N = 73) or below-average (N = 57) achievement in early arithmetic was followed from the end of Grade 1 (mean age: 86.9 months) through the beginning of Grade 4. Symbolic comparison of one- and two-digit numbers serially mediated the effect of non-symbolic comparison on later arithmetic. These results support a developmental model in which non-symbolic processing provides a scaffold for single-digit processing, which in turn influences multi-digit processing and arithmetic. In conclusion, both non-symbolic and symbolic processing play an important role in the development of arithmetic during primary school and might be valuable long-term indicators for the early identification of children at risk for low achievement in arithmetic.The nature of the relation between non-symbolic and symbolic magnitude processing in the prediction of arithmetic remains a hotly debated subject. This longitudinal study examined whether the influence of non-symbolic magnitude processing on arithmetic is mediated by symbolic processing skills. A sample of 130 children with age-adequate ( The development of arithmetic skills in primary school is of fundamental importance in modern-day societies: already at the age of seven, arithmetic abilities predict adult socio-economic status over and above the effects of intelligence and socio-economic status at birth . Severe Children’s arithmetic development has often been linked to their “number sense,” meaning the ability to deal with non-symbolic magnitudes, for example dots or other concrete objects. Typical tasks involve choosing the numerically larger of two sets of objects . This ability has been proposed to reflect the acuity of the supposedly innate approximate number system (ANS). Using a habituation-dishabituation methodology, it became apparent that 6-month-olds can differentiate between sets with a ratio of 1:2 . During It has been argued that non-symbolic magnitude processing is directly and causally related to arithmetic performance . SupportOthers have rejected the notion of a causal relation between non-symbolic magnitude processing and arithmetic, alternatively proposing that the ability to deal with abstract symbolic numbers (mainly in the form of digits) is more important for arithmetic performance. Symbolic magnitude processing is often assessed with tasks requiring participants to indicate which of two Arabic digits is numerically larger . In this vein, compared to typical development, children with dyscalculia showed lower performance in a symbolic magnitude comparison task, but not when comparing non-symbolic numerosities . Based oThe fact that non-symbolic processing skills do not explain additional variance in arithmetic performance when controlling for differences in symbolic processing does not dismiss a potentially causal relation between non-symbolic processing and arithmetic. Only recently, an alternative mediation hypothesis has proposed that the relation between non-symbolic processing skills and arithmetic might be mediated by symbolic skills . An evolN = 53) of fifth graders, For instance, If non-symbolic processing is a foundation of understanding symbolic numbers, it might be expected that it is of particular relevance in young children who are still developing their symbolic number system. Indeed, Longitudinal studies are particularly relevant in order to determine causal mechanisms during development. So far, only one such study  investigIn summary, several studies investigating the hypothesis that symbolic processing abilities serve as a mediator of the relation between non-symbolic processing skills and arithmetic did indeed report some evidence in favor of this claim. Conflicting findings might in part be due to the different measures of symbolic number processing that were employed  and differences in age groups assessed. Still, the foundational link between non-symbolic and symbolic processing is not entirely uncontested: while there is some evidence that non-symbolic processing influences the development of symbolic processing skills , other sIn addition, it might also be important to differentiate between distinct subcomponents of symbolic processing, in particular single- and multi-digit number processing. There is increasing evidence suggesting that multi-digit number processing differs from single-digit processing and is acquired later in development . MoreoveIn the current study we tested a developmental model of sequential mediation of the effect of non-symbolic processing on later arithmetic performance via processing of one- and two-digit numbers. This developmental account was investigated in a longitudinal study ranging from end of Grade 1 to beginning of Grade 4. In this important period of arithmetic development, children are introduced to the complexities of the Arabic place-value system and are expected to acquire fluent competencies in mental calculation and to store a large amount of easily accessible number facts in their long-term memory.Non-symbolic, single- and multi-digit number processing were assessed at different, sequential time points, and prior to arithmetic skills. When testing our developmental framework, we controlled for general cognitive skills that have been found to be associated with arithmetic performance and might influence its relation with non-symbolic processing, i.e., non-verbal IQ , verbal Based on the findings of previous studies, we expect that non-symbolic processing exerts an indirect effect of future arithmetic performance but may not uniquely contribute to the prediction of arithmetic performance  when considering these control variables. Note that this study design puts the hypothesis that non-symbolic processing is a foundational skill underlying symbolic processing and arithmetic at a very stringent test: while non-symbolic processing could be expected to have its strongest influence early in development, when numbers are mapped onto analog magnitudes, our longitudinal design mainly assesses whether differences in non-symbolic processing contribute to growth of symbolic processing and (in turn) arithmetic skills during the primary school years, over and above general cognitive predictors of arithmetic. If any such indirect long-term effects can be demonstrated, even though small, the hypothesis that the non-symbolic magnitude processing is a foundational skill of arithmetic should be further investigated.The study was conducted in accordance with the ethical principles of the World Medical Association Declaration of Helsinki. Data collection started in 2007 and at that time the funding agency (DFG) and local legislation did not request an explicit vote from an ethics committee for non-medical research. Legal guardians gave their written informed consent before data collection. The present sample consisted of 130 children from 19 different elementary schools and a total of 38 classrooms taking part in a longitudinal study investigating the developmental trajectories of basic numerical skills in children with typical and atypical arithmetic development .SD or more below age norm on a standardized test (SD compared to the age norm). Thus, children with low arithmetic performance in Grade 1 were overselected in our sample. As our focus was on numerical and arithmetic development, we attempted to exclude more general deficits in non-verbal IQ, working memory, attention, and reading as potential causes or confounds of arithmetic deficits. More specifically, children were not admitted to the study if they met any of the following exclusion criteria:The participants were invited to the study based on a screening of 505 children at the end of first grade. Children with arithmetic achievement of 1 zed test  were all•a native language other than German;•IQ lower than 85 as assessed by a test of non-verbal intelligence ;•SD below age norm on the German version of the WISC-IV digit span subtest  showed arithmetic skills within the typical range.The initial sample consisted of 139 children (68 boys and 71 girls), of whom 131 participated through Grade 4. One child with low arithmetic performance had to be excluded because of below chance-level performance on the non-symbolic comparison task (rendering it unclear whether this child had understood the instruction). Thus, the final sample comprised 130 children (60 boys and 70 girls) with an average age of 86.9 months at the screening (end of Grade 1). At this first assessment point, 57 children showed low arithmetic performance. At the last assessment point in Grade 4, only 39 children performed more than 1 Children’s development was followed across a 2-year primary school period from end of Grade 1 (T1) to beginning of Grade 4 (T4). The first assessment point (T1) subsumed measures that were either given at the end of Grade 1, or right at the beginning of Grade 2, interspersed only by 6 weeks school holidays. Non-symbolic processing at T1 was considered as independent predictor variable. Symbolic single-digit processing was assessed 6 months later in the middle of Grade 2 (T2) and symbolic two-digit processing was assessed after another 6 months at the beginning of Grade 3 (T3). The dependent variable arithmetic performance was assessed at the beginning of Grade 4 (T4). We additionally considered several covariates: non-verbal intelligence, attention, verbal working memory, as well as initial arithmetic performance and symbolic single-digit processing . An overview of the study design is depicted in Non-symbolic and symbolic processing were assessed by standard numerical comparison paradigms programmed with Presentation software. Children performed the tasks individually in a quiet room at their school. We obtained a combined measure of speed and accuracy for children’s non-symbolic and symbolic processing skills by calculating inverse efficiency scores (median reaction times divided by the proportion of correct responses).Children were required to indicate which of two gray displays had the larger number of yellow squares see  by pressTwo tasks assessed symbolic processing skills: single digit comparison (T1 and T2) and comparison of two-digit numbers (T3). In both tasks, children chose the numerically larger of two numbers by pressing the corresponding keyboard button as quickly as possible. The single digit comparison task consisted of 56 items with numbers from 1 to 9. Numerical distances ranged from 1 to 8 . Comparison of two-digit numbers comprised 80 items with numbers between 21 and 98. Numerical distance ranged from 4 to 37. In 30 items, both decade and unit digit were larger in one number , in 30 items, the decade digit was larger in one and the unit digit was larger in the other number , and in further 20 items, only the unit digit differed . In both symbolic processing tasks, stimuli were displayed in a 36-point Times New Roman font in black color against a white background. Item presentation was randomized and the number pairs remained on the screen until children responded by keypress. After each item, there was an interstimulus-interval of 560 ms. For both symbolic comparison measures, Cronbach’s alpha ranged between 0.96 and 0.97._) and size comparison exercises . At T4, two additional subtests targeting multiplication and division were included. Each subtest had a 2 min time limit with items being presented with increasing difficulty. Within this time limit, children were required to write down as many correct answers as possible to a list of calculations gradually increasing in difficulty. Performance in each subtest was assessed as the number of correct answers. A composite measure “arithmetic operations” was calculated as the mean of the standardized T-scores  of all subtests.Arithmetic performance was assessed by the “arithmetic operations” subscale of a standardized classroom test . At T1, Non-verbal intelligence was assessed by the German version of the Culture Fair Test , comprisChildren performed a standardized computer-based test battery encompassing different facets of attention/executive functions . AttentiThe Digit Span subtest (forward and backward combined) of the German version of the Wechsler Intelligence Scale for Children IV  was usedr = 0.324, p < 0.001 and response accuracy was close to ceiling in both symbolic comparison tasks (mean accuracy symbolic comparison with single-digit numbers at T1 = 95.5% and T2 = 96.6% and symbolic comparison with two-digit numbers at T3 = 91.3%). In order to combine response accuracy and speed, inverse efficiency scores were computed for the non-symbolic and symbolic tasks by dividing the median reaction time by the proportion of correct responses. Finally, one extreme outlier score in the non-symbolic comparison task (more than 6 SDs above the sample mean) was moved to the tail of the distribution to the second highest score to avoid overemphasizing its effect on the results.For each of the numerical processing tasks, individual median response times were calculated after removing reaction times for incorrect responses, below 200 ms and above 10,000 ms. In the non-symbolic comparison task the correlation between median RTs and response accuracy was only moderate, Means, standard deviations and intercorrelations of all relevant study variables are shown in Mediation analyses were calculated using the PROCESS macro for SPSS . In ordea1 path) and from single-digit to two-digit processing (a3 path). Importantly, neither non-symbolic processing skills at T1 nor symbolic single-digit processing at T2 exerted significant direct effects on arithmetic growth (c′ and b1 paths). The direct paths from non-symbolic to two-digit symbolic processing (a2) and from two-digit symbolic processing to arithmetic growth (b2) missed significance .Residualized change scores of arithmetic (arithmetic T4 – arithmetic T1) were considered as dependent variable, and symbolic processing of single- and two-digit numbers were introduced as mediators, so we obtained the following PROCESS model: non-symbolic processing T1 → symbolic processing (single-digit numbers) T2 → symbolic processing (two-digit numbers) T3 → residualized change in arithmetic T4-T1. We added several general cognitive covariates of both mediators and the dependent variable, namely verbal working memory, non-verbal intelligence and attention. We also included a fourth covariate, symbolic processing of single-digit numbers at T1. The full PROCESS model including the main standardized path coefficients is depicted in a1b1 path) or through symbolic processing of two-digit numbers at T3 (a2b2 path). Still, non-symbolic processing did show a significant  influence on arithmetic growth serially through symbolic processing of single-digit numbers at T2 and symbolic processing of two-digit numbers at T3 (a1a3b2 path).Indirect effects, their standard errors and confidence intervals are presented in The presented sequential mediation analyses indicated that the effect of non-symbolic processing in Grade 2 on arithmetic performance 2 years later, in Grade 4, was sequentially mediated by symbolic magnitude processing of one- and two-digit numbers. Even though this mediation effect was small, these results provide an important empirical contribution to the ongoing debate whether non-symbolic processing skills make a causal contribution to arithmetic development. Our developmental perspective on the association of magnitude processing with arithmetic see also  is consiRegarding the hypothesis that non-symbolic processing influences arithmetic via symbolic processing, the current longitudinal evidence provides support for the causal claims made based on cross-sectional studies . By coveIn contrast to a number of recent studies, we found a significant contribution of early non-symbolic processing to growth in symbolic processing skills half a year later even after controlling for a variety of general cognitive variables. A number of other longitudinal studies with kindergarten and first grade children failed to find a similar contribution . It is nThe sequential mediation model presented here also critically extends empirical evidence on the pivotal role of understanding single- and multi-digit Arabic numbers for arithmetic development. As predicted, the ability to process multi-digit numbers was scaffolded onto single-digit number processing. This finding supports the proposed developmental trajectory.p = 0.061). Future research should test the hypothesis that the ability to represent non-symbolic magnitudes facilitates the acquisition of place-value understanding.As pinpointed previously , being aThe finding that in our model single-digit processing at T2 (middle of Grade 2) did not show a direct influence on arithmetic growth from T1 to T4 is probably due to the fact that we controlled for differences in symbolic processing at the onset of the study period. This means that the single-digit processing variable actually only reflects changes in task performance through a period of about 6 months. Variance in this variable was predicted by non-symbolic processing skills half a year earlier and in turn contributed significantly to processing of two-digit numbers. Its contribution to arithmetic growth from Grade 2 to Grade 4 was, however, indirect as a mediator of non-symbolic processing.Although a strong association between performance in single- and two-digit comparison tasks was found in the present study see also , these tThere has been an ongoing discussion on how to best assess non-symbolic magnitude processing skills . In the Similarly, it has been claimed that tasks specifically tapping into cardinal or ordinal number knowledge might be better mediators between non-symbolic processing and mathematical skills. We consider it highly plausible that development of counting plays a crucial role in the mapping of number words and Arabic digits onto non-symbolic processing skills . HoweverWe would also like to remind readers that the current sample was not randomly selected: at the onset of the study period, half of the participants were selected based on below-average performance in arithmetic development. It turned out that later on the majority of children displayed typical arithmetic performance. Still, the distribution of numerical processing and arithmetic skills may not correspond to the general population and replication with more representative samples is advisable.In summary, the evidence presented in our study reveals a significant role of non-symbolic magnitude processing in the development of arithmetic during primary school: we could demonstrate that non-symbolic processing skills impacted on growth of arithmetic skills by facilitating the acquisition of symbolic number processing. This evidence indicates that non-symbolic processing should be included as one of the foundational skills in theoretical models of mathematical development. It will also be important to further specify developmental trajectories within the domain of symbolic numerical processing, by, for instance, differentiating between simple processing of one-digit numbers and more complex processes involved in multi-digit processing.As the indirect effect exerted by non-symbolic processing was small, it seems unlikely that interventions exclusively targeting non-symbolic processing would show satisfactory effects on children’s arithmetic development . Howeverhttps://osf.io/dfbqj/.The dataset analyzed for this study can be found on the Open Sciene Framework at The study was conducted in accordance with the ethical principles of the World Medical Association Declaration of Helsinki. Legal guardians of the participating children gave their written informed consent before data collection.KL developed the study concept and study design and is responsible for data collection. SF performed the data analysis and interpretation under the supervision of HF. SF drafted the manuscript. HF and KL provided critical revisions. All authors approved the final version of the manuscript for submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this article, a new concept for general pathogenesis has been proposed. Advances in molecular genetics have led to the realization that essential concepts in the framework of molecular biology are still missing. Clinical medicine is plagued by similar shortcomings: The questioning of current paradigms could open new vistas and invite challenging approaches. This article presents an unconventional idea. Foreign DNA which is regularly ingested with the essential food supply is not completely degraded. Small quantities of fragmented DNA rather persist transiently in the gastro-intestinal tract of mice and can be traced to various organ systems, except for cells in the germ line. Foreign DNA entering and persisting in mammalian cells can stochastically lead to genome-wide alterations of transcriptional and CpG DNA methylation profiles. In the course of food-ingested DNA invading somatic cells, completely new cell types can be generated which might be involved in the causation of common ailments. Projects emanating from this perception merit critical analysis and rigorous pursuit. Many of the common human diseases can be considered bad luck ailments  with uncThe invasion of foreign DNA into mammalian cells, particularly in its genomically integrated configuration, can lead to genome-wide alterations of the cells’ transcriptional and DNA methylation profiles –8.Fragments of foreign DNA, which are periodically taken up with the required food supply of mammals, survive transiently the passage through the gastro-intestinal (GI) tract. As documented in a mouse model , 10, theBased on and expanding from these two sets of experiment-based data, I submit that cells in an organism which are constantly and stochastically exposed to the intrusion of considerable amounts of food-derived foreign DNA will alter their function in the wake of altered transcription and CpG DNA methylation patterns. These alterations might become causative (co)factors for many of the common diseases, including neoplasias. Both events, fundamental changes in cell function and the occurrence of common diseases, have stochastic elements in common.In this article, two intertwined observations on the fate of foreign DNA in mammalian systems  and theiThe essential experimental findings which support the author’s proposal will be briefly summarized in the following sections. The details of these data have been published –11.In a hamster cell line transgenomic for about twelve copies of chromosomally integrated human adenovirus type12 (Ad12) DNA, the levels of cellular CpG DNA methylation are altered genome-wide . SimilarA different experimental scenario was devised to challenge the stability of cellular transcriptional and DNA CpG methylation profiles in individual cell clones of human colon tumor HCT116) cells upon the insertion of a 5.6 kbp bacterial plasmid. Single-cell clones were generated which contained either no transgenome or the 5.6 kbp plasmid DNA which carried the kanamycin resistance gene for clonal selection as sole transcribed element. In the subsequently expanded cell clones, differences in transcriptional and CpG methylation patterns between transgenomic and non-transgenomic cell clones were determined. Transcriptional and CpG methylation profiles of all transcripts investigated in five clonal cell populations of non-transgenomic controls were almost identical. This finding facilitated comparisons of the differential gene segment expression between non-transgenomic and transgenomic cell clones in 28,869 genomic DNA segments: 1343 showed differential expression with 907 regions up- and 436 regions downregulated [6 cells uFurthermore, when CpG DNA methylation profiles in the same sets of non-transgenomic versus 5.6 kbp plasmid-transgenomic HCT116 cell clones were assessed in 361,983 CpG dinucleotide positions, 3791 CpGs were found differentially methylated in the transgenomic cell clones, and 1504 proved hyper- and 2287 hypo-methylated. Hence, both transcription and CpG methylation profiles were altered in human cells by genomically inserting a 5.6 kbp DNA plasmid  to exclufragile X mental retardation gene 1 (FMR1) and the CpG hyper-methylation in its promoter region. In the region upstream of this promoter, we have identified a distinct boundary between unmethylated genome segments, as they exist in unaffected individuals, and DNA sequences further upstream from the promoter which are strongly CpG methylated. In FXS individuals, the methylation boundary is obliterated; the promoter becomes CpG hyper-methylated and inactivated. As a consequence, the FMR1 gene product fails to be produced; its lack during an individual’s development leads to the FXS syndrome. The methylation boundary is thought to exhibit a specific chromatin structure which demarcates a hyper-methylated upstream sequence from the unmethylated downstream FMR1 promoter. The boundary sequence, as it were, protects the promoter from the spreading of DNA methylation and thus helps maintain its activity [In a different biological system a similar observation was made on changes in DNA methylation patterns in cells which carried foreign DNA. One of the most frequently occurring forms of mental retardation in humans is the Fragile X Syndrome (FXS) . It is mactivity .EBV or with the human telomerase gene, the boundary is maintained. However, the region far upstream from the boundary becomes hypo-methylated by a factor of 4 [EBV DNA genome or the integrated telomerase gene [EBV [In normal human PBMCs, the far upstream genome region beyond the boundary is hyper-methylated. In PBMCs immortalized by transformation with tor of 4  in PBMCsase gene , 15. Desene [EBV .Although many questions remain as to mechanisms involved and the generality of alterations in transcriptional and CpG DNA methylation profiles, the ensemble of data presented raises the question of to what extent the introduction of foreign DNA into mammalian cells destabilizes the epigenetic landscapes in mammalian cells. By the same token, due to the mentioned epigenetic sequelae of foreign DNA intrusion, functionally completely new cell types arise and likely impact on the mechanisms causing common diseases. The stochastic nature of disease incidence is paralleled by the stochasticity of the molecular events linked to foreign DNA intrusion and intranuclear fixation.n = 84). Between 2 and 8 h after feeding, fragments of up to 1.700 bp were retrieved in PBMCs, and in spleen or liver cells up to 24 h after feeding (n = 254) both by PCR and fluorescent in situ hybridization (FISH) [IgE receptor gene [In a mouse model, we initially followed orally administered bacteriophage M13mp18 DNA to determine the fate of food-ingested foreign DNA in a mammalian organism. Fragments of this DNA were detectable in all parts of the gastro-intestinal (GI) tract of mice between 18 and 24 h after feeding (n (FISH) . About 1tor gene . The freribulose-1,5-bisphosphate carboxylase (Rubisco) gene was assessed in the mouse organism and found in different parts of the GI tract, in spleen and liver DNA [Rubisco gene could not be detected. Additionally, mice were continuously fed plasmid DNA carrying the cloned green fluorescent protein (GFP) gene for eight generations. Subsequently, DNA from tail tips or internal organs of the offspring was examined by PCR in each of the eight generations for germ-line transmission of orally administered GFP DNA. Results were consistently negative arguing against germ-line entry of orally fed foreign DNA. In controls, GFP DNA administered by intramuscular injection was detected in several organ systems remote from the injection site [In an extension of these studies, soybean leaves were fed to mice, and the distribution of the plant-specific, nucleus-encoded iver DNA . Transcrion site .This ensemble of data supports the conclusion that orally administered foreign DNA persists upon passage through the GI tract and reaches several organ systems of the test animals. Transcripts of the foreign DNA were not detectable. Foreign DNA continuously administered orally over eight generations of mice had not penetrated into their germ lines. We pursue the possibility that DNA from the food supply manages to enter the murine organism and can be recovered in fragmented form from several organ systems, except from cells in the germ-line.Foreign DNA when introduced into mammalian cells in the episomal or chromosomally integrated state can elicit genome-wide alterations of the cell’s transcriptional and DNA CpG methylation profiles –8. PotenThere is a constant flow of foreign DNA fragments derived from the broad spectrum of food-ingested DNA which can be distributed to many cells in mammalian organisms with the exception of germ cells , 10. It Hypothesis: Food-ingested DNA is capable of intruding into diverse somatic cell types, which fundamentally alters these cells and thus contributes to general pathogenetic mechanisms in the organism. In contrast to plant DNA, DNA from animal-derived food might have higher pathogenetic potential since mammalian DNA likely can more frequently recombine with human DNA due to its closer evolutionary relatedness to human DNA.Two major results have been reported:The same wake-up call can be raised for the consequences of all types of genome manipulations, e.g. by regimens of gene therapeutic approaches, by knock-out or the CRISPR-Cas9 strategies when applied directly to human organisms of all ages. Many of these approaches involve the import of foreign DNA into cells and manipulations at the genome level. At this stage, direct experimental evidence has not yet been adduced for the above-elaborated mechanisms to be operative in pathogenesis. However, an argument can be made that such evidence has not been sought vigorously enough, particularly in gene technologically altered cells.It will not be feasible to verify this unconventional concept directly in a one-step approach. I opt for proceeding with a stepwise strategy. Currently, the following goals are being pursued:It will be cogent to revisit the project of following food-ingested DNA in mice with improved technology which was not available 20 plus years ago , 10. In In addition, experiments will be repeated in which we succeeded in molecularly re-cloning food-ingested DNA fragments from the host’s spleen DNA and in determining the abutting host DNA from the integration site . The pre


Staphylococcus aureus (HA-MRSA), extended-spectrum β-lactamase-(ESBL)-producing Klebsiella pneumoniae, ESBL-producing Escherichia coli and Clostridioides difficile. The overwhelming part of the literature investigating the epidemiology of the pathogens as a function of fluoroquinolone use remain in concordence with these findings. Moreover, recent in vitro data clearly show the potential of fluoroquinolone exposure to shape the clonal evolution of Salmonella Enteritidis. The success of the international clones/STs in all these species was linked to the strains’ unique ability to evolve multiple energetically beneficial gyrase and topoisomerase IV mutations conferring high-level resistance to fluorquinolones and concomittantly permitting the acquisition of an extra resistance gene load without evoking appreciable fitness cost. Furthermore, by analyzing the clonality of multiple species, the review highlights, that in environments under high antibiotic exposure virulence factors play only a subsidiary role in the clonal dynamics of bacteria relative to multidrug-resistance coupled with favorable fitness (greater speed of replication). Though other groups of antibiotics should also be involved in selecting clones of bacterial pathogens the role of fluoroquinolones due to their peculiar fitness effect remains unique. It is suggested that probably no bacteria remain immune to the influence of fluoroquinolones in shaping their evolutionary dynamics. Consequently a more judicious use of fluoroquinolones, attuned to the proportion of international clone/ST isolates among local pathogens, would not only decrease resistance rates against this group of antibiotics but should also ameliorate the overall antibiotic resistance landscape.It is well-established that the spread of many multidrug-resistant (MDR) bacteria is predominantly clonal. Interestingly the international clones/sequence types (STs) of most pathogens emerged and disseminated during the last three decades. Strong experimental evidence from multiple laboratories indicate that diverse fitness cost associated with high-level resistance to fluoroquinolones contributed to the selection and promotion of the international clones/STs of hospital-associated methicillin-resistant  Staphylococcus aureus (HA-MRSA) the New York-Japan clone (ST5) in the 1990s (Clostridioides difficile (Enterococcus faecium (VRE)  pathogens during the last three decades. While polyclonal situations also occur a few highly successful international clones/sequence types (STs) of various MDR pathogens achieved dominance in multiple geographic regions replacing local strains and posing a serious challenge for the treatment of patients. The advent of the nowadays principal MDR clones commenced with the widespread dissemination of the first international ST of hospital-associated methicillin-resistant he 1990s  which waifficile , the intum (VRE) , and theeumoniae  and ESBLhia coli . Subsequaumannii  and most cloacae .The question arises what could have triggered the metamorphosis of the clonal landscape of MDR pathogens during this time period that resulted in the dominance of a few recently disseminated global “superbugs”?C. difficile in no the other species showed MDR major clone/ST strains greater virulence relative to minor clone isolates and this observation argues strongly against a virulence-based dissemination. Virulence factors have been rightly suspected by many authors to have contributed to the emergence of the MDR clones/STs in various pathogens. However, with the exception of the ribotype 027 clone of 1) associated with high-level resistance to fluoroquinolones was demonstrated to confer an indirect growth advantage onto the international clone/ST strains of HA-MRSA . It “was first identified in 1987” as “a minor strain accounting for less than 5% of isolates on surveys, without apparent association with large outbreaks or increased lethality”  associated with high-level resistance to fluoroquinolones . STs sufMoreover, even the fittest STs are not equal and subtle differences between their speed of replication were also reflected in clonal dynamics. The ST228 clone – that was shown to command inferior fitness associated with resistance to fluoroquinolones relative to the ST5 and ST22 clones  – was reThe observed “fitness influence” may also account for the characteristic community affiliation of community-associated (CA)-MRSA strains. The substantial fluoroquinolone-associated fitness cost demonstrated with various CA-MRSA STs  should pInterestingly fitness cost associated with high-level resistance to fluoroquinolones is not static. Some strains from the ST8 clone showed remarkable resilience and proved capable of evolving into a novel major HA-MRSA lineage in the United States .gyrA Ser84 and grlA Ser80 residues. These double-serine alterations are characteristic features of both highly successful international HA-MRSA groups of CC5 and ST22 (The ST8 clone (USA300) was previously considered CA-MRSA, however, a new lineage of the pathogen has recently emerged replacing strains from other STs in the healthcare setting . The reaand ST22 .gyrA Ser84 and grlA Ser80 (or corresponding) residues are not optimal for the function of the enzymes and their replacement by some other amino acids is associated with a modest fitness gain in the isolates, in a clone-dependent fashion [reviewed by The replacement of the double-serine residues in the DNA gyrase and topoisomerase IV enzymes involved in the binding of fluoroquinolones is crucial for the preservation of considerable fitness at high MIC values to fluoroquinolones. It was demonstrated in various species that the The question arises if the double-serine residues are not optimal for the function of gyrase and topoisomerase IV why have these residues been evolutionarily preserved?S. aureus that these serine residues (in codon 84 in gyrase and codon 80 in topoisomerase IV) confer protection against antibacterial substances of herbal and Streptomyces origin (Japanese scientists demonstrated in s origin  permittigrlA Ser80) the novel strains harbored also a gyrA Ser84 change in a similar fashion to the ST5 and ST22 clones  strains were either susceptible to fluoroquinolones or carried just one of the serine mutations  of the major international clone strains of HA-MRSA relative to minor clone isolates has serious practical implications. Since these strains multiply faster than isolates from other clones they are expected to achieve higher rates upon occupying novel niches and replacing the local pathogens. This is really what we have been witnessing for more than two decades. The replacement of minor clone strains of HA-MRSA by faster replicating international ST strains showing higher growth rates is well-documented to trigger a significant rise in the incidence of the pathogen .in vitro propagation assays that the growth rate of international HA-MRSA clone isolates is higher than that of minor ST strains. Since in many countries a large proportion of the HA-MRSA strains belong to the fluoroquinolone-selected major STs a decrease in the use of fluoroquinolones should result in a decline of these major clone strains yielding lower incidences. The overwhelming part of the literature published until 2016 investigating a link between the incidence of HA-MRSA and fluoroquinolone consumption support the existence of an association. The previous literature on the area was reviewed by one of us , with a 3-month lag time  plays a major – probably crucial – role in the clonal dynamics of MRSA in the healthcare setting in adult facilities. However, the contribution of pathogenicity to the epidemiology of MRSA is rightly considered relevant and therefore we must evaluate the significance of virulence factors relative to replication in the clonal dynamics of the pathogen.Strains from successful international clones/STs of HA-MRSA are in general less virulent than isolates of CA-MRSA which are conversely more susceptible to antibiotics.sccmec-IV/V elements (elements . These oS. aureus on the skin of patients was correctly linked to the success of the ST8 (USA300) CA-MRSA clone in the United States (The “arginine catabolic mobile element” (ACME) enhancing survival of d States . Howeverd States . MoreoveNevertheless, the carriage of ACME should certainly confer an advantage to antibiotic susceptible CA-MRSA strains facilitating transmission in a fluoroquinolone-free environment .Biofilm is also rightly considered a relevant determinant of clonal dynamics. However, observations published in the related literature show it plays only a secondary role to fitness, i.e., speed of replication.Though the production of biofilm was suggested to promote the dissemination of the ST22 (EMRSA-15) clone the general biofilm-producing capacity of the ST22 isolates remain inferior compared with those of ST228 and ST8 strains  which thBiofilm production was observed to advance the dissemination of ST59 MRSA in the community and also in the healthcare setting in pediatric wards which are supposed to be “fluoroquinolone-free” . These Spsm-mec gene carried by most strains of HA-MRSA on the SCCmec II and III cassettes is responsible for the attenuation of virulence (The primacy of fitness (speed of replication) over virulence in MRSA is also reflected by the observation that the acquisition of methicillin resistance by HA-MRSA is associated with an overall downregulation of virulence gene expression linked to a repression of the activity of the accessory gene regulator system (agr) [reviewed by irulence .psm-mec-induced suppression of multiple virulence genes allowed HA-MRSA to acquire/evolve further antibiotic resistance mechanisms without suffering significant fitness cost permitting the pathogen’s survival in “high-antibiotic” areas.It is tempting to speculate that the favorable fitness effect gained from the double-serine QRDR mutations combined with the Strains from the fluoroquinolone resistant lineage of the ST8 MRSA clone in France were reported to carry significantly fewer virulence genes than the fluoroquinolone susceptible ST5 and ST80 isolates . Thus, sS. aureus is certainly the ST121 clone harboring a large “armament” of pathogenicity factors  MDR teration  – a measK. pneumoniae observed: “.all CG258 isolates have the fluoroquinolone resistance-conferring mutations in GyrA (Ser83 to Ile) and ParC (Ser80 to Ile),” which should have contributed to the global dissemination of carbapenem resistance.Furthermore the carriage of multiple QRDR mutations proved also characteristic for ST258 isolates. K. pneumoniae sequence type, ST101, showed that almost all of the strains carry three “classic” QRDR mutations supporting further the “fluoroquinolone concept”  in the clonal dynamics of MDR K. pneumoniae strains were reported from various continents in both outpatients and the general population with higher prevalence in developing countries  strains of MDR and ST65 . Interes setting . Moreoveonal STs . MoreoveK. pneumoniae. K. pneumoniae strains in the healthcare setting  yieded spectacular results. ST131 strains had been reported to dominate the E. coli landscape in the United Kingdom prior to 2007  their study design, unfortunately, did not allow for a solid estimate. First, just the “first ten isolates per site” were collected from each participating laboratory and authors themselves admit that the study could include strains from “local epidemics” .Though virulence factors beyond doubt make a relevant contribution to the success of E. coli showed a higher level of virulence in a Galleria mellonella model than strains from some other STs , E. coli in most countries. Moreover, similarly to MRSA and MDR K. pneumoniae, the proportion of the major ST131 H30 group remains much lower among fluoroquinolone susceptible E. coli strains relative to other STs  favorable QRDR mutations and start to extensively disseminate in fluoroquinolone environments.ST405 strains of ene load  and wereene load . Howeverene load  that is ene load  preventiE. coli. However, 71.1% of the non-ST131 isolates were also resistant to ciprofloxacin that should have been associated with a considerable fitness cost in these strains preventing the carriage of a substantial virulence gene load.Interestingly ST131 H30 strains are assigned to different virotypes determined by their virulence gene profiles . InteresThough virotype C strains of ST131 H30 strains remain most frequent a few groups reported a higher incidence for virotype A isolates . VirotypK. pneumoniae (see above) – may account for the circumstance. Class 1 integrons are common in E. coli  is responsible for the rapid replication of the major international ribotypes of C. difficile when showing high-level resistance to fluoroquinolones  fitness cost upon developing resistance to fluoroquinolones. In contrast some other QRDR mutations in the same isolate were associated with a considerable loss of robustness. Authors correctly predicted that the favorable fitness effect could have facilitated the dissemination of this particular clone of C. difficile.ibotypes  – into agyrA Thre82Ile mutation with that of their fluoroquinolone susceptible parent isolates void of the genetic alteration and experienced a fitness gain of 8–22% across the isolates. This paper attests that the fitness effect associated with a favorable QRDR mutation can be substantial and is both clone-, and strain specific.gyrA Thr82Ile mutation is also reflected by a clonal shift that occurred recently in South Korea. Korean scientists demonstrated that clones which are considered minor in Europe and carry an energetically less favorable mutation will become major clones/ribotypes when they are capable of evolving the Thr82Ile gyrA mutation and will replace other ribotypes harboring energetically detrimental gyrA alterations  was also observed.Moreover, in the United Kingdom a campaign was launched in 2007 to restrict the use of fluoroquinolones to reduce the incidence of ifficile . The camof years . Interesof years . A similgyrA Thre82Ile mutation) by minor clone isolates.Furthermore The observation that the incidence of ribotype 027 strains remains significantly lower in pediatric units (where fluoroquinolones are not supposed to be used) compared with adult wards  remains C. difficile are well-known to often remain susceptible to fluoroquinolones  (Since major ribotype strains feature prominently in e of us) .K. pneumoniae, MDR E. coli and C. difficile responded to the challenge: exclusively strains from a few international/global clones/STs proved capable of developing favorable QRDR mutations and emerged commanding superior fitness and replacing minor clone/ST strains in fluoroquinolone environments. The available literature strongly suggest that the major international clones/STs of additional MDR pathogens might also have been shaped by the fluoroquinolone effect. The related literature on MDR E. faecium (VRE) was recently reviewed  might have been selected by fluoroquinolone pressure in the veterinary sphere. Isolates in this international ST also carry multiple QRDR mutations including the double-serine alterations  (C. jejuni are known to have been selected by fluoroquinolones.d by us) . ConsequA. baumannii, Enterobacter cloacae and MDR Pseudomonas aeruginosa would deserve a thorough investigation.In addition, the involvement of fluoroquinolone resistance-associated fitness cost in the selection of the global/international clones of MDR In summary fluoroquinolones have been shown to select for the major international clones/STs of various MDR pathogens and have potentially affected all species which were exposed to these antibiotics for extensive time periods. Since the evolvement of the double-serine QRDR mutations were often associated with a fitness gain – though in a clone-dependent fashion – the assumption of high-level resistance to fluoroquinolones permitted the major clone/ST isolates to acquire resistance mechanisms against additional groups of antibiotics without suffering a substantial loss of vitality. This is in sharp contrast to minor clone/ST isolates which proved less capable of evolving energetically favorable QRDR mutations and – consequently – had to apply alternative mechanisms for developing resistance to fluoroquinolones, like enhanced efflux, that requires considerable energy. This was, however, associated with substantial fitness cost resulting in compromised growth that allowed the major clone/ST isolates to replace minor clone/ST strains in fluoroquinolone environments. The faster growth rate of these newly dominant major clone/ST isolates then brought about a steep rise in the incidence of MDR pathogens.K. pneumoniae. Furthermore, it should not be an accident that the clonal landscape of multidrug-resistant pathogens remains largely stable in the hospital sector. If minor clone strains could easily reverse the fitness cost associated with resistance to fluoroquinolones by evolving compensatory mutations novel international clones of various multidrug-resistant pathogens hailing from previous minor ST strains should regularly emerge. However, this is what we have not been witnessing. What we see is just the infrequent dissemination of previous minor clone strains which with some delay proved competent to evolve the energetically favorable QRDR mutations (see above).Moreover, the available data clearly show that fitness cost suffered by minor clone/ST strains can hardly be recovered by compensatory mutations. K. pneumoniae  will confer a considerable advantage onto a particular MDR isolate vs. other competing MDR strains from the same species showing superior virulence/colonization efficiency but inferior replication capabilities. Moreover, an MDR isolate with relatively lower virulence but almost “wild-type fitness” will also command considerable advantage against a highly virulent and fit strain that remains susceptible to antibiotics. Though the latter strain should transmit more readily it can be – in contrast to our “super bug” – easily controlled in the hospital setting with antibiotics.K. pneumoniae and MDR E. coli show less virulence than isolates from many other STs/lineages of the same species (see above). C. difficile remains an exception as ribotype 027 strains are highly toxic, however, the extensive dissemination of this clone was also the consequence of substantial fitness gain conferred by the gyrA Thr82Ile mutation  in community-acquired infections (see above).Nevertheless, the situation is different in some “low-antibiotic areas” in the community. In a “low-antibiotic” sphere pathogens can afford to remain less resistant and consequently retain fitness and harbor more virulence factors. Since in these areas antibiotics will usually not restrict the bacteria the virulence factors should confer a considerable advantage onto the pathogen that is reflected in the well-established high incidence of virulent and “non-major ST” strains .The fluoroquinolone effect implies that in a fluoroquinolone-free hospital environment the incidence of the international clone/ST strains should decrease. This is exactly what has been observed with both HA-MRSA and The impact of a reduction in the consumption of fluoroquinolones on the incidence of major clone pathogens from other species remains less conspicuous since the proportion of major clone/ST bacteria is smaller among these clinical isolates. Nevertheless, some positive effect should also be observed with other species. Further epidemiological studies involving the judicious use of fluoroquinolones and investigating the clonal composition of the local pathogens are needed.Finally, the judicial use of fluroquinolones will ameliorate resistance rates only if infection control measures are complied with and poor hygiene will not compromise the expected favorable outcome.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The manuscript was based on MF’s perception and he did much of the writing. JR and AT contributed significantly to both writing the manuscript and raising its professional standard by making relevant supplements.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Post-extubation stridor is a known complication of mechanical ventilation that affects a substantial number of all critical care patients and leads to increased morbidity and mortality. Common risk factors for the development of post-extubation stridor include female gender, older age, and prolonged length of mechanical ventilation. There may be an increased incidence of post-extubation stridor in patients who require mechanical ventilation to manage the respiratory complications of COVID-19. In this case series, we analyzed nine patients from across our institution who were intubated to manage acute respiratory distress syndrome (ARDS) secondary to COVID-19 and subsequently developed post-extubation stridor. The patients were predominantly females with prolonged intubations and multiple days of prone ventilation. While the patients in this case series possessed some of the well-described risk factors for post-extubation stridor, there may be risk factors specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that make these patients more susceptible to the complication. The cuff leak test was performed on the majority of patients in the case series and did not successfully predict successful extubation in this population. Our analysis suggests that prophylactic corticosteroids given in the 24-48 hours prior to elective extubation in female COVID-19 patients who were intubated for more than six days with consecutive days of intermittent prone ventilation may be helpful in reducing the incidence of post-extubation stridor in this population. Overall, this case series elucidates the need for exceptionally close monitoring of COVID-19 patients upon extubation for the development of stridor. Post-extubation stridor affects approximately 10% of all critically ill patients and is associated with re-intubation, prolonged duration of mechanical ventilation, and increased need for tracheostomy placement . In thisWe identified nine patients from our institution who developed post-extubation stridor after intubation for ARDS secondary to COVID-19. During the interval that these patients were extubated, a total of 20 COVID-19 patients were extubated at our facility revealing an incidence of 45%. The patients were predominantly female with prolonged intubations and multiple days of prone ventilation. Four patients received systemic steroids for an indication other than laryngeal edema during their initial intubation with the remainder not receiving systemic steroids until they developed post-extubation stridor. Prior to their initial extubation, seven patients were noted to have a cuff leak. Six patients were initially extubated to either high flow nasal cannula or noninvasive ventilation with one of these patients avoiding re-intubation. In total, eight patients required re-intubation with four ultimately requiring tracheostomy , obesity (6/9 had BMI >26.5), and female gender (7/9) . There wAnother interesting correlation was that all but one of the patients in this case series required consecutive days of intermittent proning. In the 2013 Proning Severe ARDS Patients (PROSEVA) trial, which randomized 237 ARDS patients to intermittent proning, there was not a significant increase in post-extubation stridor reported in complications . It is nThere may be risk factors specific to COVID-19 that increase the prevalence of post-extubation stridor in this population. During the interval that these nine patients were extubated, a total of 20 COVID-19 patients were extubated across our institution. This is notable considering that post-extubation stridor affects less than 10% of all critically ill patients . A studySeven of the patients were noted to have a qualitative cuff leak prior to extubation, with three of them having a documented quantitative cuff leak of 30%, 24%, and 70%, respectively. Prior data correlate a successful extubation with a quantitative cuff leak of greater than 10%-15% . HoweverWith the identification of potential risk factors, a focus can shift to proposed methods of prevention. While post-extubation stridor is often treated with systemic steroids and racemic epinephrine, the utility of prophylactic treatment prior to extubation with either agent has not been formally established. A meta-analysis on the use of prophylactic corticosteroid administration to decrease post-extubation stridor concluded that the administration of corticosteroids before elective extubation was associated with a significant reduction in the incidence of post-extubation stridor . Only thLimitations of our study include the fact that our observations of stridor are mostly based on subjective assessments rather than objective findings of airway edema seen on direct visual inspection or imaging. Additionally, there are a relatively small number of patients in our case series. Post-extubation stridor in COVID-19 patients is an important clinical entity that has not yet been described in detail in literature and warrants further study. This case series elucidates the need for close monitoring of COVID-19 patients upon extubation for the development of stridor. Contributing to an increased prevalence of this complication may be risk factors specific to COVID-19, with viral-induced laryngeal inflammation being a distinct possibility. The presence of a cuff leak did not predict successful extubation and may be deferred to minimize aerosolization of the virus. To mitigate the risk of post-extubation stridor, the use of prophylactic corticosteroids in the 24 to 48 hours leading up to extubation in females with a ventilator time greater than six days and who have required consecutive days of intermittent proning may be helpful. With mortality rates of COVID-19 patients who require mechanical ventilation being exceptionally high, the reduction of any complication is paramount to the success of treating patients and reducing morbidity and mortality.


The diagnosis of pRBD was based on the 10-item RBD Screening Questionnaire. Striatal DAT binding levels and their rates of decline were compared between patients with pRBD and those without. At baseline, patients in the PD-pRBD+ group showed lower striatal DAT binding in the caudate (which was more pronounced in the less-affected hemisphere) and in the putamen. During the 4-year follow-up, patients in the PD-pRBD+ group consistently exhibited greater DAT loss than patients in the PD-pRBD− group with comparable disease duration in all four striatal subregions. These patients also exhibited a more rapid decrease in DAT binding in the caudate and a less prominent interhemispheric asymmetry in the putamen. The distinct pattern of striatal DAT depletion may contribute to a more malignant phenotype of PD associated with RBD, specifically faster progression of motor symptoms.The current study aimed to confirm whether probable rapid eye movement sleep behavior disorder (pRBD) is associated with a specific pattern of striatal dopamine depletion in an international, multicenter, prospective cohort of patients with Parkinson’s disease (PD). Two hundred and seventy  Rapid eye movement (REM) sleep behavior disorder (RBD) is a parasomnia characterized by elaborate motor manifestations related to unpleasant dreams and loss of muscle atonia during REM sleep . RBD affPrevious work investigating the pathogenesis of the disease has demonstrated that the presence of RBD is associated with more severe nigrostriatal dopaminergic lesions in patients with PD . Howeverde novo, drug-naïve PD patients.In the present study, we aimed to investigate whether the presence of RBD is associated with a more severe dopaminergic deficit and a faster decline in dopaminergic binding as PD progresses. We compared baseline and serial DAT levels in patients with and without probable RBD (pRBD) in an international, multicenter, prospective cohort study of de novo and drug-naïve PD patients. Details of the study have been published elsewhere (1. Participants meeting the following criteria were recruited between 2010 and 2015: recent diagnosis of PD (less than 2 years); no past treatment with dopamine replacement therapy; and presenting with at least two of the following: bradykinesia, resting tremor, and rigidity, or with asymmetric resting tremor/bradykinesia at screening. PD diagnosis was confirmed by imaging of striatal DAT deficits at enrollment. All patients underwent a comprehensive longitudinal schedule of clinical and imaging assessments. Data used in our study were obtained from the PPMI database2.The Parkinson’s Progression Markers Initiative (PPMI) is an international, multicenter, prospective cohort study of lsewhere  and are 123I-FP-CIT SPECT [123I-2β-carbomethoxy-3β-(4-iodophenyl)-N-(3-fluoropropyl)-nortropane] imaging was performed at participating PPMI sites and acquired images were sent to the Institute of Neurodegenerative Disorders  for quality control and data extraction3. SPECT image volumes were spatially normalized to standard Montreal Neurologic Institute space using the PMOD software . Next, the transaxial slice with the highest striatal uptake was identified and the eight hottest striatal slices around it were averaged to generate a single slice image. Regions of interest were then placed on the bilateral caudate, putamen, and the occipital cortex (reference tissue). The more affected hemisphere was defined as the contralateral one to the side of predominance of motor symptoms. Striatal binding ratios (SBRs) were calculated for each of the four striatal regions (the target region count density/the reference region count density-1). The differences of DAT binding between bilateral hemispheres were calculated to assess interhemispheric asymmetry of the striatal dopaminergic denervation.Dopamine transporter imaging took place across four visits: at screening, and 1, 2, and 4 years following the baseline scan. We included only patients with intact DAT imaging data from all four visits in this study.The presence of pRBD was assessed using the 10-item RBD Screening Questionnaire (RBDSQ) . RBDSQ sThe Movement Disorders Society-Unified Parkinson’s Disease Rating Scale Part III (MDS-UPDRS-motor) and the Montreal Cognitive Assessment (MoCA) were assessed at DAT scanning . For patThe study was approved by the institutional review board at each PPMI site. All patients signed informed consent forms prior to their participation in the PPMI study.t-test, Mann–Whitney U test, or Pearson Chi-squares, where appropriate. Repeated measures analysis of variance (ANOVA) with time as a within-subject factor and group as a between-subject factor was used to examine the effect of pRBD on the longitudinal changes in striatal DAT binding and interhemispheric asymmetry. A Greenhouse–Geisser correction was used for sphericity. Spearman’s correlation analysis was used for correlation analysis. The mean DAT binding in the putamen or in the caudate was calculated by averaging DAT binding levels of two hemispheres. The decrease rate of DAT binding was calculated as the difference between DAT levels at the last follow-up scan and at the baseline scan, divided by DAT level at the baseline scan. p < 0.05 was considered statistically significant. All data are presented as mean ± SD. Analyses were performed with SPSS version 17 .Comparisons between groups were performed using independent samples n = 135). Patients with RBDSQ scores of six or more at only one of the four visits were considered borderline (n = 37) and excluded from the analysis. The remaining 98 patients with RBDSQ scores of six or higher on more than one visit were classified as the PD-pRBD+ group. No significant differences were observed in sex, age, years of education, age of onset, and side of onset at baseline between the two groups.A total of 270 patients  were included in this study. Patients with RBDSQ scores of five or less at all four visits were classified as the PD-pRBD− group (p = 0.838). However, patients in the PD-pRBD+ group exhibited higher initial UPDRS-motor and lower initial MoCA scores compared to those in the PD-pRBD− group . During follow-up, patients in the PD-pRBD+ group also exhibited worse MoCA and MDS-UPDRS-motor scores, and tended to be treated with higher LEDD at the time of DAT scans, although the differences were not statistically significant. Demographic and clinical characteristics of the two groups are shown in As all participants were newly diagnosed (less than 2 years), the two groups were comparable in terms of disease duration  compared to those in the PD-pRBD− group. These patients also exhibited lower striatal DAT binding in the caudate, with the difference being more pronounced in the less affected side (p = 0.007) than in the more affected side (p = 0.167).At baseline, patients in the PD-pRBD+ group showed lower striatal DAT binding in the putamen  and a significant group × time interaction in the caudate .Repeated-measures ANOVA revealed main effects of time and group on DAT binding levels in all studied subregions  = 5.3, p = 0.001] and in the putamen , and a main effect of group in the putamen , suggesting that interhemispheric asymmetry decreased with time and was lower in the PD-pRBD+ group than in the PD-pRBD− group.Repeated-measures ANOVA on interhemispheric asymmetry revealed main effects of time in the caudate [r = −0.229, p < 0.001) and in the caudate , while initial MoCA scores were not significantly correlated to DAT binding in any subregions. Initial RBDSQ scores correlated to initial UPDRS-motor scores  and inversely to initial MoCA scores .Using correlation analyses, we found that initial UPDRS-motor scores correlated inversely to mean DAT binding in the putamen  and in the caudate .Significant inverse correlations were also found between initial RBDSQ scores and mean DAT binding in the putamen .The data analyzed in this study were obtained from the Parkinson’s Progression Markers Initiative (PPMI) database (The study was approved by the institutional review board at each PPMI site. All patients signed informed consent forms prior to their participation in the PPMI study.RC, XC, and PH contributed to the data acquisition and statistical analysis. RC, CX, and KW made substantial contributions to the conception and design of the work. All authors contributed to writing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Three-dimensional porous nanostructures made of noble metals represent novel class of nanomaterials promising for nonlinear nanooptics and sensors. Such nanostructures are typically fabricated using either reproducible yet time-consuming and costly multi-step lithography protocols or less reproducible chemical synthesis that involve liquid processing with toxic compounds. Here, we combined scalable nanosecond-laser ablation with advanced engineering of the chemical composition of thin substrate-supported Au films to produce nanobumps containing multiple nanopores inside. Most of the nanopores hidden beneath the nanobump surface can be further uncapped using gentle etching of the nanobumps by an Ar-ion beam to form functional 3D plasmonic nanosponges. The nanopores 10–150 nm in diameter were found to appear via laser-induced explosive evaporation/boiling and coalescence of the randomly arranged nucleation sites formed by nitrogen-rich areas of the Au films. Density of the nanopores can be controlled by the amount of the nitrogen in the Au films regulated in the process of their magnetron sputtering assisted with nitrogen-containing discharge gas. Three-dimensional 3D) percolated porous nanostructures made of noble metals and having large surface-to-volume ratio have drawn significant attention due to their remarkable physicochemical properties allowing to use them for various important applications ranging from the photo- or electro-catalysis, water splitting, hydrogen storage to bio- and chemosensing via surface-enhanced effects ,2,3,4,5.D percolaRecently, optical properties of 3D porous nanostructures have become a hot topic ,7,8,9,10State-of-the-art methods for porous nanostructure fabrication generally require complicated multi-step fabrication protocols as dealloying and soft- or hard-template synthesis ,25,26,27Herein, we applied scalable easy-to-implement nanosecond (ns) laser ablation of nitrogen-rich Au films to fabricate parabola-shaped nanobumps containing multiple nanopores inside. The nanopores were found to originate from laser-induced explosive boiling and coalescence of the nucleation sites formed by nitrogen-rich areas of the Au film, while the nanopore density can be controlled by amount of nitrogen used as discharge gas for magnetron sputtering of the Au films. Most of the nanopores hidden beneath the nanobump surface can be further uncapped using gentle etching of the nanobumps by an Ar-ion beam to form 3D plasmonic nanosponges promising for various nonlinear optical and sensing applications.Au films with a thickness of 150 ± 5 nm were deposited onto silica glass substrates without any adhesion sub-layer using a custom-built magnetron sputtering system and three discharge gases: argon, nitrogen and purified air. Deposition was performed at 10s and Au4f regions.The actual thickness and average roughness of the films were controlled by an atomic-force microscopy . Optical spectroscopic measurements performed with an integrating sphere spectrometer confirmed the identical reflectance for all Au films evaporated with different discharge gases . The surface chemical composition of the Au films was carefully studied with X-ray Photoelectron Spectroscopy (XPS). XPS spectra were collected using a Kratos Axis Nova instrument  with a monochromatic Al KPrecise pointed ablation of the Au films was performed with second-harmonic (wavelength of 532 nm), ns (pulse duration of 7 ns) laser pulses generated by an Nd:YAG laser system . The laser radiation was focused into a sub-micrometer spot on the sample surface using a dry objective  with a numerical aperture NA of 0.8  at acceleration voltage of 3 kV, gas flow of 0.15 cm ≈1 nm/s , allowinScanning electron microscopy (SEM) was performed with a Helios Nanolab 450 FIB-SEM . High-resolution surface characterization was conducted at an accelerating voltage of 5 kV and electron beam current 100 pA. Signal channels with secondary (SE) and back-scattered electrons (BSE) were simultaneously collected and analysed. Despite the lack of topographical information, the escape depth for BSE is greater than that for SE improving material/density sensitivity. This allows visualisation of the nanopores under the surface with high contrast and spatial resolution.To shed light onto the internal structure of the nanobumps (nanosponges), we involved a focused ion beam technique to prepare single cross-sectional cuts and serial cuts that were subsequently combined into a 3D reconstruction. After defining the area of interest, deposition (starting from electron beam-induced deposition to prevent surface damage and followed ion beam-induced deposition) of the Pt protective layers was performed. The thickness of the layers was chosen taking into account the morphology and smoothness of the certain laser-printed structure and varied between 150 nm (for nanobumps) and 1000 nm (for through holes). Slicing was carried out using an Ga-ion beam at an accelerating voltage of 30 kV and beam current of 30 pA. After producing subsequent FIB cut, high-resolution SEM image was automatically acquired at 5 kV and 50 pA. The resulting image had a field of view of Three-dimensional finite-difference time-domain (FDTD) calculations were undertaken to reveal local structure of the EM fields excited near the isolated plasmonic nanosponge by linearly-polarized laser pump at 532, 632 and 1030 nm. The pump wavelengths within rather broad spectral range were chosen to highlight potential applications, where the enhanced and localized plasmon-mediated fields are highly demanding including SERS substrates and enhancement of nonlinear optical effects. Representative 3D model of the nanosponge was reconstructed using high resolution top- and side-view SEM images. The linearly polarized laser radiation was modeled to pump the nanosponge from the top. Elementary cell size was ata from .In this paper we considered three types of glass-supported Au films produced via magnetron sputtering in various discharges gases—argon, nitrogen and purified air (≈80% of nitrogen). Produced films had the same thickness  of the closest nanopores growing from neighbouring randomly distributed nucleation centers. Origin of such nucleation centers will be discussed somewhat later in the paper in the context of chemical composition of the metal film fabricated with different discharge gases. Earlier coalescence was suggested as a leading mechanism of the water bubbles growing on dispersed gold nanoparticles heated by incident laser radiation ,37. As t tension . Using Nuid gold , and δh∼This time can be estimated assuming that the heat accumulated by the illuminated spot is removed mostly by the thermal conductivity. The heated volume can be estimated as a cylinder of the radius  studies . Hence, R grows with time as Noteworthy, different growth times of the pores can be also considered as alternative way to explain the large size variation of the nanopores. In particular, the pores started growing short after the onset of the melting had more time to develop than that ones started just before the resolidification. This assumption cannot be accepted because of the low growth velocity of the bubbles. Wang et al.  reportedIn a similar way, the broad size distribution can not be explained by local temperature fluctuations (pores at hot spots grow quicker) as the (t)∝T1/3 . Hence, (t)∝T1/3 . The ave(t)∝T1/3 . This caRemarkably, the density and the average size of the nanopores started to grow from randomly distributed nucleation centers were found to depend on the type of discharge gas used for the film fabrication. More specifically, negligible amount of nanopores was found for the nanobumps produced on the surface of Au films sputtered with Ar see d. The mas region revealed near-zero signal for films produced with Ar discharge gas. However, a pronounced signal for Au films fabricated with purified air and nitrogen . Analyses of the deconvolved high resolution spectra taken in the N1nitrogen b–d were nitrides ,42,43. AConsidering the chemical composition of the Au film produced with different discharge gases we can suggest the following scenario regarding formation of the nanopores upon ns-pulse laser ablation. Such laser pulse with a near-threshold fluence rapidly heats up the Au film to temperatures above 10the film , while pThe general geometry of the surface structure produced by direct laser ablation is defined by the laser irradiation parameters  as wellHere, we propose a simple approach allowing fabrication of porous plasmonic nanostructures using direct ns-laser ablation followed by Ar-ion beam etching. The nanopores were found to form through the explosive evaporation/boiling of the nitrogen-rich metal film areas exposed by a ns laser pulse. Detailed XPS and SEM analysis confirmed that the density of the nanopores correlates with the initial amount of nitrogen in the Au films that were fabricated by magnetron sputtering assisted with different discharge gases. Demonstrated unique porous nanostructures, parabola-shaped nanobumps perforated with multiple nanoholes, are expected to be useful for various applications where the plasmon-mediated EM fields are of mandatory importance as nonlinear plasmonic and chemo-/biosensing.In a broader context, advanced chemical engineering of the metal film composition suggested in this paper being combined with an adjustment of the laser ablation process  is expected to provide facile way for fabrication of unique nanostructures. Also, elaborated strategy being applied to more complicated material combinations  will further enrich the potential types of nanostructures as well as their application range ,54,55. F


Determining the relative contributions of functional genetic categories is fundamental to understanding the genetic etiology of complex human traits and diseases. Here, we present Annotation Informed-MiXeR, a likelihood-based method for estimating the number of variants influencing a phenotype and their effect sizes across different functional annotation categories of the genome using summary statistics from genome-wide association studies.Extensive simulations demonstrate that the model is valid for a broad range of genetic architectures. The model suggests that complex human phenotypes substantially differ in the number of causal variants, their localization in the genome and their effect sizes. Specifically, the exons of protein-coding genes harbor more than 90% of variants influencing type 2 diabetes and inflammatory bowel disease, making them good candidates for whole-exome studies. In contrast, <10% of the causal variants for schizophrenia, bipolar disorder and attention-deficit/hyperactivity disorder are located in protein-coding exons, indicating a more substantial role of regulatory mechanisms in the pathogenesis of these disorders.https://github.com/precimed/mixer.The software is available at: Bioinformatics online. The rapid technological advances of the last years have provided an enormous amount of genetic data, promoting the development of statistical methods aimed at unraveling the genetic architecture of complex traits Holland . ImportaRecently, we developed a model which allows the breakdown of SNP-heritability into the number of variants influencing a given phenotype (non-null variants) and the distribution of their effect sizes using summary statistics from GWAS and detailed population-specific linkage disequilibrium (LD) structure  analysis  only estHere, we extensively tested AI-MiXeR on synthetic GWAS data generated under various setups to establish scenarios where it reconstructs the underlying parameters correctly. We then applied AI-MiXeR to GWAS summary statistics for 11 complex phenotypes representing a range of diverse human traits and diseases. Our analysis suggests that both densities and effect sizes of non-null variants vary considerably across different genomic annotation categories and reveals diverse patterns of genetic architecture in different phenotypes.π) and the variance of their effect sizes . The pure (i.e. not induced by LD) effect of the kth variant (z-score) of the jth variant is then given by: jth and kth variants and z-scores). To reduce computational burden, we randomly select a subset of 106 variants of all GWAS variants to use for maximization of the likelihood function. For specific details of the model and its implementation, please refer to the following sections.We consider an additive model of genetic effects ignoring gene–environment interactions, epistasis and dominance effects. Variant effect sizes are modeled with point-normal mixture priors, where both proportion of non-null variants and distribution of their effect sizes can vary between different predefined functional genomic categories. Each functional category in the model is characterized by the proportion of non-null variants  whenever jth variant. Any variant j with Consider a quantitative phenotype standardized to mean 0 and variance 1. Let ith individual). Using (1) and this notation we can write:j observed in the GWAS (jth column of j estimated in GWAS (j: Consider now a GWAS on a quantitative phenotype. Let j and k from a multiple linear regression in an infinite population (as discussed above).where Assuming the considered phenotype is complex, i.e. it is influenced by many variants each explaining only a tiny fraction of phenotypic variance, then the variance of the simple linear regression error is approximately equal to the sample variance of the phenotype, z-score of variant j observed in GWAS:Combining In By construction . Trans-chromosome correlations as well as correlations between variant genotypes at z-scores observed in different subsets of 106 randomly selected variants.We applied the model to GWAS summary statistics on 11 phenotypes . Like inThe simulations with synthetic data demonstrate that the true parameters are estimated accurately when the proportions of heritability carried by both functional categories are comparable and each category individually carries >2% of the total heritability; if one of the functional categories carries a negligible fraction (<2%) of the total heritability the model often fails to reconstruct its parameters accurately . Selecteπexonic and πnon-exonic) can be converted into the number of non-null variants by multiplying them by the total number of variants within the corresponding annotation category. The numbers ensuing for the analyzed phenotypes are presented in The model was used to estimate exonic and non-exonic polygenicities, discoverabilities and heritabilities of 11 complex phenotypes presented in The majority of 11 analyzed phenotypes fall into the portion of parameter space where, according to our simulations, the model is expected to produce robust parameter estimates , Fig. 1.The model suggests, that despite having similar heritability, phenotypes may differ substantially in polygenicity and discoverability of non-null variants. For example both AI-MiXeR and partitioned LDSC provide comparable estimates of total and partitioned heritability for LDL and T2D (AI-MiXeR LDL: 2= 0.06) . However2= 0.06) . HoweverWe present the AI-MiXeR model, which can be used to decouple and partition a phenotype’s heritability into functional category-specific polygenicity (proportion of non-null variants in a given category) and discoverability (variance of non-null effect sizes) components and thus better characterize the phenotype’s genetic architecture. This may inform the design of future genetic studies, as efforts to improve discoverability in different genomic categories are likely to have different impact across complex phenotypes depending on their unique genetic architecture.It is widely assumed that protein-coding exons contain a higher proportion of causal variants (higher polygenicity) and have on average stronger effects (higher discoverability) on complex phenotypes compared to non-exonic regions  reported 66 exome-wide significant LDL susceptibility variants within 14 loci  and varied only the heritability (z-scores distribution only through their product [follows from formula (5)], our simulation scenario with N = 105 and N = 7 × 105 and AI-MiXeR relies on design and implementation quality of the specific GWAS. In general, model predictions for a given phenotype may differ depending on a GWAS’s sample size, as well as on the coverage of the tested variants. The sample sizes of the GWAS tested here vary by more than one order of magnitude, from approximately 5 × 10N = 489) was used to estimate LD The model underlying AI-MiXeR is sensitive to the LD structure estimates. Ideally, the LD structure should be estimated on the same sample used for association testing. However, this is mostly impractical if not impossible. Here, in the analysis of GWAS summary statistics for 11 phenotypes, we estimated the LD structure using the 1000 Genomes Phase 3 genotype panel. Inconsistencies between the LD structure of the samples used for association testing and that of the 1000 Genomes Phase 3 panel could skew the model’s results. Additionally, roughening the LD structure . However, with the current implementation, the computational cost of the optimization increases rapidly as the number of annotation categories grows. For this reason, we restricted the main analysis of this study to exonic and non-exonic categories, which we reckoned could be informative in the context of whole-exome studies. An exploratory analysis of several other functional annotation categories  for SCZ and T2D can be found in The AI-MiXeR method presented here considers predefined annotation categories allowing both different proportions of non-null variants and different effect size distributions in various functional annotation categories, which is not possible with other methods available to date . The abibtaa568_supplementary_dataClick here for additional data file.


Charcot-Marie-Tooth disease (CMT), a genetically heterogeneous group of diseases in the peripheral nervous system, is characterized by progressive and symmetrical distal weakness resulting in gait abnormality. The necessity of the diagnostic and prognostic biomarkers has been raised for both basic research and clinical practice in CMT. Since biomarkers for animal study of CMT are limited, we evaluated the feasibility of gait parameters as tool for measuring disease phenotype of CMT mouse model. Using a Trembler-J (Tr-J) mouse, a CMT type 1 (CMT1) mouse model, we analyzed kinematic parameters such as angles of hip, knee and ankle , and spatial parameters including step width and stride length (transverse plane). Regarding of kinematic parameters, Tr-J mice exhibited less plantarflexed ankle during the swing phase and more dorsiflexed ankle at the terminal stance compared to control mice. The range of motion in ankle angle of Tr-J mice was significantly greater than that of control mice. In spatial parameter, Tr-J mice exhibited wider step width compared to control mice. These results are similar to previously reported gait patterns of CMT1 patients. In comparison with other markers such as nerve conduction study and rotarod test, gait parameters dynamically reflected the disease progression of CMT1 mice. Therefore, these data imply that gait parameters can be used as useful tools to analyzed the disease phenotype and progression during preclinical study of peripheral neuropathy such as CMT. Charcot-Marie-Tooth disease (CMT) is a genetically heterogeneous group of diseases in the peripheral nervous system characterized by progressive and symmetrical distal weakness  mouse  was used as a CMT type 1 (CMT1) mouse model . The system has a narrow channel made of clear acryl for mouse walking with two mirrors on both side of the channel to reflect the motion of lateral side of the mouse, and a mobile high-speed camera, which automatically follows and records the mouse movements from three sides simultaneously. Gait patterns such as the angles of hip, knee and ankle  as well as step width and stride length (transverse plane) were used. To indicate their locations, the hair was removed and each joint was marked. Iliac crest, center of hip, knee and ankle joints, 5th metatarsal head of right side, which are regarded as center of the joints, were marked using white marking pen to calculated hip, knee and ankle joints. From the recorded images, the marked points on the right side were tracked in each frame to generate the coordination of each joint during walking.After recording the gait of each mouse, we then analyze the gait pattern using each parameter. First, we extracted the middle two steps from the whole recorded gait. This enables the analysis of steady-state gait pattern by exclusion of accelerating and decelerating periods during the whole gait. To compare kinematic patterns between Tr-J and wild-type (WT) mice, the x and y coordinates for 5 marked points were extracted on the sagittal plane video using MotoRater software . Then tht-test. The level of significance was set to p < 0.05.All values are expressed as mean ± standard error of the mean (SEM). The statistical significance of data presented were evaluated by Student’s PMP22 gene. Tr-J mouse develops CMT1 phenotype afterbirth and definite phenotypic abnormality appeared within 3 weeks of age. For determination of gait pattern of mouse model, we applied a MotoRater system which allows the record of the mouse motion from 3 sides simultaneously  in Gait analysis for five  of each group (Tr-J and WT) mice were performed at 6, 10 and 14 weeks of ages. First, we compared the kinematic patterns in sagittal plane at 14 weeks of age. Hip angles of Tr-J mice were more fluctuated compared to those of WT mice . AccordiNext, we sought to correlate the disease progression with gait abnormality. When the kinematic parameters were compared in time course from 6 to 14 weeks, flexion, extension and ROM of each angle were significantly altered according to disease progression of Tr-J mice . DorsiflThe primary signs of CMT1A patients are foot drop and/or push-off deficit, which are related to ankle angle. In follow-up evaluation, patients showed worsened foot-drop and exaggerated hip flexion than previous test  and compound muscle action potential (CMAP) of Tr-J mice were significantly lower than WT mice from the first observation (6 weeks of age). In addition, there were very slight changes according to the disease progression in Tr-J mice , step width and stride length (transverse plane). Gait disturbances of CMT1 mice were significantly different to that of WT mice in their early stage of disease. Distal muscle weakness induced changes in ankle angle, which affects to the patterns of knee and hip joint angle for the compensation. These data mostly coincide with CMT1A patients pattern except that CMT1A patients show dorsiflexor weakness in the early stage, then it carried over into the plantarflexor weakness . In addiCompared to the parameters from nerve conduction or locomotor performance, gait analysis exhibited more effective features to monitor disease progression. Long-term follow-of gait analysis with identical mice demonstrated the usefulness of gait analysis as a sensitive biomarker in disease diagnosis. Although electrophysiological analysis and rotarod performance showed a significant difference between Tr-J and WT mice, the parameters were undistinguishable within time sequences due to bottom effect. Indeed, the electrophysiological data were almost at the borderline of measuring range and the times on rod were close to zero from the first examination in Tr-J mice.In the clinical research for efficacy assessment of novel therapeutics, development of useful biomarker is prerequisite. However, very slow rate of disease progression causes very slow changes in available parameters. According to previous studies, the average disease progression determined by CMT neuropathy score (CMTNS) is rather limited (Shy et al. In preclinical study, parameters for drug efficacy need to reflect the human phenotypic symptoms. So far, no satisfactory outcome has been drawn from clinical studies, even though several treatment options for CMT have been suggested through animal studies. For example, application of ascorbic acid showed dramatic efficacy in rodent models (Passage et al.


We have used novel rapid methods to structurally characterize the complementary-determining regions (CDRs) of more than 180 million human and mouse B-cell receptor (BCR) repertoire sequences. These structurally annotated CDRs provide unprecedented insights into both the structural predetermination and dynamics of the adaptive immune response. We show that B-cell types can be distinguished based solely on these structural properties. Antigen-unexperienced BCR repertoires use the highest number and diversity of CDR structures and these patterns of naïve repertoire paratope usage are highly conserved across subjects. In contrast, more differentiated B-cells are more personalized in terms of CDR structure usage. Our results establish the CDR structure differences in BCR repertoires and have applications for many fields including immunodiagnostics, phage display library generation, and “humanness” assessment of BCR repertoires from transgenic animals. The software tool for structural annotation of BCR repertoires, SAAB+, is available at  B-cell receptors (BCR) are the major components of the adaptive immune system. These are immunoglobulin molecules that bind to foreign substances known as antigens. Each individual has a huge BCR repertoire, where each individual BCR has a specific binding site composed of the complementary-determining regions (CDRs) capable of recognising a specific antigen. Drug discovery and immunodiagnostics inspired by the adaptive immune system rely on our ability to accurately interrogate the structural diversity of the binding sites of the BCR repertoire. Here we report our novel rapid pipeline, SAAB+, which has enabled us to interrogate how the structure of the CDR changes in BCR repertoires along the B-cell differentiation axis. By analysing human and mouse BCR repertoires at an unprecedented scale, we observed species-specific structural predetermination and detected CDR dynamics across multiple stages of B-cell differentiation. We showed that naïve repertoires share the highest number and diversity of CDR structures, a pattern which was highly conserved in all B-cell donors. Our results suggest that increased B-cell differentiation is associated with a personalization of CDR structure usages. Finally, we established the differences in CDR usages between humans and mice, analysis with immediate relevance for BCR repertoire “humanness” assessment and rational immunotherapeutic engineering. B-cells are essential components of the adaptive immune system in jawed vertebrates. They play a key role in recognizing foreign molecules (antigens) via membrane-bound B-cell receptors (BCR), and antibodies (secreted BCRs). Successful recognition of a broad array of structural motifs (epitopes) on antigens relies on the enormous sequence and structural diversity of BCR repertoires, generated by the rearrangement of V(D)J gene segments in the two variable domain chains (heavy and light), each consisting of four framework and three complementary-determining (CDR) loop regions ,2. Upon Next-generation sequencing of immunoglobulin genes (Ig-seq) has become an essential technique in immunology ,6. For evice versa Reviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: In this study, Kovaltsuk et al. study the structural diversity of BCR repertoires along human and murine B-cell ontogeny.Structural diversity along B-cell ontogeny has so far not been investigated. To achieve this goal, the authors improved on a previously published software for the structural annotation of antibodies (SAAB --> SAAB+) and leveraged two large-scale mouse and human BCR repertoire datasets that comprise different B-cell stages and isotypes. Applying SAAB+ to these datasets, the authors found differences in structural templates used by mice and humans and also a restriction in structural diversity across B-cell ontogeny. Previously, antibody repertoire analyses were limited to sequence-only analysis. Here, Kovaltsuk and colleagues point the way towards structural analyses on entire repertoires across adaptive immune cell thus complementing sequence-only analyses.This is a very well executed study and i have only minor comments:- Methods:-- It has not really become clear to this reviewer what the advancement of saab+ over saab is?-- Can the authors make this more explicit and dedicate a larger portion on this also to the main text. Can the authors also indicate ho how much time it took to annotate the two datasets (plus hardware requirements).- Wording:-- line 36: system(without s)-- for example line 42: in this work, the authors mix paratope with CDR. While the CDRs contain the paratope, the paratope is mostly one a portion of the CDR and a function of the epitope. Thus, it would be great if the authors could adjust ALL relevant portions of the text accordingly.-- line 142: the authors state "these results confirm a structural basis for self-tolerance". Can the authors explain this further or remove this sentence from the manuscript?- Data availability: Has the structural annotation of the two datasets also been deposited? If not, it would be great if the authors could do so so that other groups can build off of their efforts.- Results-- The authors performed subsampling experiments to account for the smaller number of PC (antigen-experienced) sequences. However, in antigen-experienced repertoires, the sequence similarity among populations will also likely be higher. Have the authors accounted for that? Or, in other terms, to what extent is the restriction in structural diversity a consequence of the increase sequence similarity?-Discussion-- the authors state that structural diversity becomes restricted with B cell ontogeny. Would this mean that the recognition potential become more restricted as well? What does this mean for the memory response which is supposed to be a trade-off between increased diversity and increased affinity?- Figure aesthetics:-- the figures are fine but not the most attractive. I don't have anything specific against them but if the authors could dedicate a few hours to making them a bit more visually pleasing, the readers would be very thankful i think.Reviewer #2: N/A. Withdrawal due to late-perceived conflict of interest per PLoS policy.Reviewer #3: The authors analyzed heavy chain sequences of human and mouse B-cell repertoiresthat were obtained from Observed Antibody Space database. They used antibodymodelling tools (SCALOP and FREAD) to annotate H1, H2 and H3 loops by theirputative structure/conformation. Using the structural annotation the authorsshowed that paratopes in naive B-cell populations are structurally distinct fromparatopes in mature B-cells. Furthermore, they observed different paratopeusage between human and mice.Overall, this is a well executed work that will be appreciated byresearchers involved in immunotherapy and antibody engineering.I would like to raise following points:1. Due to the limitation of modelling tools the H3 loop structure predictionswere carried out only for loops shorter than 16 amino acids. As a result less than50% of observed human heavy chain H3 loops were annotated. The authors shoulddiscuss what impact (if any) may the missing annotations have on theconclusions regarding changes of paratope usage upon B-cell differentiation.2. What precluded a similar analysis of light chain sequences? Could authorsdiscuss what can we expect: similar findings or, on the contrary, learnsomething about distinct roles of heavy and light chains?**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Computational Biologydata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********what does this mean?). 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