TOMM40). The highly variant ‘523’ poly-T repeat is associated with age-related cognitive decline and age of onset in Alzheimer’s disease, but whether it plays a role in modifying the risk or clinical course of PD it yet to be elucidated. The TOMM40 ‘523’ allele length was determined in 634 people with PD and 422 healthy controls from an Australian cohort and the Parkinson’s Progression Markers Initiative (PPMI) cohort, using polymerase chain reaction or whole genome sequencing analysis. Genotype and allele frequencies of TOMM40 ‘523’ and APOE ε did not differ significantly between the cohorts. Analyses revealed TOMM40 ‘523’ allele groups were not associated with disease risk, while considering APOE ε genotype. Regression analyses revealed the TOMM40 S/S genotype was associated with a significantly later age of symptom onset in the PPMI PD cohort, but not after correction for covariates, or in the Australian cohort. Whilst variation in the TOMM40 ‘523’ polymorphism was not associated with PD risk, the possibility that it may be a modifying factor for age of symptom onset warrants further investigation in other PD populations.Abnormal mitochondrial function is a key process in the pathogenesis of Parkinson’s disease (PD). The central pore-forming protein TOM40 of the mitochondria is encoded by the translocase of outer mitochondrial membrane 40 homologue gene ( The pivotal role of mitochondrial dysfunction in PD is supported by several parkinsonism-causing toxins, genetic mutations and retrotransposon insertions4, which specifically impair mitochondrial function. Insights from these toxins and mutations imply that mitochondrial dysfunction in the pathogenesis of PD can arise from a wide array of biological processes, such as bioenergetic disturbances, nuclear and mitochondrial DNA mutations, impaired fusion and fission, defective mitophagy, and abnormal morphology and size5. For example, a significant cause of mitochondrial dysfunction in PD is the inhibition of mitochondrial complex I, an ETC defect that leads to severe oxidative stress and ROS- and caspase- mediated dopaminergic cell death5. Notably, the latter impairment is a major pathological feature of PD induced by familial PTEN-induced kinase 1 (PINK1), alpha-synuclein (SNCA) and Daisuke-Junko-1 (DJ-1) gene mutations or the toxin 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP)3. Thus, mitochondrial dysfunction is thought to cause neuronal stress and degeneration, eventuating in this neurodegenerative disease, PD.Parkinson’s disease (PD) is increasingly known as a multifaceted neurodegenerative disorder with a heterogeneous and burdensome symptom presentation and progression. Abnormal or deficient mitochondrial functioning is widely implicated as a key process in the selective neuronal death and pathogenesis of PD. Mitochondrial dysfunction, resulting in a loss of electron transport chain (ETC) efficiency or decline in ATP-synthesising capacity, appears to elicit dopaminergic cell death via a number of mechanisms, including reactive oxygen species(ROS)—generation, impaired ATP production and disrupted calcium homeostasisTOMM40) gene encodes the pore-forming subunit (TOM40) of the protein-transport channels in the mitochondrial outer membrane, thus playing a fundamental role in mitochondrial functioning6. The TOM40 protein facilitates the import of approximately 1500 externally synthesised proteins and peptides into the mitochondria, and plays a key role in the mitophagy degradation pathway8. Altered or abnormally functioning TOM40, mediated by genetic changes or atypical protein expression, is thought to contribute to mitochondrial dysfunction and protein accumulation in Alzheimer’s disease (AD) and PD8. This may occur through different mechanisms: (1) mitochondrial import impairment, which may prevent essential proteins and peptides from reaching their designated mitochondrial targets, or allow unwanted and mutant proteins to aggregate in the mitochondria; or (2) mitophagy disruption, which may enable damaged and malfunctioning mitochondria to accumulate. Given evidence for mitochondrial dysfunction in PD, genetic variants affecting TOMM40 expression may have risk and disease-modifying effects.The translocase of outer mitochondrial membrane 40 homologue (TOMM40 is apolipoprotein E (APOE), which is in a region of strong linkage disequilibrium (LD), or non-random association, with TOMM40 variation11. Though the extent to which common variants in TOMM40 can have APOE-independent effects on disease risk or disease-modifying effects remains uncertain, polymorphisms in TOMM40 have been independently associated with a range of primarily cognition-based neurodegenerative diseases, including AD, frontotemporal dementia and dementia with Lewy bodies, as well as non-pathological ageing15. One of these variants is situated in intron 6 of TOMM40 and is a homopolymer of repeating thymine (T) nucleotides known as rs10524523 or ‘523’16. The ‘523’ variant is polymorphic, with lengths that vary from 11 to 39 T residues in Caucasians17. To better categorise and describe this diverse structural variant, three allelic groups for the homopolymer have been established previously, according to the number of constituent T residues: ‘Short’ , ‘Long’  and ‘Very Long’ (T ≥ 30)18. Although the functional effects of the ‘523’ variation are mostly unknown, increasing length of the poly-T repeat is thought to increase TOMM40 expression20. Therefore, the effect of the TOMM40 ‘523’ variant and altered TOM40 protein levels may contribute to mitochondrial dysfunction in PD21. While a vast number of published studies report conflicting roles of the various TOMM40 ‘523’ variant lengths in AD and ageing populations26, there have only been two previous studies within PD cohorts with differing findings. The first was a study of a relatively large Polish PD cohort which found no significant associations between TOMM40 ‘523’ and PD risk or age of onset27. Subsequently, a significant overrepresentation of the L/VL ‘523’ genotype was reported in a Swedish PD cohort compared to healthy controls, though it should be noted that these findings are yet to be published in full28. Thus, with conflicting findings in these two European cohorts, further investigation in other populations is required.Downstream of TOMM40 in mitochondrial import and mitophagy, both plausible sources of PD-causing mitochondrial dysfunction, the ‘523’ variant in TOMM40 is therefore a potential risk factor and disease modifier in PD. This study investigated the distribution of ‘523’ alleles and their association with disease risk and age of symptom onset in two independent PD cohorts, one Australian and one international , using PCR-based and whole genome sequencing approaches.Due to the essential role of 29. Clinical and demographic data, including the age of symptom onset, were recorded in the database. All PwP were examined by a movement disorder neurologist prior to inclusion in the study for verification of the diagnosis in accordance with the UK Brain Bank criteria for idiopathic PD and reported no family history of PD30, while healthy controls were confirmed to have no history of any neurological disorders. This study was approved by the Sir Charles Gairdner Hospital Human Research and Ethics Committee . Written informed consent was obtained from all participants, in accordance with the Australian National Health and Medical Research Council research guidelines.The first cohort comprised of 235 home-based people with PD (PwP) and 231 healthy controls of European heritage from the Perron Institute for Neurological and Translational Science PD Database, as previously reportedhttp://www.ppmi-info.org/data). This cohort comprised of 368 PwP and 172 healthy controls, and only individuals of European heritage were included in order to reflect the composition of the Australian cohort.The second cohort was derived from the international Parkinson’s Progression Markers Initiative (PPMI) database  and stored until DNA extraction. Alternatively, blood was collected from the medial cubital vein. DNA was extracted and purified from these samples using QIAamp DNA mini kits , according to the manufacturer’s protocol. DNA concentration was determined using absorbance readings calculated by a NanoDrop One Microvolume UV–Vis spectrophotometer .‘523’ variant in the Australian cohort was completed using fluorescently labelled primers, as previously described19. The forward primer sequence was 5′-/6-FAM/-TGCTGACCTCAAGCTGTCCTC-3′ and the reverse primer was 5′-GAGGCTGAGAAGGGAGGATT-3′, synthesized by Integrated DNA Technologies Pty Ltd . Endpoint PCR was performed using 5 μL of 5 × MyFi Reaction Buffer , 1 μL of MyFi DNA Polymerase (Bioline), 0.2 μL of each forward and reverse primer (20 μM) (IDT), 0.25 μL of 1% dimethylsulfoxide , 50 ng of genomic DNA, and 11.35 μL of dH2O ; to a final volume of 25 µL. Optimised PCR conditions were as follows: 1 cycle at 94 °C for 3 min, 27 cycles at 94 °C for 15 s, annealing at 65 °C for 20 s and extension at 70 °C for 30 s, and 1 cycle at 70 °C for 5 min. Applied Biosystems SimpliAmp Thermal Cyclers  were used for endpoint PCR cycling. Post PCR products were stored at 4 °C until capillary fragment separation, which was conducted by the Australian Genome Research Facility . Electropherograms were analysed using Peak Scanner Software . ‘523’ allele lengths were determined according to a previously established method by Linnertz et al.19. Briefly, the highest intensity peak(s) in each peak cluster between 160–190 bp were identified and sized, and 150 bp (accounting for flanking regions and primers) was subtracted from the peak sizes to determine poly-T allele lengths. Alleles were grouped using the convention established by Roses et al.9: Short , Long  and Very Long .PCR-amplification of the http://www.ppmi-info.org/data) in binary alignment map (BAM) format that had been aligned to the human reference genome GRCh38 using the Burrows-Wheeler transform alignment algorithm31. Resultant BAM files were analysed using the Integrative Genomics Viewer32 in order to calculate the length of the poly-T repeat, as previously demonstrated33. Alleles were grouped using the convention established by Roses et al.9: Short , Long  and Very Long .Whole genome sequencing (WGS) data were obtained from the PPMI database (available at APOE ε were determined using the single nucleotide polymorphism (SNP) and PCR-restriction fragment length polymorphism (PCR–RFLP) analyses. APOE ε2/ε3/ε4 genotypes were determined by sequencing two SNPs (rs429358 and rs7412) using the MassARRAY system  at the Australian Genome Research Facility 34. For PCR–RFLP analyses, endpoint PCR reactions were prepared to a final volume of 10 µl using primer sequences previously described35. Reactions contained 7.2 µl dH2O , 2 µl MyFi reaction buffer , 0.05 µl MyFi DNA polymerase , 0.375 µl forward and, 0.375 µl reverse primer  at 200 ng/µl, and 25 ng DNA. The PCR amplification protocol followed, being an initial hold temperature of 95 °C for 4 min 30 s and 35 cycles of denaturation at 95 °C for 30 s, annealing 60 °C for 30 s and extension at 72 °C for 1 min 30 s. Restriction enzyme reactions were prepared to a final volume of 20 µl, containing 6.3 µl dH2O , 0.2 µl acetylated BSA , 2 µl C buffer , 0.5 µl Hhal  and 10 µl PCR product. Reactions were incubated for 4 h at 37 °C prior to polyacrylamide gel fractionation. Restriction enzyme digested products were fractionated on 12% (w/v) 29:1 polyacrylamide gel  in 1 × TBE. Electrophoresis fragment separation was performed at 100 V for 3 h on the DCode Universal Mutation Detection System . Gels were stained in 1 × TBE containing SYBR Gold nucleic acid gel stain  for 4 min before visualization using a BioRad Chemidoc MP Imaging System. APOE ε genotype data relating to the PPMI cohort was obtained from the online database (available at www.ppmi-info.org/data).Genotyping of p-value of < 0.05 was employed for all statistical tests. Variables were described using mean and standard deviation , or frequency and percent , as appropriate. Normality was assessed and subsequent clinical characteristics were analysed using Independent Samples T-Test, Mann–Whitney U, or Chi-square, as appropriate. For cross-sectional analysis, Chi-square, stratified Mantel–Haenszel tests and binary logistic regression models were used to evaluate the association between TOMM40 ‘523’ genotypes and risk of PD in the Australian and PPMI cohorts. Binary logistical regression models were run both with and without correction for the APOE ε4 status ) and patient sex. Analysis was also run considering all combinations of ‘523’ length category and APOE ε genotype, to examine for interactive effects without a priori assumptions. The aforementioned analyses were performed separately in the PPMI and Australian cohorts, and after combination of the cohorts. Following this, binary logistic models were carried out when considering populations of APOE ε3/ε3 carriers only, as previously examined in this fashion and stated as a requirement for replication studies13.The Australian and PPMI cohorts were analysed separately, and together, using IBM-SPSS software . A significant nominal TOMM40 on age of disease onset, correcting for APOE ε allele status and sex. Again, GLMs were carried out when considering populations of APOE ε3/ε3 carriers only. Residual plots were examined for all models and no violations were noted. Correction for multiple comparisons was conducted using Bonferroni pairwise comparisons, where appropriate.Generalised linear models (GLMs) were also constructed in order to study the interaction of TOMM40 ‘523’ S allele carrier status  and APOE ε4 status  were combined to produce 5 groups. Mean comparisons were then analysed using the Kruskal–Wallis one-way analysis of variance and univariate GLMs. This analysis was repeated in the combinations of TOMM40 ‘523’ S allele carrier status and APOE ε2 status, and TOMM40 ‘523’ VL allele carrier status and APOE ε4 status.Subsequently, the load combination of TOMM40 ‘523’ genotype as well as both the TOMM40 ‘523’ and APOE genotype. To compare the survival curves, the log rank test was applied, placing weight on longer survival periods37. Allelic stratification by TOMM40 ‘523’ was also run using Kaplan–Meier analysis. Additionally, all distributions of ages at onset adjusting for sex were compared via Cox proportional hazard regression models.Finally, Kaplan–Meier curves for age at PD symptom onset were estimated, stratified by A summary of the cohorts included in this study is presented in Table TOMM40 ‘523’ genotyping was obtained following optimisation of a PCR-based assay, with representative electropherograms presented in Supplementary Fig. 1. Genotyping of the TOMM40 ‘523’ variant in the PPMI cohort was completed using a WGS method, previously reported as an alternative method to fragment analysis and Sanger sequencing33. The distribution of the ‘523’ alleles in PwP compared to healthy controls for both cohorts are presented in Fig. 9 (Supplementary Table 1). APOE ε genotypes of the Australian cohort were further validated using PCR–RFLP, with representative samples ε2/ε3, ε2/ε4, ε3/ε3 and ε3/ε4 presented in Supplementary Fig. 2. APOE ε genotype and allele groupings for both cohorts exhibited similar distributions, however there was an absence of individuals with the ε2/ε2 genotype in the Australian cohort (see Supplementary Table 2).In the Australian cohort, ‘523’ distribution and no significant difference was observable . Each distribution was also compared to distributions on Webstr database, similarities were seen between the current study cohorts and both the Gtex (predominantly European self-reported ancestry) and 1000 genomes European distributions. Thus, the current cohort were considered reflective of a population of European descent and the two cohorts could be combined to increase the sample size and power to detect ‘523’ effect.Both groups consisted of individuals with self-reported of European heritage, though country of origin information was not available for all cases. To determine whether cohorts could be appropriately combined, Levene's Test for Equality of Variances was used to compare the TOMM40 allele distributions between cohorts, case–control analyses were completed in both individual cohorts and in a combined cohort. When examined genotypically, the TOMM40 ‘523’ variant was not significantly associated with the risk of PD in either the Australian cohort , or the PPMI cohort , or when cohorts were combined . Moreover, when combined, allelic distribution of the S , L  and VL  binned groups was not significantly different between PD and controls . Analysis considering all combinations of ‘523’ genotypes and APOE ε genotypes also failed to show any significant differences between PD and control groups. As with prior findings, analysis utilising stratified Mantel–Haenszel tests did not find significant differences.As there were no significant differences in APOE ε4 status and patient sex into account, which again exhibited no significant association between TOMM40 ‘523’ genotype and PD risk. Models considering all combinations of ‘523’ and APOE ε genotypes also returned non-significant results. Binary logistic regression models were used subsequently to assess this association allelically. Neither naïve nor covariate corrected models, in either cohort or the combined cohort, exhibited a significant allelic association between TOMM40 ‘523’ and disease risk (Table APOE ε3/ε3 carriers (Supplementary Table 3), and again failed to show any significant associations between TOMM40 ‘523’ alleles or genotypes and risk of PD.Binary logistic regression models were analysed comparing each genotype or allele to all other genotypes or alleles Table . GenotypTOMM40 ‘523’ on age of disease onset, GLMs were constructed, with data from naïve and corrected models reported in Table TOMM40 ‘523’ alleles or genotypes and age of PD symptom onset was observed. However, in the PPMI cohort, carriage of the TOMM40 ‘523’ S/S genotype was associated with a 2.58 year later age of symptom onset (p = 0.040). This difference was not significant after controlling for APOE ε4 load and sex in multivariable GLMs, although a trend for later age of onset was still observable . It is important to note, however, that there was a lack of co-carriage of the S/S genotype with the APOE ε4/ε4 genotype in this cohort (see Supplementary Table 4) and that APOE ε4/ε4 homozygotes had a mean age of symptom onset almost 4 years earlier than other genotypes, although this was not statistically significant (p = 0.314). Thus, significance may have been lost due to opposing directionalities of these two genotypes on age of symptom onset. Carriage of the S/S genotype was also associated with a 2.69 year later age of PD symptom onset in the PPMI cohort (p = 0.032) after correcting for APOE ε2 load and sex. However, when adjusting for multiple comparisons, model-derived estimated means revealed the S/S genotype did not result in a significantly different age of PD symptom onset when compared to individual genotypes (p = 0.326).To investigate the influence of TOMM40 ‘523’ and age of PD symptom onset (Table APOE ε3/ε3 homozygotes (Supplementary Table 5), none of which revealed a significant association between TOMM40 ‘523’ and age of symptom onset. Finally, when considering the load combination of the TOMM40 ‘523’ S allele and APOE ε4 or APOE ε2, no significant differences were found in the mean age of symptom onset between groups in either cohort or in the combined cohort according to the copy number of these alleles.Subsequent analysis of the PPMI cohort exhibited no other statistically significant associations (at the 0.05 level of significance), either allelically or genotypically. Furthermore, when the cohorts were analysed as a combined cohort, no significant associations were exhibited between TOMM40 ‘523’ genotype, in the Australian cohort , respectively. Subsequently, Kaplan–Meier curves for the age at which symptoms of PD began were analysed, stratified by TOMM40 ‘523’ and APOE genotype as in Roses et al.13. Again, neither the Australian cohort (p = 0.550, Fig. p = 0.867, Fig. p = 0.660, Fig. TOMM40‘523’ allelic stratification also failed to produce significant results. Finally, all analyses completed using the Kaplan–Meier method were re-run using Cox proportional hazard regression models, which adjusted for sex, and no significant results were noted.Kaplan–Meier curves for the age at which symptoms of PD were first reported were stratified by ort Fig. A, the PPort Fig. B and theort Fig. C. No or TOMM40 ‘523’ structural variant as a potential PD risk factor and modifier of the age at symptom onset. The distribution of ‘523’ poly-T alleles did not vary between PwP and healthy controls, or in the two independent cohorts examined. The similarity in ‘523’ allelic distribution between the PPMI and Australian cohorts is worth noting as this study utilised two different approaches in the calling of the TOMM40 ‘523’ variant, establishing a PCR-based and a WGS-based assay for use in the Australian and PPMI cohorts, respectively. Assay development for the TOMM40 ‘523’ variant is generally considered to be difficult, as poly-T variants are challenging to sequence19. Despite optimisation within this study, PCR stuttering was observed in the PCR-based assay similar to previous reports19, which is a standard complication when amplifying repetitive genomic sequences, and highlights the need for further optimisation of the assay. By comparison, calling from WGS files revealed a similar distribution as reported previously33. While it has been presented as having fewer challenges in optimisation and sequencing, calling of ‘523’ variants was an arduous process and the aforementioned study has stated that the correlation between ‘523’ calling by WGS and PCR-based methods decreases with increasing size of the ‘523’ allele33.The current study aimed to investigate the TOMM40 ‘523’ variant is implicated in PD risk and found no association between carriage of allelic variants and the risk of developing PD. When binned, genotypic and allelic frequencies appeared to be similar between controls and PwP in both independent cohorts, and when the cohorts were combined. This agrees with previous work in a Polish PD cohort which showed no association of ‘523’ alleles, genotypes or haplotypes with the risk of PD or age of symptom onset27. In contrast, in a Swedish population, a higher frequency of the L/VL genotype was observed in PD patients compared to controls28, though these findings are yet to be published in full. Given that these studies were conducted in different ethnic groups, and that ‘523’ allele frequencies are ethnic-specific18, the contradictory findings in the studies to date may indicate that ‘523’ is a risk factor in some populations, but not in others. As this is only the third study to investigate the role of TOMM40 ‘523’ in PD risk, further studies should be performed in other ethnically diverse populations.The present study then examined whether the 39. Previous studies have implicated TOMM40 ‘523’ length in the age of onset of AD, particularly in carriers of the APOE ε3 allele22, but mixed findings exist. For instance, although Roses et al.9 reported that longer ‘523’ allele lengths were associated with risk of AD and earlier age of onset, the initial findings were not replicated in other populations40. Such varied findings may be a result of varied methodology and varied consideration of the influence of the APOE ε locus13, which is well-established as the strongest genetic predictor of AD. Most studies to date report no association of APOE ε variation with susceptibility to PD42, as was also the case in the current study. While the present findings did not show any positive evidence of interactive effects between TOMM40 ‘523’ and APOE ε genotypes in relation to PD risk, they do suggest that the two loci may have small independent and opposing effects on determining the age of onset of the symptoms. While initial regression analyses in the PPMI cohort showed that age of onset was significantly delayed by carriage of the S/S ‘523’ genotype, this was not replicated after correction for covariates, or in the Australian cohort. However, it is possible that this difference is caused by a country- or geographical-specific effect, as seen in other genetic studies43. It is worth noting that only one prior study has examined this association27, though it is not clear whether this study completed rigorous analysis of the interactive effects of TOMM40 and APOE, as was conducted in the current study. As such, further in-depth analysis in larger PD cohorts is required to determine the significance of the present findings.As mitochondrial involvement in PD is thought to be a key contributor to neuronal dysfunction and degeneration, it is plausible that a genetic variant that modulates mitochondrial function could modify age of symptom onset. Furthermore, AD and PD share a number of clinical, pathological and molecular features including toxic protein accumulation and mitochondrial dysfunction in the form of respiratory chain defects, oxidative stress, mitochondrial DNA damage and morphological abnormalities‘523’ allele length are poorly understood due to a scarcity of research in this area. While several studies have suggested that the VL allele increases TOMM40 mRNA expression and the S allele represses expression44, others have found no significant differences in mRNA levels between S and VL variants45. A recent study demonstrated that overexpression of TOMM40 in vitro was associated with greater mitochondrial membrane potentials, respiratory rates, spare respiratory capacities, ATP levels, amyloid-beta resistance, and protein uptake16. On the other hand, another study observed a correlation between TOM40 protein deficits and enhanced oxidative stress, reduced ATP production and abnormal complex I protein concentrations in the brains of PD patients and in alpha-synuclein overexpressing murine models21. As the literature currently stands, further elucidation of the biological consequences of up- or down-regulated TOMM40 expression is required to give insight into its potential role in PD risk and disease modification.Currently, the functional effects of variation in TOMM40 ‘523’ variant in two independent PD cohorts. TOMM40 plays an essential role in mitochondrial import and mitophagy, and the ‘523’ polymorphism has been associated with age of onset of AD and with age-related cognitive decline. While this study clarifies that TOMM40 ‘523’ is not in itself a predictor of PD risk, it raises the possibility that, as in AD, it may be a genetic marker for the age of symptom onset in PD. Whilst not conclusive, our findings in the large international PPMI cohort suggest that carriage of the S/S ‘523’ genotype may be protective in terms of delaying the age of symptom onset, and may warrant further investigation in other populations. Importantly, the effect of co-carriage of the APOE ε4 allele, which appears to have an opposing effect on age of onset, must be considered in future studies. Though not significant in modulating risk of PD, future studies should consider the possible role of TOMM40 ‘523’as a determinant of age of symptom onset, and symptom trajectory. Given a phase 3 clinical trial of a therapeutic for the prevention and delay of onset of AD was recently conducted involving participants stratified by TOMM40 ‘523’46, the findings reported herein are noteworthy, and may allow the design of symptom-focused studies in PD for much-needed improvements in patient outcomes and care.Overall, this study aimed to investigate the risk and disease-modifying role of the Supplementary Information


Diversity in metacommunities is traditionally viewed to consist of the diversity within communities ( Diversity in metacommunities is traditionally viewed to consist of the diversity within communities that is complemented by the differences between communities so as to result in the total diversity of the metacommunity  nor environmental diversity alone but rather the combination of both.The effect of this combination on (joint) diversity becomes apparent through the associations between the two components “trait”  and “community affiliation” . Associations rank between complete association  and the absence of association . Particularly complete association underlines the role played by the “flanking” or “marginal” distributions, since in this case, the joint diversity equals the overall  trait diversity (when differentiation of communities for types is complete) or the diversity of communities . The criterion is also known as the principle of transfers  of their effective numbers into components relating to the marginal variables “trait” and “community affiliation.” Herewith, one component (the leading component) is distinguished by realizing a minimization criterion. As applied to community affiliation as leading component, for example, the criterion determines the minimum number of communities required to realize the observed joint diversity given the observed trait distribution. The criterion does not rely on the concept of effective numbers at the outset, but it is amenable to an interpretation in terms of such numbers. The minimally required number lays the basis for a unique factorization of the joint diversity into the leading  and the associated marginal component (trait). The relation of the factorization to a more recently proposed method of partitioning diversity is demonstrated that avoids the arbitrariness inherent in averaging diversities within communities , the joint diversity equals the overall  type diversity  equals the product Consequently, equating the product with the observed joint diversity effective number C as a leading component, for which the number community-centered perspective of the factorization. One then arrives at the multiplicative decomposition (or factorization) of joint diversity in the formThe latter fact asks for distinction between a leading and an associated component of the factorization, where the leading component reflects the desirable properties. The above demonstrations suggest For Rényi-diversities , one would then aim at finding the minimum number of types required to obtain the observed joint diversity in the first place. In this way, the contribution of types to the ecological diversity of a metacommunity is assessed on the basis of the observed partition of the metacommunity into its constituent communities. This done, the associated effective number of communities is to be specified. Since community determinants are frequently considered to be less dynamic than the traits of the community members existing on or adapting to these determinants, this may be the more relevant perspective to be taken in many ecological studies.The apparent asymmetry in the above treatment of the community and type component T and C. All of the conclusions obtained so far apply analogously. Thus, one first determines the minimum number In essence, this perspective simply marks the dual analogue of the previous community-centered approach to the decomposition of joint diversity into community and type components. It therefore results from just reverting the roles of For Rényi-diversity, indicator of structural characteristics under the community-centered perspective when considering the deviation of The minimum number The latter suggests consideration of varies from 1 towhile structural characteristics vary from complete differentiation to complete absence of differentiation among communities.When compared with the observed number of communities, N again be the largest natural number with N, one infers that generally whilevaries from 1 tothe marginal type frequencies start with monomorphism and end with maximum polymorphism .Characterization of the structural aspects encoded in This result is obvious for Rényi-diversities, since there always The structural characteristics inherent in the factorization of joint diversity illustrate more vividly the conceptual advantages of the present approach, which focuses on the numbers of communities and types that essentially determine metacommunity diversity. By “essential,” two issues are addressed, factors and their interaction. The factors are specified by the minimally required number of communities (which depends on their differences in type composition) and the associated number of types . Their interaction is “orthogonal” in the sense of an independent operation in producing the joint diversity.The factorization reminds of the common approaches to partitioning diversity in such a way that the total (metacommunity) type diversity Chiu et al.  destabilize polymorphisms and by this imply tendencies toward monomorphism.Since in self-incompatibility systems, heterotypic combinations are preferred irrespective of community affiliations, potential differences in community affiliation between types tend to be equalized. This situation is characterized by small It remains to demonstrate how the two perspectives are connected. Since both perspectives rest on the same joint diversity entclass1pt{minimaThe latter observation becomes evident when considering the fact that Often, the spatial distribution of communities does not show a sufficient degree of fragmentation to allow for determination of a unique subdivision into subcommunities. As a consequence, more than one subdivision may become relevant, each of which may show a different partitioning of the same underlying overall type diversity. Similarly, different criteria for subdivision, such as the spatial distribution of environmental variables, may be relevant in comparative causal analyses of observed genetic or species variation. In this case, the states of each variable give rise to a subdivision into “communities,” which in turn enables comparison of the effects of the various variables on the observed type variation.structure , which is established especially in population genetics and implemented in methods such as An assignment of the above kind specifies a joint distribution of types and community affiliations and therefore allows computation of the joint diversity together with its factorization into In most cases, the models underlying an analysis of latent factors are based on probability laws that specify likelihoods or posterior probabilities of the assignments. These probabilities serve as the primary qualification criterion that is to be maximized . This allows consideration of the most likely structural characteristics indicated by


General knowledge questions are used across a variety of research and clinical settings to measure cognitive processes such as metacognition, knowledge acquisition, retrieval processes, and intelligence. Existing norms only report performance in younger adults, rendering them of limited utility for cognitive aging research because of well-documented differences in semantic memory and knowledge as a function of age. Specifically, older adults typically outperform younger adults in tasks assessing retrieval of information from the knowledge base. Here we present older adult performance on 421 general knowledge questions across a range of difficulty levels. Cued recall data, including data on the phenomenology of retrieval failures, and multiple-choice data are available. These norms will allow researchers to identify questions that are not likely to be known by older adult participants to examine learning or acquisition processes, or to select questions within a range of marginal accessibility, for example. Comparisons with young adult data from prior databases confirms previous findings of greater knowledge in older adults and indicates there is preservation of knowledge from early adulthood into older adulthood.The online version contains supplementary material available at 10.3758/s13428-020-01493-2. A standard categorization of long-term, declarative memory is as either episodic or semantic Tulving, . Many, iIn contrast, the nature of semantic memory or the knowledge base is such that assessing the contents of this system can be challenging. The assumption is that the contents include pre-experimentally acquired information that is relatively stable over the lifespan and can be accessed across contexts Tulving, , 1985. IInterestingly, GK has been found to predict recent and current event knowledge , so having an accurate assessment of what someone knows is important, to control for differential effects of prior knowledge. For example, researchers examining marginal knowledge , to item selection effects , to changes in reading habits among younger cohorts  and multiple-choice questions. The former typically require more effortful search strategies in memory, whereas the latter, because the answer is provided, are more sensitive to discrimination among related foils. Older adults, in episodic tasks, generally show more marked deficits in tests that offer less environmental support, such as cued-recall, than tests such as recognition , were normed in a cued-recall test. The questions were divided into four sets ranging from 70 to 148 questions each. For each question, participants had the option of providing an answer, indicating they could not remember (DR), or indicating they did not know (DK) the answer. Specific guidelines on when and how to use DR and DK were not provided  platform . Fifty-seven participants completed the first set of questions  consisted of 55 older adults recruited on MTurk (see Table n = 32) and at Claremont McKenna College . Five participants reported English was not their first language (see Table Participants tested in the lab were community-dwelling older adults (ages 60+). For the Set C questions, the participants were 67 older adults recruited from the Waterville, Maine, community who participated in two experimental studies examining the phenomenology of retrieval failures .As mentioned above, four different sets of questions were used. Two sets (A and B) were developed for the purposes of gathering the present normative data; the other two (C and D) were originally used in experimental tasks in our labs. The encoding phase of the experimental tasks was similar to the norming task, in that participants provided responses to open-ended questions about a variety of topics or responded DR or DK. The questions in all sets covered a variety of topics, ranging from literature to sports, geography, history, science and technology, pop culture, and music (see the https://github.com/el-cms/Open-trivia-database) and the online version of the Encyclopedia Britannica, which includes an online quiz platform (www.britannica.com/quiz). Set B included 134 questions from Burke et al. . If you do not remember the answer, please type DR ("Don't Remember"). Please do not leave any questions blank.”Importantly, no additional instructions were provided on when and how to use these options. They were further told some of the general knowledge questions were quite difficult and were informed that the purpose of the study was simply to assess what people know. Because MTurk participants are paid by the number of jobs they complete online, we were reasonably confident participants would not spend additional time searching for correct answers online.Participants who were tested in the lab were tested individually. The study was programmed using E-Prime . Errors were coded as errors of omission  or errors of commission . DR and DK responses were coded as such. Table Analyses are reported at the item level. As noted above, for each item between 54 and 67 participants responded . Responses were hand scored by a research assistant. Responses were scored as correct if the participant provided the correct answer or a clear misspelling or minor morphological/wording variation , indicating a wide range of question difficulty was successfully obtained. Errors ranged from 0 to .82 . The proportion of DR responses was overall low ; however, the range of 0 to .56 does indicate some questions in the pool might be reflective of marginal knowledge. DK response rates ranged from 0 to .93 . Very few questions were left blank overall , indicating participants were generally following instructions. Rank was calculated in order of increasing difficulty; ties were left as such in the database.To examine whether the rate of responses varied as a function of item difficulty, we binned all questions into quartiles, using accuracy as a proxy for difficulty. By binning the items into quartiles, we provide a basic stimulus selection framework for researchers, who might be interested in identifying items with a specific level of difficulty or items that elicit a high rate of DR or DK items. Because the data are necessarily ipsative and not independent , we focus on the descriptive statistics rather than providing any inferential statistics. Thus, the 421 questions were divided into quartiles ranging in overall accuracy from most difficult , to moderately difficult , to moderately easy , to easiest . As can be seen in Figure The variable rates of DR and DK responses suggest that questions that are in a moderate difficulty range elicit the highest proportion of DR responses whereas questions that are too difficult elicit the highest rate of DK responses. This reflects the fact that general knowledge varies in accessibility as well as in availability .Mean response times (RT) in milliseconds as a function of response  were calculated for each participant. DK responses were faster  than all other response types, which were similar . The rank order in the present study was correlated to the orders in previous studies: Compared to Tauber et al.  and to Nelson and Narens  there appears to be both generational and longitudinal stability overall.As was reported by Tauber et al. , we compd Narens  norms weM = .24, SEM = .02), intermediate in the Nelson and Narens’ norms , and lowest in the Tauber et al. norms , F = 82.98, MSE = .014, p < .001, ηp2 = .58 . This suggests that there is greater overlap in the knowledge base between today’s older adults and college students from the late 70s than between older adults and current young adults. Furthermore, although the knowledge base does increase over the lifespan (as evidenced by the higher accuracy among our older adults), the information that was known by young adults at the time of Nelson and Narens’ study appears to be preserved in older adults but very obscure to today’s younger adults. In concert with Tauber et al.’s conclusions about the need to regularly update general knowledge norms for younger adults due to fluctuations in the relevance of information, these findings point to the need to have age-appropriate norms for older participants as well. We acknowledge that these analyses are predicated on the strong assumption that age (or cohort) is the main factor that distinguishes our sample from the college students tested in the Nelson and Narens and Tauber et al. studies. Clearly, other factors, such as overall educational achievement, individual and group differences in information-seeking behaviors, and even personality characteristics such as conscientiousness, might contribute to the age-related differences. We return to these points in the General Discussion.In addition to examining overall ranking of difficulty as Tauber et al.  did, we Because general knowledge questions are used across a variety of tasks, in addition to providing cued-recall performance, we administered a multiple-choice version of the task to a set of new participants, all from online sources. Multiple-choice questions provide additional environmental support and, on tests that do so, such as item recognition, older adults often show reduced performance deficits relative to younger adults Craik, . Thus, pA total of 201 participants were recruited. All participants were recruited on MTurk, using the same restrictions described above. Data from five participants were omitted because they self-reported their age under 55 (three from Set A and one each from Sets C and D), leaving 49 participants in each set. All but four participants (two in Set B and two in Set C) reported being native speakers of English . We also confirmed that these participants were not pressing the same key on every trial nor were they responding quickly.What is the last name of the criminal who was killed by FBI agents outside of a Chicago movie theater”, to which the correct answer is Dillinger, names of criminals who were called “Public Enemies” by the FBI, just as Dillinger was, were listed as foil choices. The The same sets of questions were used. For each question, three alternative, incorrect responses were added. For set A, the foils were developed in the lab or were selected from the online quizzes where provided. For the questions in Set B, the Burke et al.  foils weThe procedure was very similar to that used in Experiment ρ(419) = .81, p < .001, although not perfect, indicating some variability in performance as a function of the demands on the retrieval process. To examine whether item difficulty based on the cued-recall task also predicted accuracy in multiple-choice, we used the quartile bins based on cued recall accuracy from Experiment SEM = .02) for the most difficult questions to .51 (SEM = .02), to .69 (SEM = .02), to .86 (SEM = .02) for the easiest questions, F = 212.00, MSE = .03, p < .001, ηp2 = .60. At all levels of difficulty, performance was above chance, estimated at .25 given four alternatives . Thus, the questions that were difficult to answer in one format were similarly difficult in the other format.Item-level information is presented in Table ρ49 = .81, F = 8.84, MSE = 16433694, p < .001, ηp2 = .06. Correct responses  were faster than errors , F = 31.34, MSE = 12122700, p < .001, ηp2 = .07. The interaction was significant, F = 18.93, MSE = 12122700, p < .001, ηp2 = .12. For easy and moderately easy questions, correct responses were faster than errors for the easier items ; however, as difficulty increased, the pattern reversed, such that errors were slightly faster than correct responses for the most difficult items t(105) = 2.0, p = .047, d = 20. RTs for moderately difficult items did not differ as a function of accuracy, t(104) = 1.44, p = .15, d = .14.RTs to correct and incorrect multiple-choice questions were examined in a one-way ANOVA as a function of question difficulty using the cued-recall rank as a between-items factor Fig. . This anF = 1018.79, MSE = .014, p < .001, ηp2 = .71  = 19.41, MSE = .014, p < .001, ηp2 = .12. The interaction  x 4  mixed ANOVA. Unsurprisingly, accuracy was higher in the multiple-choice format  than in the cued-recall format , n = 23). Thus, there appear to be some GK items that are more affected by incorrect foil presentation than others; although we acknowledge a very small set in our database fit this description. Overall, items that are more accessible in a cued-recall task also seem to be more accessible in a multiple-choice task. One caveat of this conclusion is that different participants completed the two tasks; future studies aimed at more directly assessing the relative accessibility or availability of specific units of knowledge might use alternative methods, such as directly comparing free recall, cued-recall, and recognition in a within-participants design. Alternatively, methods such as Buschke’s . Although significant, the effect was not particularly large, accounting for less 6% of the variance. This pattern held in all sets , except for Set A , which included the most difficult questions, suggesting that education predicts general knowledge level across a range of difficulty, but not when items are largely inaccessible. There was no systematic relation between rate of DR responses and education (r = .01). Years of education were negatively correlated with DK responses for all sets , in which DK was the modal response. This suggests that some items are simply not known and do not depend on educational attainment. Note that the items in Set D were specifically selected based on young adult norms to be especially difficult. In the multiple-choice task, there was an overall modest positive correlation between education and accuracy . Within each set of questions, the correlation was only significant for participants in Set C , which had the highest rate of correct answers overall. Thus, education, not surprisingly, does affect accuracy and presumably the knowledge base of participants.One important way in which the samples tested here and those included in the previous studies differ concerns student status and overall educational attainment. Whereas participants in Tauber et al.  and NelsAccording to several theories of intelligence


Substituent effects were interpreted in terms of the Hammett equation, showing a good correlation for carbons para- to the substituent group, not for the meta- ones. Similarly, the Lynch correlation shows the additivity of the substituent chemical shifts in the case of both H and C nuclei, again with the exception of the carbons in the meta- position. Density Functional Theory (DFT)-predicted 1H and 13C chemical shifts correspond closely with experimentally observed values, with some exceptions for C NMR data; however, the correlation is valid only for the aromatic moiety and cannot be extended to the heterocyclic ring of the chroman-4-one scaffold.The attribution of  Some time ago, pioneering studies demonstrated that Hammett parameters can be correlated, not only with reaction rates and equilibrium constants, but also with ultraviolet absorption spectra, infrared frequencies, and NMR chemical shifts [P Hammett parameters were successfully correlated with the chemical shifts of 1H and 13C NMR spectra in solution of a series of tetrabutylammonium iron(III) porphyrin complexes, in turn revealing how the electronic effect of the substituents affected the electronic configuration [F and σR) were employed as a molecular descriptor to characterize the substituent effects [Linear free-energy relationships (LFER) establish a direct correlation between thermodynamic parameters and kinetic data, offering insights into reaction mechanisms. LFERs create a connection between several measurable quantities and well-known parameters, Hammett substituent constants  of a given substituent on the electron density at the portion of molecule under study. Accordingly, there should be a direct correlation with any property affected by the electron density at the considered site. Therefore, the rationalization of substituent effects propagation in a molecule leads to a deep understanding of structure-property relationships and shines light on reaction mechanisms, two essential concepts in chemistry ,9,10.1H and 13C chemical shift values [0 is the chemical shift value of the unsubstituted compound [Frequently, NMR spectroscopic parameters are used to understand the transmission of substituents electronic effects within organic molecules, with a specific focus on routinely used t values ,12,13. TWhen good linear correlations are obtained applying Equation (1), the substituent effect on the chemical shift values can be considered electronic in origin, and the electron density around the nucleus under study is mostly affected by the electron-donating or -withdrawing ability of the substituent itself.In recent years, computational chemistry, in particular Density Functional Theory (DFT), has provided strong support to experimental chemists, giving insight into the properties and reactivity of different molecular entities. This is particularly apparent in the realm of organic chemistry, where the behavior of a given molecular group  can be tuned by the nature of the attached substituents, if any. On one hand, it has been demonstrated that DFT can reproduce nicely the experimental data and, on the other one, it may have a predictive value as well, e.g., in those cases where experimental data are not available ,15,16,17Herein, we report the application of these methods to 2,2-dimethylchroman-4-one derivatives, for which substituent effects have not been previously assessed. The chroman-4-one  ring system occupies an important position among oxygen heterocycles, as it is featured in a wide variety of compounds endowed with biological, medicinal, and synthetic interest ,22,23,24With the objective of understanding the factors underlying the transmission of substituents effect within the chromanone system, a library of substituted 2,2-dimethylchroman-4-one derivatives  has been studied and their chemical structures are shown in 1H and 13C NMR spectroscopic data of 2,2-dimethylchroman-4-one derivatives from Series 1–3 [Lynch correlations to evaluate the proportionality relationships with respect to the appropriate substituent chemical shift (SCS) in monosubstituted benzene; and (ii) Hammett correlations to show the resonance and inductive transmission of substituent electronic effects. Finally, prediction of the 1H and 13C nuclei chemical shifts by using DFT calculations has been also undertaken, followed by an analysis of the existing correlation among theoretical and experimental data.Based on our previous work and after an accurate survey of the literature, we decided to collect the ries 1–3 ,31,32. I1H chemical shifts of chroman-4-one derivatives belonging to Series 1, 2 and 3  displays a deshielding effect for all the studied compounds, as can be inferred from the data collected in the tables. Thus, this signal is located at ca. 2.70 ppm in the 1H-NMR spectra, as a result of the effect of both the carbonyl and the alkoxy groups belonging to the chroman-4-one scaffold. However, the substituents attached to the phenyl moiety  of the 6-substituted derivatives (Series 1) are those expected for this substitution pattern, with values of J5,7 from 1.6 to 2.4 Hz and J7,8 from 8.0 to 8.6 Hz. The J constants of compounds belonging to Series 2 (substituted in the 7-position) show values of J5,6 from 8.0 to 8.8 Hz and J6,8 from 1.0 to 2.4 Hz, while those of Series 3 (substituted in the 5-position) show values of J6,7 from 8.0 to 8.8 Hz and J6,8 from 1.0 to 2.4 Hz.The experimental nd 3 see  are repond 3 see  —SM—. Thety R in  do not p13C chemical shifts of the 2,2-dimethylchroman-4-one derivatives from Series 1, 2 and 3 are reported in 13C chemical shifts of the methylene group in the 3-position and the methyl groups in 2-position were observed, as apparent from the data collected in the tables. A “normal” substituent effect on the 13C chemical shift of the carbonyl group was found for the compounds in Series 2, as can be judged from the deshielding effect on moving from electron-releasing to electron-withdrawing substituents, viz. 190.9 ppm for -NMe2 group vs. 195.0 and 201.3 ppm for -NO2 and -Cl substituents, respectively. On the other hand, no substituent effect on the same group was observed for derivatives from Series 1 and 3, due to a noticeable spread of the data. In addition, the substituent effect is nicely observed on ortho- and para- carbons for all the 2,2-dimethylchroman-4-one derivatives studied, while the same effect on the meta- carbons showed no linear trends due to the spread of the data (see below).The 1H and 13C chemical shifts of the investigated 2,2-dimethylchroman-4-ones according to the Lynch correlation was carried out [EXP is the substituent chemical shifts (SCS) of 1H or 13C nuclei observed in 2,2-dimethylchroman-4-one derivatives, while Δδ(Benzene) is the SCS of 1H or 13C nuclei observed in monosubstituted benzene; a is the slope that compares the substituent effect on both aromatic moieties and b is the intercept. As an example, 1H nuclei of chroman-4-one derivatives belonging to Series 1, while the same analysis for the 13C nuclei of the aromatic ring is shown in Analysis of substituents effect on the ried out . This ana values < 1) than in the monosubstituted benzene ring and this is particularly apparent for compounds belonging to Series 1.The obtained Lynch correlations are collected in 13C NMR data depicts a different scenario, since the correlation cannot be extended to all the carbon atoms. In particular, the carbon atoms meta- with respect to the substituent R show a spread along the vertical axis for all the series and, accordingly, were not further considered in the data set  − 0.04, with a R-square value of 0.90, while for 13C nuclei chemical shifts (carbons meta- to the substituent R were not considered), the Lynch correlation afforded the following relationship: Δδexp = 1.0 Δδ(Benzene) − 0.1, with a R-square value of 0.90. These results reinforce the conclusions for each series previously described.Notably, cumulative Lynch correlations of ries see  in SM co13C chemical shifts as the probe to analyze this type of LFER. This choice was based on the advantages of carbon atoms over their hydrogen counterparts because 13C nuclei span over a larger chemical shift range and smaller dependence on medium effects is commonly observed. Thus, looking for a correlation involving 13C chemical shifts of chroman-4-one derivatives grouped in Series 1–3, different Hammett parameters have been screened, including σp+, σm and σI values [p+ Hammett parameters, when carbon atoms in the para- position with respect to the substituent R  were considered. The choice of the σp+ Hammett parameter is related to the specific position of the analyzed carbons, as well as to the presence in the chromanone scaffold of both the carbonyl and alkoxy groups attached at positions C-4a and C-8a, respectively. Accordingly, the attached R substituent can interact directly with the alkoxy or carbonyl groups through resonance effect [p+ Hammett parameter [0 around 160 ppm) Series 2 and 3 show very similar δ0 values (around 120 ppm), as a consequence of the different arrangement of the R substituent with respect to the carbonyl and the alkoxy groups of the chroman-4-one skeleton.Linear Hammett correlations for 2,2-dimethylchroman-4-one derivatives have been also investigated, adopting solely e effect ,35,36. Tarameter . Notably13C chemical shifts of carbon atoms in the meta- position , indicating that the chemical shifts are sensitive to the inductive effect of the substituent attached to the aromatic ring. A similar behavior was observed for the C-8a of compounds in Series 2 with both Hammett parameters. On the other hand, no correlation (R-square consistently < 0.4) with either σm or σI Hammett parameters was observed for C-8 of Series 1, C-5 of Series 2 and for both C-7 and C-8a of Series 3. This behavior can be attributed to the fact that the electronic effect of the substituent is poorly transmitted to meta-carbons, resulting in modest chemical shift changes (<3 ppm range). Accordingly, subtle non-electronic effects on the chemical shift may have a role, preventing to obtain a correlation with Hammett parameters.Next, we analyzed Hammett correlations of tion see , by choo1H and 13C chemical shift values have been collected in 1H chemical shift values (δTHEOR) and the experimental ones (δEXP), the corresponding plots being shown in The results of DFT calculations for all the 2,2-dimethylchroman-4-one derivatives belonging to Series 1, 2, and 3 in terms of predicted ortho-, meta- or para-) with respect to the substituent group R, nice linear correlations between the predicted and experimental H-NMR chemical shifts have been obtained with slopes near unity (between 0.96 and 1.16) and R-square ≥ 0.95 . Thus, the nitro group is markedly tilted with respect to the aromatic plane in the modeled conformer of 3f , to Series 3  and, finally, to Series 2 , increasingly higher chemical shifts values can be expected. Moving to para-hydrogens  reported in 1e and 1j, which clearly deviate from the set of the other chroman-4-ones, albeit no specific geometric features can be related to such anomalous behavior. Furthermore, it is important to point out that the reported data have been calculated by considering the average chemical shift of the non-equivalent hydrogens , as obtained from the calculation carried out on the most stable conformer of each compound.On the other hand, methylene groups of chroman-4-one derivatives give a worse linear correlation d than th13C chemical shifts, and the plots are shown in ipso-, meta-, and para- with respect to the substituent R for all the examined Series, with some exceptions in the case of ortho-carbons. The R-square values of the linear correlations are shown in ipso-carbons in Series 1–3 and the obtained slope value (0.88) is lower than unity. Indeed, the presence of a certain spread of data was found, resulting in a quite poor linear correlation (R-square = 0.75). On contrast, good linear correlations were obtained for the ortho-carbons in Series 2 and 3 (not in Series 1), as well as meta- and para-carbons in Series 1–3  of Series 1–3 and the plots are given in It is worth noting, however, that in the case of bons see c. FinallThe synthesis and full characterization of the 2,2-dimethylchroman-4-one derivatives belonging to Series 1–3 have been reported in previous studies ,31,32.3)4Si) calculated at the B3LYP/6-311+G level of theory as reference.DFT calculations were carried out with the Gaussian09 (rev. D.01) software  . All the1H and 13C NMR Lynch correlations, an estimate of the substituent effect transmission on the nuclei of 2,2-dimethylchroman-4-ones compared to the case of monosubstituted benzenes can be inferred. The data reported for the first time in this work clearly reveal that in all cases good linear correlations were found. Indeed, 1H NMR Lynch correlations showed slope values of 0.90 (for Series 1), while Series 2 and 3 consistently gave a slope around 0.95. Likewise, application of Lynch equation to 13C NMR data showed good linear correlations and the slopes were found to be between 0.94 and 1.05, demonstrating again that the substituent effect is well transmitted, similarly to the case of monosubstituted benzenes. However, the carbons meta- to the substituent R showed no correlation.From the analysis of the p+ Hammett parameters and the carbon atoms in the para-position with respect to the substituent R belonging to Series 1–3 and, in all cases, the ρ values were positive indicating that the chemical shifts are sensitive to the electronic effects of the substituents attached to the aromatic ring. Indeed, the resonance effect of electron-donating groups causes upfield chemical shifts, while that of electron-withdrawing groups causes downfield chemical shifts. However, for the case of the 13C nuclei in meta- position with respect to the substituent R in Series 1–3, the correlation with σm and σI Hammett parameters demonstrated that these carbon atoms are not sufficiently sensitive to the substituent inductive effect. This behavior can be attributed to the fact that the chemical shift changes on meta- substitution are in every case of comparable magnitude (<3 ppm range), therefore subtle non-electronic effects on the chemical shift may have to be postulated.On the other hand, good linear correlation fittings were observed between the σortho- to the substituent). These results demonstrate that the prediction of the chemical shifts of the aromatic hydrogen and carbon atoms using the adopted theoretical approach is accurate, in turn leading to a good estimation of the desired values.Finally, nice linear dependences between the theoretically predicted and experimental hydrogen and carbon NMR chemical shifts were obtained with slopes near unity, with only a few exceptions in the case of C-NMR (notably for carbon atoms


Staphylococcus aureus (SA) belonging to the clonal complex 398 (CC398) took a special place within the species due to its spread throughout the world. SA CC398 is broadly separated in two subpopulations: livestock-associated methicillin-resistant SA (MRSA) and human-associated methicillin-susceptible SA (MSSA). Here, we reviewed the global epidemiology of SA CC398 in human clinical infections and focused on MSSA CC398. The last common ancestor of SA CC398 was probably a human-adapted prophage φSa3-positive MSSA CC398 strain, but the multiple transmissions between human and animal made its evolution complex. MSSA and MRSA CC398 had different geographical evolutions. Although MSSA was present in several countries all over the world, it was mainly reported in China and in France with a prevalence about 20%. MSSA CC398 was frequently implicated in severe infections such as bloodstream infections, endocarditis, and bone joint infections whereas MRSA CC398 was mainly reported in skin and soft tissue. The spread of the MSSA CC398 clone is worldwide but with a heterogeneous prevalence. The prophage φSa3 played a crucial role in the adaptation to the human niche and in the virulence of MSSA CC398. However, the biological features that allowed the recent spread of this lineage are still far from being fully understood. Staphylococcus aureus (SA) is a leading cause of morbidity worldwide [orldwide . During orldwide . Recent orldwide ,3,4. SevIn this review, we first discussed the evolution of SA CC398 and its epidemiology in clinical infections in human and then focused on methicillin-susceptible SA (MSSA) CC398.Although a history of exposure to livestock has been identified as one of the main risk factors for human infections with CC398, an increasing number of cases are emerging in patients with CC398 infection with no such history ,6,7. EpiMicroarray studies of CC398 have shown a distinction between livestock-associated infections and those associated with human-to-human transmission in terms of their distribution of mobile genetic elements (MGEs) and antibiotic resistance [tet(M) gene, and methicillin resistance with the presence of SCCmec, while Hu-MSSA CC398 had erythromycin resistance with erm(T) gene.SA CC398 was then separated into two subpopulations: a human (Hu) clade and a livestock-associated (LA) clade. The two clades are characterized by different prophages. LA-MRSA CC398 isolates commonly carry phages φ2, φ6, or φAvb ,10,11. Bmec, leading to the emergence of human MRSA CC398 strains. At some point, ancestral MSSA CC398 strain jumped to livestock, which was accompanied by the loss of IEC and the acquisition of tet(M), conferring resistance to tetracycline and later on by the acquisition of SCCmec, conferring resistance to methicillin, leading to the emergence of livestock-associated MRSA CC398. However, a more recent quantitative time-scaled phylogeny, indicated that both Hu and LA-SA CC398 emerged in parallel around 1970 [The advent of whole-genome sequencing (WGS) has allowed the reconstruction of the evolution of this clone. WGS analyses revealed a possible human origin for LA-CC398, followed by the emergence of methicillin and tetracycline resistance driven by antibiotic pressure in animal . Indeed,und 1970 . Ward etund 1970  supporteund 1970 . In the und 1970 .Finally, in view of the frequent transfers of CC398 between humans and animals, with some differences according to the country, a detailed analysis of the dissemination of CC398 between individual countries with a same sampling methodology should be done, to better understand the evolution and the spread of this clone.We proposed a model of evolution of SA CC398, into the two different subpopulations: animal and human clade . The aniThe limited molecular epidemiology of large collections of MSSA has probably precluded an accurate assessment of the global spread of CC398 , human SA variety of MRSA CC398 infections has been documented, ranging from relatively minor or localized infections (SSTI) to more serious or invasive infections (including bloodstream infection (BSI), osteomyelitis, pneumonitis, and endocarditis) . DespiteThe analysis of the publications that have specified the clinical diagnosis of SA CC398 infections is summarized in In France, since the first description of MSSA CC398 by Armand-Lefevre et al., 16 publications with data on MSSA CC398-related infections have been published, and the proportion of CC398 among total MSSA infections ranged from 1.9 to 38.3% depending on the date of the study and type of infections ,36,37,78A population-based survey of patients with MSSA infective endocarditis 2008) and MSSA BSI 2006–2007) revealed that 7 of 170 (4.1%) MSSA isolates belonged to CC398 . Valenti revealed08 and MSConcerning BJI, a retrospective study in 2008 found that only 1.9% of MSSA were ST398 . Valour SA lineage responsible for infections in France.In patients with diabetic foot infection (DFI), MSSA ST398 represented 26 of 166 (15.7%) and was the main lineage isolated. Moreover, it was identified exclusively in cases with osteomyelitis . These rSA BSI episodes in a multicenter study in The Netherlands that only retrieved two MSSA CC398 (0.3%) isolates during the period 2010–2011 [SA isolates in intensive care units  in this country [In Europe, MSSA CC398 represented 2.1% of the 565 MSSA isolates with data of spa typing from invasive infections (mainly BSI) collected in 26 countries during the period 2006–2007 . These i010–2011 . Rijnder country . In Belg country . In sout country . In Gree country . Then, S country .SA lineage in China with a prevalence varying from 5.5 to 26.6% [As observed in France, MSSA CC398 is the most prevalent to 26.6% ,62,63,64to 26.6% . These rto 26.6% . In patito 26.6% ,58. The spa type t571 has been described in Colombia as causing infection in a woman without livestock association and in Ecuador from a patient with BSI [MSSA CC398 infections are also observed in America. In South America, MSSA ST398 with BSI ,72. Fromwith BSI . Anotherwith BSI . In DomiIn the United States, most cases of MSSA CC398 infections are reported in New York City ,76,77. IFinally, MSSA CC398 was retrieved all over the world but with different prevalence depending on the country. France and China were countries where this clone has been most studied and where it is most frequently isolated from human infections. However, most countries do not perform MSSA surveillance, and the disease burden of MSSA CC398 at the national and international level is therefore largely unknown.In France and China, respectively, the two most affected countries, MSSA CC398 is responsible for BSI, ranging from 4.1 to 13.4%, and from 5.4 to 14.9% of all BSI ,59,60,61SA CC398 isolates were significantly more frequent in osteomyelitis than in SSTI patients with diabetic foot infection, suggesting a particular tropism of this clone for bone [BJI related to CC398 included principally prosthetic joint infection (PJI) and diabetic foot osteomyelitis (DFO) . MSSA CCfor bone . Howeverfor bone .SA pneumonia in pediatric inpatients was due to MSSA CC398 [MRSA CC398 seems to be significant in respiratory tract infections (RTI) . It coulSA CC398 . RTI wasSA CC398 ,20,50. TSA CC398 ,68.SSTI seems to be the predominant infection linked to MRSA CC398. However, this over-representation could be biased due to the specific surveillance of MRSA infections in several countries, which lacks for MSSA. Indeed, in a French study of 105 clinical isolates of MSSA CC398, SSTI was the most represented infection (35% of cases), followed by BSI with 26.5% of cases . Howeverp < 0.01) [In the study of Bonnet et al., MSSA CC398 BSI were associated with an intravascular device in 56.3% of the cases vs. 31.6% in non-CC398 MSSA BSI . Similar < 0.01) . Moreove < 0.01) . These d < 0.01) ,37. More < 0.01) . PrevaleSA infections, many case reports of patients with MSSA CC398 described patients with predisposing conditions . However, some studies showed different characteristics from patients with MSSA CC398 and non-CC398. Indeed, it has been reported that BSIs due to MSSA CC398 more frequently affected fragile patients with a history of neurological disease [Like most  disease . Similar disease . In the  disease .p = 0.5) [p = 0.035) and lower treatment failure rates  than other CCs [As mentioned above, MSSA CC398 causes a wide range of infections, including BSIs. Many studies have reported MSSA CC398 deaths in France. A lethal necrotizing pneumonia caused by Panton–Valentine leukocidin (PVL) producing MSSA CC398 was described in a previously healthy 14 year-old girl . AnotherWhereas MRSA CC398 were frequently multiresistant to antibiotic, including tetracycline, secondary to the wide use of this antibiotic in the pig industry, MSSA CC398 was more often only reported as resistant to erythromycin .ermT gene [ermT was absent in MRSA CC398 from animal origin [Uhlemann et al. reported that MSSA CC398 isolates retrieved in humans in New York (USA) were almost always resistant to erythromycin (97%) and clindamycin (97%) [rmT gene . Interesl origin .tet(M). Indeed, only four MSSA CC398 studies showed tetracycline resistance with the presence of tet(M) gene [Tetracycline resistance was rarely described in MSSA isolates but was frequent in MRSA CC398 due to the presence of (M) gene ,31,40,42SA CC398 were initially distinguished by their peculiar resistance to digestion by SmaI, the restriction enzyme most frequently used for PFGE typing [sak (coding for a staphylokinase), chp , scn , sea  and sep . However, only a small proportion of the MRSA CC398 isolates retrieved from infected patients contains the φSa3-associated IEC [SA CC398 had a higher capacity for binding to human cytokeratin-10 than LA SA CC398, mediated by the clumping factor B (ClfB) and very likely important for nasal colonization of humans [SA to that of a naive SA animal colonizer increased the transcription of clfA (encoding the clumping factor A precursor) and fnbA (encoding the fibronectin binding protein), which in turn increased the bacterial virulence in a rat model of infectious endocarditis [SA CC398.E typing . InvestiE typing ,35. HoweE typing . This liE typing . Most ofated IEC ,85,86,87ated IEC . Interescarditis . This stThe PVL-encoding gene is usually absent in MSSA CC398 genomes, but the spread of a PVL-producing CC398 subpopulation in China confirmed the capacity of this clone to acquire virulence genes ,61,62,63bbp, clfA, clfB) were reported in the genomes of isolates responsible for DFI [Other virulence factors have been described in CC398 isolates. The presence of genes encoding hemolysins and intracellular adhesion proteins, cap5, together with three genes encoding MSCRAMM  are still far from being fully understood.The spread of the MSSA CC398 ancestral clone, and/or the transmission of a human-adapted clone from animals, played a role in the emergence of