar studies in other countries and facilitate further interdisciplinary research in this area.Figure S1Ethno cultural differentiation of Czechia.(TIF)Click here for additional data file.Figure S2High resolution version of Czech surname space based on surnames co-occurrence in micro-regions.(PDF)Click here for additional data file.Figure S3The Czech surname space based on surnames co-occurrence in micro-regions: node size proportional to the degree of particular surnames.(PDF)Click here for additional data file.Figure S4Surname degree versus surname population size.(PDF)Click here for additional data file.Figure S5High resolution version of Czech surname space based on surnames co-occurrence in municipalities.(PDF)Click here for additional data file.Text S1Tests of behaviour of i,jJ and i,jD with respect to differing population size.(PDF)Click here for additional data file.Text S2th century.Ethno-cultural differentiation of Czechia and main migratory trends over the second half of 20(PDF)Click here for additional data file.


Recent studies suggest that the incidence of idiopathic pulmonary fibrosis (IPF) is rising. Accurate epidemiological data on IPF, however, are sparse and the results of previous studies are contradictory. This study was undertaken to gain insight into the various methods used in the epidemiological research of IPF, and to get accurate and comparable data on these different methodologies.A systematic database search was performed in order to identify all epidemiological studies on IPF after the previous guidelines for diagnosis and treatment were published in 2000. Medline (via Pubmed), Science Sitation Index (via Web of Science) and Embase databases were searched for original epidemiological articles published in English in international peer-reviewed journals starting from 2001. After pre-screening and a full-text review, 13 articles were accepted for data abstraction.Three different methodologies of epidemiological studies were most commonly used, namely: 1) national registry databases, 2) questionnaire-based studies, and 3) analysis of the health care system’s own registry databases. The overall prevalence and incidence of IPF varied in these studies between 0.5–27.9/100,000 and 0.22–8.8/100,000, respectively. According to four studies the mortality and incidence of IPF are rising.We conclude that there are numerous ways to execute epidemiological research in the field of IPF. This review offers the possibility to compare the different methodologies that have been used, and this information could form a basis for future studies investigating the prevalence and incidence of IPF. Diagnostics and treatment of idiopathic pulmonary fibrosis (IPF) is currently undergoing changes. In the most recent guidelines , the diaEpidemiological data of IPF are sparse, and moreover, changes in classification of idiopathic interstitial pneumonias (IIP) and diagnostics make the comparison of the data from various decades difficult. Until the 1990s, idiopathic pulmonary fibrosis covered a heterogeneous group of different interstitial diseases. The ATS/ERS consensus statement of diagnosis and treatment in 2000  and clasNational IPF registries are being set up ,8, and wOur aim was to review the literature of IPF epidemiology that has been published after the previous international statement on IPF in 2000. Therefore, we performed a systematic literature search in September 2012 with the help of an information specialist at the University of Turku/Medical library. Relevant literature was identified through the databases Medline (via Pubmed) and Science Citation Index (via Web of Science) starting from 01/01/2001 onwards. Only articles published in English were included in the study. The search was limited to human adults, using the terms idiopathic pulmonary fibrosis, interstitial lung diseases and epidemiology, combined with geographical areas . The initial search resulted in 214 articles. Two authors  independently pre-screened titles and abstracts, and data extraction resulted in 28 articles. All articles containing demographic data, incidence, prevalence or mortality rates of IPF or interstitial lung diseases (ILD), also including IPF patients, were accepted.The 28 eligible articles were then full-text reviewed by four authors . By consensus, only international peer-reviewed original articles were included, leading to the exclusion of seven review articles and one comparative report of ILD registries in three European countries . Two stuAn additional search was conducted in June 2013 to complement the systematic analysis. Embase was searched using the same queries as initially. 942 articles were found and titles were pre-screened by one author (JK). One additional article – published after September 2012 – was added to this systematic review. A summary of the selection process of articles is presented in Figure After the final evaluation, 13 articles were selected for data abstraction Table .From the 13 articles chosen for this review, we identified three commonly-used methods for the evaluation of IPF epidemiology in a population, and used this knowledge to categorize epidemiological studies in order to simplify the comparison of heterogeneous population data (Table In Flanders—the northern part of Belgium—20 centers of respiratory medicine took part in the registration of ILD patients between the years 1992–1996 . The tweIPF was the second most common ILD 20%) after sarcoidosis. Of the 72 patients with IPF, 21 (29%) had biopsy-proven UIP, 10 patients had bronchiolitis obliterans organizing pneumonia (BOOP), nine had desquamative interstitial pneumonia (DIP), three had lymphocytic interstitial pneumonia (LIP) and in 29 cases (40%) the histology was not specified. The cases with biopsy proven IPF/UIP were identified according to the ATS/ERS 2000 guidelines . Again m0% after The Italian register for diffuse infiltrative lung disorders (RIPID) was established in 1998 . The meaBoth of these national registry studies rely considerably on the activity of physicians in reporting patient numbers and data, and studies performed in this way should always be interpreted knowing of this bias, that results in underestimation of patient numbers. Registry cohorts like these, are, however important in the determination of patient clinical course and the possible differences in the management of patients in different centers.Two methodologically similar articles were found from Greece and Spain, where a standardized questionnaire was sent to respiratory medicine centers in order to obtain information on patients’ clinical data, procedures used to establish the diagnosis and the exact diagnosis.6 inhabitants. A standardized questionnaire was sent to the centers. An ATS/ERS consensus statement from 2002 was applied for classifying ILDs. A total of 511 cases of ILD were registered. Idiopathic pulmonary fibrosis was the most frequent disease, covering 197 patients/38.6% of all ILD patients; thus, according to our calculation, this results in an incidence of 2.94/100,000. The diagnosis was established by a surgical lung biopsy in 31.5%, and clinical criteria including HRCT scanning in 68.5%.In Spain, a registry of incident cases of ILD was set up from October 2000 to September 2001 [In Greece, departments of Pneumonology were contacted, and pneumonologists were asked to complete a questionnaire for all ILD patients who were alive in 2004 , as well as for every new incident case diagnosed during 2004 . CentersDue to the low response rates and lack of monitoring of the centers that have attended the study, data derived from these two studies should be interpreted with caution.In Finland, the prevalence of sporadic and familial IPF was evaluated during the years 1997–1998 . All FinIn Norway, the incidence and prevalence of physician-diagnosed and hospitalised cryptogenic fibrosing alveolitis (CFA) was studied during 1984–1998 . Thus, tIn the United Kingdom, a computerized longitudinal general practice database  was analyzed to estimate the incidence of IPF and sarcoidosis between 1991–2003 . The terIn the United States, the annual incidence and prevalence of IPF was estimated by utilizing a large health care claims database from 01/1996–12/2000 . IPF patIn another study conducted in the United States, mortality to pulmonary fibrosis (PF) was studied from 1992–2003 . The NatClinical records were also utilized in Japan to gain epidemiological data of IIP . All IIPIn Olmsted County, Minnesota, during a nine-year period (01/1997–12/2005) residents were screened, and those with possible IPF were identified . These c—was also screened for diagnoses of IPF-CS between 2000–2008. Cases with a co-existing connective tissue disease, and individuals who had co-existing diagnoses of extrinsic allergic alveolitis, asbestosis, pneumoconiosis and sarcoidosis were excluded. The search resulted in a total number of 2,074 incident cases which equates to an incidence of 7.44/100,000. The majority (63%) of new cases were men. The mean age at diagnosis was 74.3 years. After adjusting for age, sex and health authority, the estimated annual increase in the incidence of IPF-CS was 5%. The overall median survival for this cohort was 3.03 years. The advantage in the methodology used here is the combination data derived from of two different and independent registries, but the results are handicapped with the lack of reevaluation of the diagnostic accuracy of these registries.In the United Kingdom, death registers and general practice databases were used to investigate mortality (1968–2008) and incidence (2000–2008) of IPF . The diaIn Taiwan, a large population-based study was conducted to collect epidemiological information of IPF among Asians . The datThe estimated prevalence, based on the broad case definition, ranged from 0.7 in 1998 to 6.4 in 2007. According to the narrow case definition the estimated prevalence was 0.5 in 1998 and 4.9 in 2007. The annual incidence was calculated to be 0.6 in 1998 to 1.4 in 2007 . The incidence ranged 0.5 in 1998 to 1.2 in 2007  or from IPF registries ,34. The In addition to the identification of epidemiological parameters, our primary aim was to identify plausible and reliable methods in studies related to IPF epidemiology, as the field of IPF medicine is undergoing considerable change due to specific drugs that are entering clinical use, and many nationwide registry databases which are currently being built. Various and very heterogeneous methods are being used by IPF researchers, and so far the data from these studies is not comparable between countries due to this variation. We identified three categories or strategies for collecting IPF epidemiological data: nationwide registries for IPF or ILD, such as the Italian registry; questionnaires for health-care professionals; and systematic searches from existing databases. From these methods, questionnaires seemed to produce the lowest prevalence rates compared to national registries and/or searches from pre-existing databases. The coverage of pre-existing databases might be high in cases such as those shown in the study of Navaratnam , but theThe number of incidental and prevalent cases varied greatly in the presented studies (prevalence from 0.5  to 27,9/Changes in diagnostic criteria may also explain some of the observed variation. Studies implemented before the ATS/ERS 2000 guidelines  have shoThe international disease classifications have also reflected the lack of a clear, systematic classification of ILD:s: only in the latest version of ICD-10 from 2013, there has been a separarate subclassification for IPF. It is to be expected, that studies performed after this disease classification has been taken into use, and in countries where the population registries are most reliable, will yield high-quality data on IPF epidemiology. There are already preliminary reports on several national registry studies, which hopefully will yield comparable data on IPF epidemiology in different geographical and cultural areas ,40,41.Overall, it can be concluded that the existing databases present an overestimation of patient numbers, whereas registries and questionnaire-type studies result in an underestimation of patient numbers. The acquirement of accurate epidemiological data is crucial in elucidating the natural course and prognosis of IPF in different populations, and can lead to a better understanding of the disease etiology. However, if epidemiological data are collected, it should include a broad systematic screening of registries in the health care system, and a specific reevaluation of the patient records, especially the evaluations of pulmonary pathologists and radiologists. Novel electronic databases will hopefully offer more comparable data in this setting in the near future.BOOP: Bronchiolitis obliterans organizing pneumonia; DIP: Desquamative interstitial pneumonia; HRCT: High resolution computed tomography; IIP: Idiopathic interstitial pneumonias; ILD: Interstitial lung diseases; IPF: Idiopathic pulmonary fibrosis; LIP: Lymphocytic interstitial pneumonia; NSIP: Nonspecific interstitial pneumonia; PF: Pulmonary fibrosis; UIP: Usual interstitial pneumonia; VATS: Video-assisted thoracic surgery.None of the authors have competing interests related to this work., revised the manuscript, RK: presented the original idea for this work, reviewed the original articles included in this work and participated in writing the manuscript, MM; reviewed the original articles included in this work, and contributed in the writing of the manuscript. All authors read and approved the final manuscript.JK: reviewed the original articles included in this work, collected data from these articles, and drafted and wrote the manuscript; E-RS: reviewed the original articles included in this work, and wrote the manuscript, UH:The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2466/13/53/prepub


Non-covalent interactions of aromatics are important in a wide range of chemical and biological applications. The past two decades have seen numerous reports of arene-arene binding being understood in terms Hammett substituent constants, and similar analyses have recently been extended to cation-arene and anion-arene binding. It is not immediately clear why electrostatic Hammett parameters should work so well in predicting the binding for all three interactions, given that different intermolecular forces dominate each interaction. This review explores such anomalies, and summarizes how Hammett substituent constants have been employed to understand the non-covalent binding in arene-arene, cation-arene and anion-arene interactions. Non-covalent interactions of aromatic π-electron density have been extensively studied . There imeta to the carboxylic acid functional group are termed σm, and are generally recognized as describing the movement of electrons via the σ-framework (inductive effects). The σp Hammett constant is obtained from substitution para to the –CO2H group, and it describes the movement of electrons via the σ- and π-framework (inductive and resonance effects). Hammett constants were developed to help explain trends in the reactivity of meta- and para-substituted benzoic acid derivatives and related compounds. For instance, the electrophilicity of meta- and para-substituted benzoic esters, the nucleophilicity of meta- and para-substituted anilines, and the solvolysis of meta- and para-substituted benzyl halides . A . A ‡, ofsyn and anti 1,8-di-o-tolylnaphthalenes where each tolyl group was substituted para to the naphthyl ring (p), and this led to the conclusion that charge-transfer effects should be expected to minimally contribute to the overall aromatic interaction as compared to the electrostatic contribution . Not. Not1]. –-substituted benzene complexes  and electron-donating  groups. All complexes were defined with the anion in the plane of the aryl CH and outside of the periphery of the aromatic ring. As would be expected, the presence of electron-donating substituents decreased the binding energy and lengthened the anion-arene distance, and electron-withdrawing substituents increased the binding energy and shortened the anion-arene distance meta- or stituent , 44, 46,stituent . The facstituent , 35, 37 m or Σσm, while others show σp or Σσp to be best. Why would the inductive contributions of a substituent, as measured by σm or Σσm, sometimes be a better predictor of the binding energies than the combination of the inductive and resonance effects, as measured by σp or Σσp? Certainly one cannot turn off the resonance withdrawing/donating abilities of a substituent, so why should σm or Σσm ever outperform σp or Σσp? Even more perplexing are the reported correlations with Σ∣σm∣ [Total, which equals (Σσm + Σσp) [m∣ value the authors suggest the absolute value of ∣σm∣ contains information about the electronic and polarizability properties of a substituent [Total suggest it contains information about both induction and resonance effects, but why is induction counted twice: once in the σm value and again in the σp value [m∣ value [Of equal interest to discussions about what forces dominate the various interactions, and the role of Hammett constants in predicting the binding energies, is why some studies show the best correlations with σth Σ∣σm∣  or σTotam + Σσp) . In the stituent , but thiσp value ? Note thm∣ value . We are m, while for others it is σp, and for others still it is some manipulation of these parameters such as ∣σm∣ or (σm+σp). In some cases the differences between the cited studies can be attributed to the investigation of different substituted aromatics. Still, this is not always the case, and significant work remains to determine why Hammett constants have performed so well in predicting the binding energies of the interactions discussed here, and why sometimes σm gives the better correlations, while in other instances σp performs best. Finally, as noted by Hunter, Cockroft and coworkers, the importance of dispersion in gas-phase interactions may be due to the lack of a desolvation term [The correlation of Hammett substituent constants with arene-arene, cation-π and anion-π binding energies has allowed researchers to comment on the forces that govern the interactions; however, we believe these studies have produced as many questions as they have addressed. At a few places in this review we have noted that some studies observe the best correlations with σion term , 39, and


The membrane proximal external region (MPER) of HIV-1 is an important target of broadly cross-reactive mAbs. CAPRISA participant, CAP206, developed anti-MPER antibodies early that became cross-neutralizing at 18 months post-infection. This coincided with neutralization of the C4 HIV-2/HIV-1 chimera containing the W670 residue, suggesting changes in an antibody paratope may have resulted in the acquisition of breadth. A neutralizing mAb (CAP206-CH12) was isolated, providing an opportunity to determine which somatic hypermutations contribute to breadth.http://www.dulci.org). Batch transient transfections were used to generate recombinant antibodies. Neutralization breadth and potency was tested against autologous, Tier 2, and HIV-2/HIV-1 MPER chimeric viruses using the TZM-bl assay. Binding to MPER peptides was assessed by ELISA and surface plasmon resonance (SPR).The putative CAP206-CH12 reverted unmutated ancestor (RUA) was inferred using SoDA  where changes near the CDRs in CAP206-CH12 were reverted back to the germline sequence (11 in the heavy and 5 in the light chain). This CAP206-CH12_IP did not neutralize the C4 chimera suggesting that changes responsible for the affinity-matured CAP206-CH12 neutralizing capacity were among these 16 residues. Chimeric pairs of the light chain IP with the heavy chain of CAP206-CH12 or CAP206-CH12_RUA showed binding to MPER with differences in binding kinetics.The reduced binding and neutralizing activity of CAP206-CH12_RUA and CAP206-CH12_IP compared to CAP206-CH12 suggests a correlation between affinity maturation, neutralization breadth and potency. Ongoing work will assess the affinity maturation of CAP206-CH12 by determining moieties in the antibody paratope associated with effective epitope recognition and the effects of somatic mutations on the evolution of neutralization.


Phonons, which are collective excitations in a lattice of atoms or molecules, play a major role in determining various physical properties of condensed matter, such as thermal and electrical conductivities. In particular, phonons in graphene interact strongly with electrons; however, unlike in usual metals, these interactions between phonons and massless Dirac fermions appear to mirror the rather complicated physics of those between light and relativistic electrons. Therefore, a fundamental understanding of the underlying physics through systematic studies of phonon interactions and excitations in graphene is crucial for realising graphene-based devices. In this study, we demonstrate that the local phonon properties of graphene can be controlled at the nanoscale by tuning the interaction strength between graphene and an underlying Pt substrate. Using scanning probe methods, we determine that the reduced interaction due to embedded Ar atoms facilitates electron–phonon excitations, further influencing phonon-assisted inelastic electron tunnelling. A fundamental understanding of electron–phonon interactions in graphene is crucial to developing graphene-based electronic and photonic devices. Here, the authors control the properties of phonons in graphene by tuning the interaction strength between graphene and an underlying platinum substrate. Graphene, which is known to exhibit linear energy dispersion near the Fermi level, is considered a highly desirable material for future electronic1247910111011In the following, we propose a simple method to fabricate deformed graphene for easy modification of the graphene–substrate interaction with local control. We choose nanobubbles as the structural ‘knob', that is, an on-off switch for phonon control, and exploit the fact that noble-gas ions can penetrate below the first atomic layer of a graphite surface to form interstitial defects by low-energy impinging1314+ ions, protrusions emerged as a result of certain modifications in graphene but not in Pt(111), as evident in +-ion irradiated graphene can in general be attributed to several origins such as adatom adsorption, vacancy-induced deformation1314+-ion sputtering with subsequent annealing. This is evidenced by the fact that the generally observed resonance peak near the Dirac point, which is characteristic of vacancies in graphene, is indeed absent in the differential conductance (dI/dV) spectrumIn our study, graphene nanoislands were first grown on a Pt(111) substrate. The large-scale topography  as well I/dV spectra of the nanobubbles obtained near their apexes share the common gap-like feature centred at the Fermi level . As shown in K projection occurs at 68–70 meV for all finite-size dots, whose energy range corresponds well with the phonon energy of 67.3 meV in an infinite plane with momentum K. However, the larger the dot size is, the narrower the calculated distribution of the K projection becomes, and this behaviour explains why the depth and the edge slope of the gap-like feature in the dI/dV are observed to increase for larger bubbles. In addition, the calculation of the root mean square amplitude of the phonon modes  maps. The spatial dI/dV map in 2I/dV2 map in I/dV maps because of the gap, and a strong contrast is observed at around ±70 meV in the d2I/dV2 maps, which is manifested as a peak and dip in d2I/dV2 spectra in I/dV and d2I/dV2 maps coincides with that of the nanobubble in the topograph. We also plotted the bias voltages of the largest peak in the d2I/dV2 spectra, Vph, shown in Vph shows little variation from 64 to 70 meV. However, the intensity of phonon signal increases as a function of the distance between the graphene and Pt layers, as plotted in To examine the spatial distribution of excited phonons around graphene nanobubbles, we obtained the spatial dI–V behaviourWe successfully demonstrated phonon excitations in graphene via local control by generating nanobubbles of various sizes by means of Ar-ion irradiation. The reduced graphene–metal interactions due to Ar atoms embedded in between the graphene and metal substrate were shown to promote phonon excitations and enhance electron tunnelling above the phonon energy. We expect that our demonstrated control over phonon excitations down to the nanoscale will pave the way for advancements in graphene-based nano-electromechanical devices such as electrical switches based on the induced nonlinear + ions , and the sample was annealed at 1,000 K to remove additional impurities adsorbed onto graphene. Our STM measurements were performed using a Unisoku low-temperature STM at 2.8 K, and scanning tunnelling spectroscopy was performed using a conventional lock-in technique with a modulation bias voltage at a frequency of 1 kHz and an amplitude of 10 mV.A Pt(111) sample was cleaned by means of repeated cycles of Ar-ion sputtering and annealing under ultrahigh vacuum. Graphene nanoislands were prepared by exposure of the clean Pt(111) surface to ethylene followed by annealing at 1,100 Kab initio simulation package2425−1.Our first-principles calculations are based on the DFT employing the generalized gradient approximation with the projector-augmented-wave method as implemented in the Vienna We computed the Hessian matrix of the atoms within the graphene nanobubble using the adaptive intermolecular reactive empirical bond order (AIREBO) potential for carbonHow to cite this article: Kim, H.W. et al. Nanoscale control of phonon excitations in graphene. Nat. Commun. 6:7528 doi: 10.1038/ncomms8528 (2015).Supplementary Figures 1-7, Supplementary Note 1 and Supplementary References


Charge noise is critical in the performance of gate-controlled quantum dots (QDs). Such information is not yet available for QDs made out of the new material graphene, where both substrate and edge states are known to have important effects. Here we show the 1/f noise for a microscopic graphene QD is substantially larger than that for a macroscopic graphene field-effect transistor (FET), increasing linearly with temperature. To understand its origin, we suspended the graphene QD above the substrate. In contrast to large area graphene FETs, we find that a suspended graphene QD has an almost-identical noise level as an unsuspended one. Tracking noise levels around the Coulomb blockade peak as a function of gate voltage yields potential fluctuations of order 1 μeV, almost one order larger than in GaAs/GaAlAs QDs. Edge states and surface impurities rather than substrate-induced disorders, appear to dominate the 1/f noise, thus affecting the coherency of graphene nano-devices. There has been no report on the charge noise for graphene nano-devices where the carrier channel size is of 10-nm order although researchers have fabricated various graphene nanostructures including single graphene quantum dots (GQDs)192021222324262728292 was etched away to a depth of about 50 nm. We estimate our graphene nanoribbon was suspended about 50 nm above the substrate. By tilting the sample holder of the SEM to a specified angle, we can observe the nano-device from the side to check whether the graphene nanoribbon was suspended. As shown in After dipping the sample in the BOE for 40 seconds, SiOWe used the standard lock-in method to probe the electronic signals to make the suspended nanoribbon work in the Coulomb blockade region. We applied a dc voltage to the back gate to tune the Fermi energy of the graphene. The V-shaped current-voltage relation was observed, and a transport gap was found in the range 0.5–6.0 V . We thenWe measured the current fluctuation using a spectrum analyzer (SR785). A schematic of the circuit used is shown in I(f) is the noise spectrum measured. SCB(f) is the background noise induced by the measurement system. Here we used the spectrum at C as SCB.We calculated the magnitude of the current fluctuation ΔI by integrating the spectrum from 1 to 9 Hz; SD = 200 μV to the source and measured the current from the drain. Changing the back gate voltage from 4.283 to 4.297 V, we obtained a Coulomb peak. I at different back-gate voltages along the Coulomb peak and calculated the integral in −1|dI/dV|Δε given in Ref. Next, we applied a dc voltage VThe potential fluctuation Δε, calculated from the peak in We also measured the current noise of regular (unsuspended) graphene nanoribbon devices for comparison. The regular graphene device was fabricated in a similar way as described above. The only difference was that the regular samples were not dipped in BOE after making electrodes. Similar noise measurements of regular devices were performed under the same experiment conditions. To investigate the influence of the temperature on the potential fluctuation, we measured the same Coulomb peak at different temperatures, from 240 mK to 1 K. From 2* in graphene nano-devices. Notably, we also used the method described in Ref. We measured different Coulomb peaks from several devices, both suspended and unsuspended. For E, the density of disorders in the substrate is nS. The effect of edge states and substrate on the low frequency noise of the device is proportional to S. In the GFET experiment, the removal of the substrate decreases the noise by one order of magnitudeIt is well known that the edge states can greatly affect the properties of graphene devices. The edge states change the electron distribution, resulting in the formation of puddles in graphene nano-devices3738Although this simple model can qualitatively explain the present experiment, more studies are still needed to investigate how the edge states or surface impurities affect the 1/f noise, and how to decrease the noise level in the graphene devices.In summary, we have fabricated both suspended and unsuspended graphene nano-devices and measured their 1/f noise along the Coulomb peaks to obtain the charge noise level. Suspending the QD from the substrate had no observable effect on the charge noise of a GQD. The edge states closely surrounding these nano-devices are argued to increase the charge noise to 1 μeV and become the dominant charge noise source instead of that from the substrate. More studies are needed to improve the electronic performance and increase the quantum coherence of future graphene nano-devices.2 layer away. To avoid graphene from being wrinkled, we used a critical-point dryer to dry the sample instead of blowing with N2.Graphene flakes were produced by mechanical cleaving of bulk graphite and deposited on a highly doped silicon substrate covered by 100 nm of silicon dioxide. The doped silicon substrate worked as a back gate. Graphene flakes were found using an optical microscope, and few-layer flakes were selected using Raman spectroscopy40X.X.S. fabricated the samples. X.X.S., H.O.L., J.Y., T.Y.H., G.C. and G.P.G. performed the measurements. X.X.S., M.X., T.T. and G.C.G. provided theoretical support. Data are analyzed by X.X.S., H.O.L. and J.Y. The manuscript is prepared by X.X.S., H.W.J. and G.P.G., G.P.G. supervised the project. All authors contributed in discussing the results and commented on the manuscript.Supporting information


The ability to control light-matter interaction is central to several potential applications in lasing, sensing, and communication. Graphene plasmons provide a way of strongly enhancing the interaction and realizing ultrathin optoelectronic devices. Here, we find that photoluminescence (PL) intensities of the graphene/GeSi quantum dots hybrid structures are saturated and quenched under positive and negative voltages at the excitation of 325 nm, respectively. A mechanism called plasmon-gating effect is proposed to reveal the PL dependence of the hybrid structures on the external electric field. On the contrary, the PL intensities at the excitation of 405 and 795 nm of the hybrid structures are quenched due to the charge transfer by tuning the Fermi level of graphene or the blocking of the excitons recombination by excitons separation effect. The results also provide an evidence for the charge transfer mechanism. The plasmon gating effect on the PL provides a new way to control the optical properties of graphene/QD hybrid structures. It has been found that by electric gating and thermal annealing, the dynamic conductivity of graphene device would change largely, and the Fermi energy of graphene is effectively tuned. As a result, the guided light can be modulated within a broad operation spectrum212278Thus, we naturally consider whether the SPP enhanced PL properties of the graphene/GeSi QDs hybrid structure can be controlled by introducing an external electric field. It is also easily considered whether the PL quenching, based on the carrier transfer or the energy transfer of excitons, can be directly indicated by tuning the graphene Fermi level with a bias voltage. The controlling PL properties of QDs are very essential to its application in improving optical properties. In this work, we fabricate the graphene/GeSi QDs hybrid structure devices to explore their optical properties by adding external electric field. It is indicated that the vertical electric field nonlinearly determines the enhancement or quenching of PL intensity at the different excitation wavelengths. At the excitation of 325 nm laser line, which is the resonant excitation wavelength of graphene plasmon−1) to G-band (~1580 cm−1) is larger than two. The 2D-band is quite shape and cannot be decomposed into two or more components. The characteristics indicate that the graphene on the GeSi QDs is monolayer one. And the low intensity of D-band (1341 cm−1) demonstrates the good quality of graphene sheet28The schematic diagram of the graphene/ten-layer GeSi QDs hybrid structure device demonstrates that the positive voltage was added on the Al back electrode, and the In point electrode was connected with graphene on the top of the device . Figure In our previous publicationNext, the PL spectra of graphene/GeSi QDs hybrid structures are obtained with the other exciting wavelengths for different bias voltages. At the exciting wavelengths of 405 and 795 nm, it is well known that there is no SPP enhanced absorption in graphene2 layer between the first layer GeSi QDs and the graphene. Thus, the SPP can influence the topmost layer GeSi QDs. The resonant excitation of graphene SPP can enhance the absorption at surface region so that the generation of electron-hole pairs in the topmost layer QDs is enhanced. For the hybrid structure, there are two competing mechanisms. One is the SPP absorption mechanism to enhance the PL intensity, and another is the electron transferring from QDs to the graphene, which reduce the PL intensity. Under the positive voltage, the oblique band alignment shown in At the excitation of 325 nm and under positive biased voltage, the results look quite interesting. From Similar to photogating effect in the graphene covered PbS QDs structure, in which the photogenerated charges can transfer to the graphene and the oppositely charged carriers remain trapped in the QD layer under the build-in field at the QD/graphene interfaceIn summary, we have fabricated a graphene/GeSi QDs hybrid structure device and measured the PL properties under varying bias voltages at different excitation wavelengths. At all three exciting wavelengths, the PL intensity of the hybrid structure is found to be quenched with increasing negative biased voltage on the graphene/QDs structure, which can be explained by the electron transfer model. Under positive biased voltage, different dependences of PL intensity of the hybrid structure on the voltages are observed at three excitation wavelengths. At the excitation of 325 nm laser line, a plasmon-gating mechanism is proposed for the observed PL intensity dependence on the positive biased voltage. At the excitations of 405 and 795 nm, the PL intensity of the hybrid structure is found to be quenched with increasing positive bias voltage, which is explained by excitons separation effect. The gating of the plasmon resonance excitation and its effect on the photoluminescence provides a new way to control the optical properties of graphene/QD hybrid structures.10 layers ordered GeSi QDs sample was grown on pit-patterned Si (001) substrates with periods of 100 nm by solid source Molecular Beam Epitaxy. Large-scale graphene layer was fabricated by chemical vapor deposition (CVD) on a Cu substrate. The structure of graphene was confirmed by Fourier transform infrared (FTIR)-Raman spectra with the excitation of 325 nm laser line. Surface morphology of the sample was characterized using atomic force microscopy (AFM). Then the monolayer graphene was transferred on to the surface of GeSi QD sample to form graphene/GeSi QDs hybrid structures with the aid of polymethylmethacrylate (PMMA).2. Two electrical lines from the probes were pressed on the In electrode and the back side. The vertical electric fields can added to the sample in the vacuum chamber using an external 2400 Keithley Source Meter. We provide stable applied bias voltage from −100 V to 100 V.In order to add the vertical electric field on the hybrid structure, the In and Al contacts are deposited as the front and back electrodes. The back electrode was formed by thermal evaporation of 300 nm Al on the back of the silicon wafer, and the front electrode was formed by alloying In contact at the top of graphene layer with about 4 mm2. The PL spectra were recorded by an extended InGaAs detector using the standard lock-in technique.The PL measurements under different voltages were performed in a He-flow cryostat with the 325 nm line of HeCd laser, and 405 and 795 nm lines of diode lasers as the excitation source. The spot size of laser can be focused to 1 mmHow to cite this article: Chen, Y. et al. Plasmon-gating photoluminescence in graphene/GeSi quantum dots hybrid structures. Sci. Rep.5, 17688; doi: 10.1038/srep17688 (2015).