DNA methyltransferase 1 (DNMT1) is the enzyme primarily responsible for propagation of the methylation pattern in cells. Mutations in DNMT1 have been linked to the development of adult-onset neurodegenerative disorders; these disease-associated mutations occur in the regulatory replication foci-targeting sequence (RFTS) domain of the protein. The RFTS domain is an endogenous inhibitor of DNMT1 activity that binds to the active site and prevents DNA binding. Here, we examine the impact of the disease-associated mutation A554V on normal RFTS-mediated inhibition of DNMT1. Wild-type and mutant proteins were expressed and purified to homogeneity for biochemical characterization. The mutation increased DNA binding affinity ~8-fold. In addition, the mutant enzyme exhibited increased DNA methylation activity. Circular dichroism (CD) spectroscopy revealed that the mutation does not significantly impact the secondary structure or relative thermal stability of the isolated RFTS domain. However, the mutation resulted in changes in the CD spectrum in the context of the larger protein; a decrease in relative thermal stability was also observed. Collectively, this evidence suggests that A554V disrupts normal RFTS-mediated autoinhibition of DNMT1, resulting in a hyperactive mutant enzyme. While the disease-associated mutation does not significantly impact the isolated RFTS domain, the mutation results in a weakening of the interdomain stabilizing interactions generating a more open, active conformation of DNMT1. Hyperactive mutant DNMT1 could be responsible for the increased DNA methylation observed in affected individuals. In mammals, DNA methylation occurs predominately on cytosine bases within CpG dinucleotides. Proper DNA methylation patterns are important for normal cellular function. Aberrant methylation patterns have been observed in several diseases including cancer and neurodegenerative disorders -3.S-adenosylmethionine to the 5-carbon of cytosine. DNA methylation patterns are established during germ cell development by DNMT3a and DNMT3b. For this reason, these isozymes are often referred to as de novo methyltransferases. They are aided by DNMT3L, a catalytically inactive regulatory factor that enhances their activity. Methylation patterns are maintained through multiple rounds of cell division by the maintenance methyltransferase DNMT1. This isozyme exhibits specificity for methylation of hemimethylated DNA, DNA in which only one strand of the duplex contains methylcytosine [DNA methylation patterns are established and primarily maintained by a family of proteins known as DNA methyltransferases (DNMTs). Humans have three active enzymes that methylate DNA: DNMT1, DNMT3a, and DNMT3b. These enzymes all catalyze the transfer of a methyl group from the methyl-donating co-factor cytosine . For exacytosine .DNMT1 is a large multidomain protein consisting of a C-terminal methyltransferase domain and an N-terminal regulatory region connected by glycine-lysine repeats . The N-tDNMT1 have been reported that result in adult-onset neurodegenerative disorders in humans [Over the past decade, several mutations in n humans -22; all Methylation profiling has shown drastic changes to the normal DNA methylation pattern in affected individuals ,24. Howe2.2.1.Expression constructs for truncated forms of human DNMT1 were previously created . Wild-ty2.2.v/v) glycerol at −80 °C. Proteins were quantified using A280 and calculated extinction coefficients. Size-exclusion chromatograms and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) of pure proteins and domains are shown in Following previously published procedures, wild-type and mutant proteins and domains were expressed in Rosetta 2(DE3) pLysS competent cells . All pro2.3.DNA binding affinity was measured by fluorescence polarization using a fluorescently tagged 18 base pair hemimethylated duplex DNA as previously described . In shorPolarization values were normalized and averaged for visualization.2.4.S-adenosylmethionine, and 0.5 μM RFTS-containing DNMT1. Samples were removed and flash frozen 2, 8, and 20 min after enzyme addition, subsequently digested with Sau3A1  and separated on 18% PAGE. The gel was stained using ethidium bromide and imaged with a Bio Rad Gel Doc EZ system.Activity of RFTS-containing DNMT1 was assessed using a previously described endonuclease-coupled DNA methylation assay . Assays 2.5.m). Fluorescence data were normalized for visualization.DSF was used to examine the thermal stability of RFTS-containing DNMT1 proteins as previously described . Assays 2.6.http://bestsel.elte.hu, accessed on 4 April 2023) [m. RFTS-containing DNMT1 data were collected at a concentration of 1 μM (0.14 mg/mL), while RFTS domain data were collected at a concentration of 5 μM (0.15 mg/mL) in 20 mM KPi (pH 7.5) buffer.CD data were recorded on a Jasco J-1500 spectrometer using a 0.1 cm path length cuvette. Spectral accumulation parameters included a scanning rate of 50 nm/min with a 1.0 nm bandwidth over a wavelength range of 190–260 nm; spectra were initially collected at 25 °C. Each spectrum at 25 °C was obtained from an average of 3 scans, corrected for buffer contributions, and smoothed using the Jasco Spectra Manager software, version 2. Secondary structure analysis of the resulting CD spectra was performed using BeStSel (il 2023) ,28. All 3.Escherichia coli as an N-terminally truncated form of human DNMT1 that begins with the RFTS domain, termed RFTS-containing DNMT1 (amino acids 351–1616). Previous work has shown that truncating the extreme N-terminus of DNMT1 does not impact the protein’s behavior in in vitro assays [E. coli. We were able to obtain pure wild-type and mutant truncated proteins and protein domains for biochemical characterization , the RFTS mutation seems to decrease the thermal stability of RFTS-containing DNMT1. Additionally, this loss of stability is also observed in melting curves obtained by differential scanning fluorimetry (DSF) (m of the mutant protein. The DSF data were obtained with a slightly faster temperature ramp (2.4 °C per minute), yet the difference in relative stabilities is still observed. Taken together, these data indicate a decrease in relative thermal stability for A554V RFTS-containing DNMT1 even though the mutation did not impact the relative stability of the RFTS domain alone. We suggest this difference is due to a weakening of normal RFTS-mediated inhibition in A554V RFTS-containing DNMT1. Loss of the interdomain stabilizing interactions would make it easier to unfold the mutant protein, even if interactions within the isolated domain were not significantly impacted.We again used changes in CD to track the unfolding of the RFTS-containing DNMT1 proteins. For these proteins, large changes in CD signal were observed at the 193 nm peak and near the minimum at 208 nm as temperature was increased ,b. Similelengths . Thus, wry (DSF) . This da4.DNMT1 is the enzyme primarily responsible for maintenance of the DNA methylation pattern following cell division. The C-terminal methyltransferase domain is responsible for the catalytic activity of DNMT1; however, the first ~1100 amino acids of the protein are critical for proper regulation, targeting, and activation . DNMT1 iE. coli. The CD spectrum of the A554V RFTS domain was very similar to that of the wild-type protein, indicating that the mutation did not significantly impact the structure of the domain. In addition, A554V RFTS-containing DNMT1 was able to bind and methylate DNA in our studies, indicating that the folding of the larger protein was not severely impacted either. Similarly, previous work has shown that ACDA-DN-associated mutations G589A and V590F, found in the same region of the domain as A554V, did not significantly impact the folding of DNMT1 [E. coli [Here, we report our investigation of the impact of A554V, a mutation associated with development of ADCA-DN, on normal RFTS-mediated autoinhibition of DNMT1. Mutant RFTS-containing DNMT1 and the RFTS domain were able to be expressed as soluble proteins in of DNMT1 . This isof DNMT1 ,22. Furt[E. coli . Taken tNormal RFTS-mediated autoinhibition of DNMT1 activity is impacted by the disease-associated mutation. A554V RFTS-containing DNMT1 bound DNA ~8-fold tighter than the wild-type enzyme. This increase in DNA binding affinity is even larger than that reported for the other ADCA-DN-associated mutations. Those mutations resulted in ~2.5–3.5-fold increases in affinity , suggestCD investigations revealed that mutant RFTS-containing DNMT1 had decreased thermal stability relative to its wild-type counterpart, while the relative stability of the RFTS domain was unaffected by the mutation. Moreover, the CD spectrum of the A554V RFTS domain was very similar to that of wild type, indicating that the mutation did not significantly affect the structure of the isolated domain. A554 is found in the helical bundle at the C-terminus of the RFTS domain . The intWhile the CD spectra of wild-type and mutant RFTS domain were very similar, noticeable differences were observed in the RFTS-containing DNMT1 proteins. Secondary structure analysis suggests that introduction of A554V results in a ~2% loss in helical content in DNMT1. Since the same change in secondary structure content was not observed in the purified RFTS domains, we suggest that this change in structure comes from a region outside of the RFTS domain itself. The RFTS domain is near a region of DNMT1 termed the autoinhibitory linker . RemarkaMethylation analysis of individuals with ADCA-DN showed increased DNA methylation. Normally unmethylated promoters and CpG islands were shown to be methylated in affected individuals. In addition, hypermethylated gene bodies and intergenic regions were further methylated in ADCA-DN patients . HyperacSupplement_DNA_Switzer


To address the challenge of declining pro-environmental behaviors in adolescence, this paper uses the theoretical foundations for subjective wellbeing to verify the influence of three latent dimensions of subjective wellbeing  on adolescents' pro-environmental consumption behaviors (PECBs). Furthermore, we explore the moderating effects of nations and regions in the relationship between subjective wellbeing and PECBs in adolescents.Based on the international data from the Programme for International Student Assessment 2018 (PISA 2018), we construct a unique dataset that includes 57,182 samples related to the subjective wellbeing and PECBs of 15-year-old students from eight countries/economies. Specially, we employ an ordered probit model to test our hypotheses.Both adolescents' life satisfaction and positive emotions can significantly improve their PECBs, while there is a significant negative association between negative emotions and PECBs. At the nation's level, adolescents' life satisfaction and negative emotions in developed countries/economies significantly impact PECBs. In contrast, the positive emotions of adolescents in developing countries/economies have more substantial effects on PECBs. At the regional level, the impact of all three dimensions of adolescents' subjective wellbeing on PECBs is more significant in rural than urban areas.This paper provides a new perspective for understanding adolescents' PECBs from the insights of subjective wellbeing. Previous studies have examined the effects of life satisfaction or happiness on PECBs in adults. This paper examines the impact of subjective wellbeing on adolescents' PECBs from life satisfaction, positive emotions, and negative emotions, which suggests that promoting adolescents' subjective wellbeing can be an effective strategy for encouraging PECBs. From a comparative research perspective, we further analyze the differences between the nations at different levels of development, the rural and urban areas, providing a valuable reference for policymakers and practitioners in promoting pro-environmental behaviors among adolescents. Adolescents and youth are the largest generations in history, at over 1.8 billion . The choPro-environmental consumption behaviors (PECBs) are actions taken by individuals that prioritize environmental sustainability in their consumer choices , 18. A lResearch in positive psychology suggests that those with higher levels of subjective wellbeing are more willing to give more money or time to engage in socially beneficial behavior, while those with lower levels of subjective wellbeing are often under urgent pressure to make the modest lives and are overwhelmed by the need to defend the public interests –43. But Some scholars suggest that happier people are more willing to pay more or change their consumption behaviors to protect the environment , 44–48. In response to a growing call for research, several scholars have explored whether adolescents' increased happiness or life satisfaction would fare better in PECBs . NonetheOn the one hand, although a growing body of literature has focused on the relationship between subjective wellbeing and PECBs, subjective wellbeing is regarded as a one-dimensional structure in the existing literature . SubjectOn the other hand, the existing research that has been conducted usually focuses on one country or region, and there is a lack of international and cross-regional comparative studies. Moreover, extensive research has focused on developed countries, whereas research on those in developing countries has yet to be explored, especially the comparative studies during different development levels of nations. In addition, comparative studies of PECBs in adolescents in different areas, such as urban and rural areas, remain a puzzle.To shed light on these puzzles, using a sample of 57,182 adolescents across eight countries/economies, we explore whether adolescents' PECBs are associated with all of the three components of subjective wellbeing by the ordered probit model. Furthermore, we explore the differences between developing and developed countries/economies, and between rural and urban areas.This paper is structured as follows. Section 2 presents the literature overview of subjective wellbeing and PECBs and proposes the hypotheses. In Sections 3, we describe the data sources, variables, and empirical model. Section 4 reports the general results and the mechanism analysis of the moderating effects. In Section 5, we further discuss the findings. Finally, conclusions are drawn in Section 6.Adolescents' lifetimes, in particular, will face the environmental consequences of actions taken or not taken today. Their active engagement in understanding the state of our environment (and the importance of protecting it) is critical to achieving sustainable development globally . PECBs aFor adolescents, United Nations Environment Programme (UNEP) suggests they can influence the market by buying or not buying . PECBs aLife satisfaction is a subjective measure of one's overall sense of fulfillment and contentment with their life , 45, 54.Moreover, individuals with high life satisfaction may be more likely to be influenced by social norms that promote PECBs. Ouyang et al.  suggesteH1a. Adolescents' life satisfaction is significantly and positively correlated with their PECBs.Positive emotions are subjective experiences that are typically pleasant and enjoyable. These emotions include feelings such as happiness, joy, contentment, gratitude, and hope , 56. ResNotably, the influence of positive emotions on PECBs has been identified in the literature. Yuan , BrionesH1b. Adolescents' positive emotions are significantly and positively correlated with their PECBs.Negative emotions are subjective feelings that can cause one to be miserable and sad, such as hate, afraid, anger, jealousy, and sadness , 67. NegAlthough research has suggested that negative emotions, through fear, anger, guilt, shame, etc., help to evoke environmentally responsible behaviors . In factH1c. Adolescents' negative emotions are significantly and negatively correlated with their PECBs.Nations worldwide are usually divided into developed and developing countries/economies based on their economic development, wealth, and wellbeing. The level of development of a country or economy is linked to the long and healthy life, access to knowledge, and standard of living of its people, which are also significant predictors of PECBs , 79.In developed countries/economies with high levels of material wealth and availability of resources, people might be more selective about the products they purchase , 47, 80.Conversely, in developing countries/economies where poverty, inequality, and environmental degradation are pressing issues, individuals' subjective wellbeing may be more tied to stable access to basic needs of survival , 80. TheH2a. Compared to the group of developing countries/economies, the effect of adolescents' life satisfaction on PECBs is more substantial in developed countries/economies.H2b. Compared to the group of developing countries/economies, the effect of adolescents' positive emotions on PECBs is more substantial in developed countries/economies.H2c. Compared to the group of developing countries/economies, the effect of adolescents' negative emotions on PECBs is more substantial in developed countries/economies.Just as there are significant differences in levels of development between countries, there are essential differences between rural and urban areas in terms of population density, economic activity, infrastructure, lifestyle, education, health care, and the environment , 82.In rural areas, communities have stronger social connections and a sense of community compared to urban areas. Ouyang et al.  argued tH3a. Compared to the group in the urban area, the effect of adolescents' life satisfaction on PECBs is more substantial in rural regions.H3b. Compared to the group in the urban area, the effect of adolescents' positive emotions on PECBs is more substantial in rural regions.H3c. Compared to the group in the urban area, the effect of adolescents' negative emotions on PECBs is more substantial in rural regions.The data for this paper comes from two different sources. One is the individual dimension data, 2018 Programme for International Student Assessment (PISA 2018), from Organization for Economic Co-operation and Development (OECD). The other is the national dimension data, the human development index (HDI), from the United Nations Development Programme (UNDP).The questionnaire from PISA 2018 is a comprehensive and rigorous international standardized assessment for adolescents, with both the multi-item measurements of subjective wellbeing and PECBs . PISA 20Table 3). Respondents are asked whether they have participated in PECBs in the questionnaire. We assign a value to each question answer. “No” is given a matter of “0”; “yes” is given a value of “1”.In the PISA 2018 student questionnaire, three question items are related to PECBs (see Table 3). Life satisfaction is measured by the four-point Likert scale of 10 question items, ranging from 1  to 4 . Positive emotions are measured by the four-point Likert scale of five question items, ranging from 1 (Never) to 4 (Always). Negative emotions are measured by the four-point Likert scale of four question items, ranging from 1 (Never) to 4 (Always). All three scales are tested reliability and validity. Cronbach's α for these scales are all >0.8, indicating that the reliability of scales is acceptable , which indicate the suitability of factor analysis. Factor loadings (FL) of each item above 0.5 indicate good construct validity . In order to test the moderating effect of the level of development of countries or economies, we classify them as developing countries/economies and developed countries/economies. The HDI, produced by UNDP since 1990, consists of three components: a long and healthy life, access to education, and a decent standard of living . The HDITo explore the impact of the level of national development on the PECBs of 15-year-olds, we set the data collection period from birth to the year the adolescents were surveyed, i.e., 2004 to 2018. We take the average value of the HDI for each country or economy over a total of 15 years, from 2004 to 2018. The results show that Hong Kong, Ireland, Spain, and the UAE have a mean HDI above 0.800 for these 15 years, while Bulgaria, Mexico, Panama, and Serbia have HDIs between 0.700 and 0.799. According to existing studies , HDI aboAs for the regions, in PISA 2018, the communities in which students' schools are located are divided into five categories by population see . We furtThe individual demographic characteristics, such as gender and grade, are controlled. Moreover, according to Ouyang et al. , we furtGiven that the dependent variable PECBs is an ordered variable, according to Ouyang et al. , Wang ani represents the individual. PECBsi, the dependent variable, stands for the PECBs of individual i. Subjectivewellbeingi, the set of the core explanatory variables, represents the subjective wellbeing of individual i. Xi is the set of control variables. Lastly, εi is an error term.where The regression results of the effect of the three dimensions of subjective wellbeing on PECBs in adolescents are reported in Furthermore, at the individual level, the grade level, gender, and environmental knowledge of adolescents all significantly impact PECBs. The research sample for this paper is 15-year-old school students of the same age and in grades ranging from 7th to 12th. The results show that the higher the grade level of the adolescents, the better their performance on PECBs. At the same time, there is a significant positive correlation between adolescents' environmental knowledge and PECBs. The findings indicate that the promotion of environmental knowledge and academic knowledge helps adolescents to develop sustainable consumption behaviors that are conducive to environmental protection, which is consistent with Carmi et al. , Latif eEmpirical p-value in β = 0.173 , p < 0.001) to developing countries/economies . Thus, H2a is verified.We further examine whether the impact of adolescents' subjective wellbeing on PECBs differs between developing and developed countries/economies. At the same time, we test the significance of the differences between the three groups. The results show that the differences between all three groups are significant at the 0.001% level . Nevertheless, the impact of negative sentiment on PECBs is not significant in developing countries/economies. Thus, H2c is verified. Notably, on the dimension of positive emotions, adolescents' subjective wellbeing in developing countries/economies  has more substantial positive impacts on their PECBs than developed countries/economies . Thus, H2b is not verified.The effect of negative emotions on PECBs also confirms this result. The significant inhibitory effect of negative emotions on PECBs is more strongly manifested in developed countries/economies (Empirical p-value in On the other hand, we explore the moderating effect of region on adolescents' subjective wellbeing on their PECBs. The results of this paper show that life satisfaction and positive emotions are significantly associated with greater PECBs. In contrast, negative emotions are associated considerably with lower PECBs in adolescents, even after controlling for environmental knowledge and sociodemographic variables. There are consistently significant correlations between the three dimensions of subjective wellbeing and PECBs in developed countries/economies. However, in developing countries/economies, life satisfaction and positive emotions significantly affect PECBs, while negative emotions do not correlate substantially with PECBs. Interestingly, the significant impact of adolescents' subjective wellbeing on PECBs is more substantial in developing countries/economies than in developed countries/economies. More specifically, in developed countries/economies, adolescents' life satisfaction and negative emotions significantly impacted their PECBs more than in developing countries/economies. However, the positive sentiment of adolescents in developing countries/economies has a more significant impact on PECBs than in developed countries/economies.Furthermore, life satisfaction, positive emotions, and negative emotions are all significantly associated with PECBs in rural and urban areas. Notably, all these significant correlations are more substantial in rural than urban areas. The results indicate that adolescents in rural areas may have more opportunities to participate in community-based activities and get close to nature, which promotes sustainability and environmental protection. This can give them a sense of purpose and fulfillment, which may further reinforce their commitment to pro-environmental behaviors.These findings contribute to the existing literature in several ways. Firstly, our study offers new ideas for approaching the challenge of declining environmental behavior in adolescents , 12. OurThe finding that subjective wellbeing positively influences PECBs has important practical implications for promoting sustainable consumption and environmental protection. This paper contributes to understanding the psychological mechanisms underlying environmentally friendly behaviors. It suggests that promoting subjective wellbeing can be an effective strategy for encouraging PECBs in adolescents. Our findings also highlight the importance of fostering subjective wellbeing as a critical component of sustainable consumption policies and programs. By improving adolescents' subjective wellbeing, policymakers and practitioners can create an environment that fosters pro-environmental attitudes and behaviors, ultimately leading to more sustainable lifestyles and a healthier planet.More specifically, in developed countries/economies, more attention should be paid to the positive emotions of adolescents. In comparison, in developing countries/economies, more attention should be paid to the life satisfaction of adolescents and the impact of negative emotions on PECBs. Additionally, focusing on PECBs for adolescents in urban areas is essential. Policymakers, schools, and families should create more opportunities for young people to connect with nature and participate in environmental activities.The limitations should also be considered when interpreting the results of this paper. Firstly, our study uses cross-sectional data and could not provide any evidence of causality. There are few international research projects on adolescents' subjective wellbeing and pro-environmental behavior. We look forward to more long-term follow-up projects to examine the causal relationship and long-term effects between adolescents' subjective wellbeing and PECBs. The second limitation concerns the measurement of adolescent PECBs used in this paper are self-reported rather than actual pro-environmental behaviors. Moreover, all PECBs are measured on dichotomous yes or no scales. Future research would benefit from a more detailed classification and more accurate frequency measurements on PECBs. Last but not the least, the idiosyncratic attributes inherent to various nations, encompassing elements like political structures, societal contexts, and cultural legacies, exert a profound impact on the behaviors exhibited by adolescents. Consequently, a meticulous examination that takes into account the unique characteristics of each nation becomes of paramount importance, thereby highlighting a pivotal trajectory for forthcoming research undertakings. Last but not least, the distinctive attributes inherent to various countries/economies, encompassing elements like political structures, societal contexts, and cultural legacies, profoundly impact adolescents' behaviors. Consequently, a meticulous examination that considers the unique characteristics of each country/economy becomes paramount, thereby highlighting a pivotal trajectory for forthcoming research undertakings.In conclusion, our study confirms that all three components of subjective wellbeing, life satisfaction, positive emotions, and negative emotions, are significantly associated with adolescents' PECBs. Moreover, the effect of adolescent subjective wellbeing on PECBs differs substantially between developed and developing countries/economies, and between rural and urban areas. The findings of this paper provide new insight into the challenge of declining pro-environmental behaviors in adolescence.https://www.oecd.org/pisa/data/2018database/.Publicly available datasets were analyzed in this study. This data can be found here: MZ: conceptualization, formal analysis, writing—original draft, writing—review and editing, visualization, funding acquisition, and project administration. WZ: formal analysis, data curation, and visualization. YS: conceptualization, formal analysis, writing—original draft, writing—review and editing, funding acquisition, and supervision. All authors contributed to the article and approved the submitted version.


Membrane identity and dynamic processes, that act at membrane sites, provide important cues for regulating transport, signal transduction and communication across membranes. There are still numerous open questions as to how membrane identity changes and the dynamic processes acting at the surface of membranes are regulated in diverse eukaryotes in particular plants and which roles are being played by protein interaction complexes composed of peripheral and integral membrane proteins. One class of peripheral membrane proteins conserved across eukaryotes comprises the SEC14-like phosphatidylinositol transfer proteins (SEC14L-PITPs). These proteins share a SEC14 domain that contributes to membrane identity and fulfills regulatory functions in membrane trafficking by its ability to sense, bind, transport and exchange lipophilic substances between membranes, such as phosphoinositides and diverse other lipophilic substances. SEC14L-PITPs can occur as single-domain SEC14-only proteins in all investigated organisms or with a modular domain structure as multi-domain proteins in animals and streptophytes . Here, we present an overview on the functional roles of SEC14L-PITPs, with a special focus on the multi-domain SEC14L-PITPs of the SEC14-nodulin and SEC14-GOLD group . This indicates that SEC14L-PITPs play diverse roles from membrane trafficking to organism fitness in plants. We concentrate on the structure of SEC14L-PITPs, their ability to not only bind phospholipids but also other lipophilic ligands, and their ability to regulate complex cellular responses through interacting with proteins at membrane sites. PI and 3,4,5)P3 . They ar3,4,5)P3 . It is a3,4,5)P3 . Here, w2Lipids can be distributed in the cell through vesicle-independent trafficking, which includes spontaneous lipid transfer, flip-flop exchange within bilayers, lateral diffusion or single-lipid transfer by lipid-transfer proteins, that act, for example at membrane contact sites . On the Arabidopsis thaliana (Arabidopsis) the PITP/LNS2 domain can for example be found in two phosphatidate phosphohydrolase proteins involved in galactolipid synthesis and necessary to maintain membrane structure by lipid remodeling due to phosphate starvation (The first group is simply named the phosphatidylinositol transfer protein (PITP)-superfamily, defined through its e.g. phosphatidylinositol transfer protein and Lipin/Ned1/Smp2 (PITP/LNS2) domain (InterPro accession number (IPR): IPR031315). Such a domain is thought to promote the exchange of phospholipids at the membrane contact sites of the endoplasmic reticulum (ER) and the plasma membrane by non-vesicular lipid transport . Proteinarvation . These eArabidopsis thaliana.The second PITP-superfamily and subject of this review is defined through its SEC14 domain (IPR001251), named the SEC14-like phosphatidylinositol transfer protein (SEC14L-PITP)-superfamily. SEC14L-PITPs are able to recognize, bind, exchange and transfer small lipophilic molecules between membranes by non-vesicular transport . Additio310-helices; 2x distinct domains) (The SEC14 domain forms a characteristic hydrophobic phospholipid-binding pocket at its carboxy (C)-terminus . Yeast Sdomains)  and was domains) . An idendomains) . That isdomains) . A uniqudomains) , while odomains) . The alpdomains) . The abidomains) . The opedomains) . This fidomains) . The lipdomains)   . Phosphodomains)   . The SECdomains) . This sidomains)   . Upon a domains) .While the functions of several single-domain SEC14-only proteins are studied well, only a few multi-domain SEC14L-PITPs are characterized. At the same time, their abundance in higher eukaryotes highlights their importance. This raises several fundamental questions: Which are the pathways that single- and multi-domain SEC14L-PITPs are integrated in? What are the functions of the different domains within the multi-domain SEC14L-PITPs? Which protein-protein and protein-ligand interactions are relevant for the functions of SEC14L-PITPs? Herein, we review SEC14L-PITPs and focus on structure and function of single- and multi-domain SEC14-PITPs, especially in plants. We highlight particularly the subgroup of SEC14-GOLD proteins. We analyze which role might be played by subdomains to allow binding with phospholipids of the membrane and protein-protein interaction.4In addition to Sec14p and its homologs in yeast , single-A. thaliana, a functional role and structural characteristics are known for CPSFL1, also known as PITP7   . The memer PIPs) . For exaer PIPs) . The Pleembranes . Patientcer risk . Anothercer risk . The humcer risk . Its exacer risk .5.1A. Thaliana SFH1, also known as CAN OF WORMS1 (COW1), which is involved in root hair biogenesis by controlling the tip-directed gradient of PIP2 (and PI(4)P) P) . Sfh1 loongation . Essentiongation . The SEC mutants . The polongation . Two furongation . These tct sites . SEC14-n5.2Marchantia polymorpha and vascular plants (A well-studied family of multi-domain SEC14L-PITPs is the SEC14-GOLD family. All SEC14-GOLD proteins contain a Golgi dynamics (GOLD) domain (IPR009038) at their C-terminus Table 1.r plants . It has r plants . The GOLr plants ., Drosophila melanogaster, Mus musculus, and Caenorhabditis elegans, but not in any of the checked plant and yeast species. It is assumed that the PRELI/MSF1 domain could function in protein association to membranes as well as in transferring lipids  p45, a hTAP2 homolog, especially binds PIP3in vitro and localizes with it in secretory vesicles, the cytoplasm and the extracellular space  P and PIP2, both still have the ability to associate with all other PIPs , a Na+/H+ antiporter localized at the plasma membrane , named after patella, the Latin word for small plate, referring to PATL1 localization at the developing cell plate . Analysi1 (PIN1) . The roldivision . Distincmeristem . Proteinmeristem . PATLs ameristem Figure 4her PIPs . All domher PIPs  (Figurene motif . A hint ne motif  (Figuremembrane . Its clomembrane  (Figuremembrane . patl2 lenotypes . Interesenotypes . Vitaminenotypes . In thisenotypes , indicatenotypes . PATL2 ienotypes  (Figureenotypes . This inenotypes . Interesenotypes . Moreoveenotypes  (Figureenotypes . The SlTrganelle . Additiorganelle . Interesrganelle .Taken together, PI/PIP and α-Toc binding, cell plate and membrane localization, phosphorylation, increased expression during stress responses, and interaction with membrane and trafficking proteins indicate that PATLs are basic cell regulators adapting the cell/organism during cell division and growth and to altered environmental influences and in response to external stimuli. As regulatory proteins PATLs may be involved in membrane trafficking, e.g., by initiating vesicle formation. Furthermore, they might play a role in protecting the cell from ROS and radical damage, by offering α-Toc as an antioxidant or through regulating the activity of membrane proteins.6The SEC14 domain is the common feature of SEC14 proteins. The domain is capable to recognize, bind, transport, and exchange single lipophilic molecules between membranes inside a lipid-binding site. Unlike other lipid-binding domains, the SEC14 domain not only binds phospholipids, but also other lipophilic substances, e.g., α-Toc, carotenoids. This ability leads to further cellular and physiological effects when these lipid transfer activities are required. This characteristic and the presence of SEC14L-PITPs in higher eukaryotes indicates a conserved function and highlights the need of controlling lipid signaling and membrane trafficking. Especially, the presence of additional domains in multi-domain SEC14L-PITPs of higher multi-cellular eukaryotes indicates an increasing variety of functions due to enhanced possibilities for protein interaction, cellular localization or enzyme activities. These additional domains could be involved in the sensing of the lipid environment at the membrane or in changes of lipid-signaling pathways leading to environmental adaptation. The idea is supported by the findings that multi-domain SEC14L-PITPs are able to influence cell division, vesicle formation, lipid signaling, environment responsiveness and organism development, which directly affects the organism fitness/health. The SEC14 domain is also of special interest, since mutations in the SEC14 domain result in defects in development and in the plant-stress response and tolerance, as well as in neurodegenerative diseases and an increased cancer risk in humans.Not all complex tasks of SEC14L-PITPs are understood right now and many interesting questions remain to be answered. For example, are SEC14L-PITPs regulated by post- transcriptional regulation? What are the effects of protein phosphorylation? What is the function of the plant-specific regions of SEC14L-PITPs? How do SEC14L-PITPs influence protein activity and metabolic reactions? In which pathways do yet uncharacterized proteins of this kind play a role? Identifying the functions of SEC14L-PITPs on cellular levels can help to understand the adaptation of regulatory pathways to environmental changes.KM and PB wrote the article and prepared figures. All authors contributed to discussion. PB and RI revised the manuscript. All authors contributed to the article and approved the submitted version.


It has been observed that, in samples subjected to deformation, the changes in texture are minimal and the rise in temperature is relatively low. Therefore, the influence of these two factors on the deformation mechanism can be disregarded. As the strain rate increases, the dominance of dislocation slip decreases while deformation twinning becomes more prominent. Notably, at a strain rate of 2000 s−1, nanoscale twin lamellae are activated within the grain with a size of 500 nm, which is a rarely observed phenomenon in pure Ti. Additionally, martensitic phase transformation has also been identified. In order to establish a correlation between the stress required for twinning and the grain size, a modified Hall–Petch model is proposed, with the obtained value of This study investigates the microstructures and deformation mechanism of hetero-structured pure Ti under different high strain rates (500 s The usage of pure Ti has been steadily increasing in the fields of chemical, biomedical, and aerospace engineering. This is primarily due to its exceptional corrosion resistance, excellent biological compatibility, and impressive high specific strength ,2. As a Previous studies have indicated that the deformation mechanism of pure Ti are affected by various factors, including its inherent characteristics  and experimental conditions  ,6,7,8,9.In addition to strain rate, the deformation mechanism of pure Ti also has a strong grain size dependency . StudiesTherefore, this study focuses on hetero-structured pure Ti samples with a hierarchical grain size. A room temperature split Hopkinson pressure bar test was conducted to examine the dynamic mechanical responses of the samples at different strain rates. Microstructural evolution during deformation was characterized using electron backscattering diffraction (EBSD) and transmission electron microscopy (TEM) techniques. Microstructures and deformation mechanism of hetero-structured pure Ti were systematically investigated, with a particular focus on exploring the influence of grain size on twinning under high strain rates.−1, 1000 s−1, 2000 s−1). In this study, a split Hopkinson pressure bar (ALT1000) compression test was conducted to analyze the mechanical responses of the sample. The compression was performed along the ND  axis of Ti samples at room temperature, and the compression rate was controlled by adjusting the gas pressure. This allowed for data collection on the dynamic mechanical responses of the samples at different strain rates , twinning only occurs in coarse grains due to the notable influence of grain size on twinning behavior in pure Ti [−1, twinning appears in some smaller grains, as shown in −1, 1000 s−1, and 2000 s−1, the −1 shows a similar −1, suggesting insignificant dislocation accumulation as the strain rate increases from 1000 s−1 to 2000 s−1. This observation is consistent with the minimal increase in the proportion of LAGBs shown in on twins . For pure c-axis . As depi pure Ti ,23. Whene sample . Specifi−1, 1000 s−1, and 2000 s−1, respectively. The pole figures reveal a bimodal basal texture, with the highest texture strengths measured at 13.5, 12.8, and 14.6, respectively. There is no significant change in the texture type when compared to the original sample. These results indicate that the strain rate has a minimal effect on the texture. In other words, the influence of texture on the change in deformation mechanism can be ignored.−1. It can be observed that the deformed sample contains a high density of dislocations, which is a typical characteristic of plastic deformation. Twin boundaries are commonly observed within coarse grains, as illustrated by the yellow dotted lines in −1, the microstructure of the sample is shown in −1, twinning remains suppressed in the ultrafine grains. On the other hand, within the coarse grains, a prominent twin boundary is present, accompanied by a significant distribution of dislocations that form a ring-like pattern. In the magnified region  phase, ultimately resulting in the formation of fine needle-like martensitic structures [When the impact strain rate reaches 2000 sructures ,30.For the hetero-structured pure Ti investigated in this study, the grain sizes are generally below 3 μm and contain a significant number of ultrafine grains, resulting in a relatively small grain size  [Numerous studies have demonstrated that dislocations play a pivotal role in the initiation of twinning in pure Ti ,38,39. Fin curve , the corMNm−3/2) . ExtensiMNm−3/2) ,42,43. TIn summary, our experimental results and empirical model greatly enhance our understanding of the twinning mechanism at ultrafine grain level, as well as establish a correlation between grain size and critical shear stress. These findings also offer potential avenues for strengthening mechanisms through twinning in HCP structured materials with ultrafine grains.−1 to 2000 s−1, dislocation activities are the primary deformation carrier, but the dominance of dislocation slipping reduces and the dislocation configurations undergo changes. Conversely, there is an increase in the percentage of deformation twinning with higher strain rates.The mechanical responses and deformation mechanism of hetero-structured pure Ti samples are closely related to the strain rate. As the strain rate increases from 500 s−1, 1000 s−1, 2000 s−1), the changes in texture are relatively minimal, and the degree of temperature rise is low . Consequently, the alterations in these two influential factors are insufficient to induce changes in the deformation mechanism.It has been found that under different high deformation rates (500 s−1, martensitic phase transformation is identified in the deformed Ti sample. Moreover, nanoscale twin lamellae are observed within the ultrafine grain, which can be attributed to the high flow stress. A modified Hall–Petch model is proposed, and the obtained When subjected to a strain rate of 2000 sThis study investigates the influence of deformation rate on the microstructures and deformation mechanism in hetero-structured pure Ti. The results can be summarized as follows:


This study aimed to i) determine the load-velocity relationship in the seated chest press in older adults, ii) compare the magnitude of the relationship between peak and mean velocity with the relative load, and iii) analyze the differences between sexes in movement velocity for each relative load in the chest press.Thirty-two older adults  performed a chest press progressive loading test up to the one-repetition maximum (1RM). The fastest peak and mean velocity reached with each weight were analyzed. Quadratic equations were developed for both sexes and the effectiveness of the regression model was analyzed through a residual analysis. The equations were cross-validated, considering the holdout method. The independent samples t-test analyzed i) the differences in the magnitude of the relationship between peak and mean velocity with the relative load and ii) the differences between sexes in the peak and mean velocity for each relative load.r2 = 0.97, standard error of the estimate (SEE) = 4.5% 1RM; mean velocity: r2 = 0.96, SEE = 5.3% 1RM) and men  without differences (p>0.05) in the magnitude of the relationship between peak and mean velocity with the relative load. Furthermore, there was no overfitting in the regression models due to the high and positive correlation coefficients (r = 0.98–0.99). Finally, men presented higher (p<0.001) lifting velocities than women in almost all relative loads, except for 95–100% 1RM (p>0.05).It was possible to observe very strong quadratic load-velocity relationships in the seated chest press in women (peak velocity: Measuring repetition velocity during the seated chest press is an objective approach to estimating the relative load in older adults. Furthermore, given the velocity differences between older women and men at submaximal loads, it is recommended to use sex-specific equations to estimate and prescribe the relative loads in older adults. Human aging is a continuous process characterized by progressive loss of muscle mass and reduced ability to produce and apply force in motor tasks such as walking, climbing stairs, and standing up from a chair –3. ThereIn a geriatric setting, a common practice to determine the resistance training load (intensity) is through the direct measurement of the one-repetition maximum (1RM), which consists of displacing the maximum weight possible in a single lift , 7. Althr2: ~0.91–0.94; standard error of the estimate (SEE): ~5.7% 1RM), suggesting that load-velocity regression equations are accurate to estimate the relative load in geriatric settings  following the cut-off scores described by Mendes et al. ), musculIn a cross-sectional study design, untrained older adults went to a fitness health club five times over three weeks from 2 p.m. to 4 p.m. at an ambient temperature of 22–24°C. Four sessions (two each week) were dedicated to familiarization and one to implement the test. In the first week, the participants underwent two sessions with an interval of 48 hours to adapt to the test procedures (focus on exercise technique). Anthropometric measurements were also performed during this period and the correct position was identified for the seated chest press machine  for each participant . In the -1) or pedaling on a stationary bicycle at self-selected velocities (50–70 rpm) and resistance levels (1–5), followed by 5 min of upper extremity joint mobilization in a seated position . Then, the participants started the test sitting down with the handgrips in the middle of the chest, shoulders abducted, elbows flexed at ~90° (angle adjusted using the machine arms) and grabbing the handles of the machine with a neutral grip  [The anthropometric measurements included body mass  and height . The data was collected using a valid and highly reliable linear velocity transducer  , 35, whi-2) ) was notr2) assessed the predictive ability of the regression equations, the SEE  calculated the prediction accuracy, and Pearson’s correlation coefficient (r) assessed the relationship between variables. The magnitude of correlation was interpreted as: 0.00–0.10, negligible; 0.10–0.39, weak; 0.40–0.69, moderate; 0.70–0.89, strong; 0.90–1.00, very strong [The sample size was estimated using the t-test for two independent groups . Twenty-y strong . The verr2) between peak and mean velocity with the relative load and ii) the differences between sexes in the estimated peak and mean velocity for each relative load. Hedge’s g effect size compared the magnitude of sex differences in the estimated peak and mean velocity for each relative load. The effect size (g) was interpreted as trivial (0.0–0.2), small (0.2–0.6), moderate (0.6–1.2), large (1.2–2.0), very large (2.0–4.0), and extremely large (> 4.0) [p < 0.05. Statistical analyses were performed in Microsoft Office Excel®  and SPSS v27 . The figures were designed in GraphPad Prism v7 .Individual quadratic regression equations were established for each participant to estimate the peak and mean velocity associated with each relative load. The normality and homogeneity of the data  were evaluated using the Shapiro-Wilk test and Levene’s test, respectively. The independent samples t-test analyzed i) the differences in the magnitude of the relationship ( (> 4.0) . Finally (> 4.0) . The sigr2 of 0.93 and 0.95, respectively, while the raw peak velocity data adjusted for linear and quadratic regression produced an r2 of 0.93 and 0.94, respectively. In men, the raw mean velocity data adjusted for linear and quadratic regression produced an r2 of 0.90 and 0.91, respectively, while the raw peak velocity data adjusted for linear and quadratic regression produced an r2 of 0.90 and 0.91, respectively.The quadratic regression model initially provided the best curve fitting on both velocity variables in women and men and was therefore chosen for further analysis. In women, the raw mean velocity data adjusted for linear and quadratic regression produced an While conducting residual analysis in the regression models, the assumption of extreme values was violated due to the presence of large residuals, which consequently led to their removal. Therefore, in women, 17 observations (92 to 75) were removed for the peak velocity analysis  and 12 . On the r2 = 0.96–0.97) and men (r2 = 0.97–98). Figs After confirming the assumptions in the quadratic regression models, the results revealed a very strong relationship between the relative load and velocity variables in older women (p > 0.05) in the magnitude of the relationship between the estimated peak and mean velocity with the relative load in older women  and men .Finally, there were no differences (p < 0.001), except for 95 and 100% 1RM (p > 0.05). There was greater variability at 90–100% 1RM in both velocity variables in women and men and at 20–40% 1RM in men. In women, there was similar variability using the peak and mean velocity . On the other hand, in men, there was higher variability using peak velocity than mean velocity .Tables The following sex-specific equations were obtained to estimate the relative load from peak velocity:Additionally, to estimate the relative load from mean velocity, the sex-specific equations obtained were the following:r = 0.977–0.990) and the trivial differences between both subsets, the data suggest no overfitting presence.This study analyzed the predictive ability of the movement velocity to estimate the relative load in the seated chest press exercise in older women and men. The main findings can be synthesized as follows: i) there was a very strong quadratic load-velocity relationship in the seated chest press in older women and men; ii) both velocity variables presented a very strong relationship with the relative load, without differences between them; iii) men presented higher velocities than women in almost all relative loads, except for 95 and 100% 1RM. Therefore, these results indicate that measuring intended maximal concentric velocity during the seated chest press is a practical approach to accurately determine the relative load in older women and men.r2 (0.83) and higher SEE (6.10% 1RM) values than those observed in the present study. Two possible explanations for these differences can be considered. One possibility could be that the use of a resistance machine instead of free weights allowed a linear cable movement, which increased its stabilization and avoided oscillations adding noise to data collection. Another possibility could be that the statistical approach used in the present study included testing the assumptions of the regression models through residual analysis. This is a procedure suggested by the specialized literature [In previous research, Marcos-Pardo et al.  analyzedterature , which iThe results of this study showed no differences in the magnitude of the relationship between peak and mean velocity with the relative load, suggesting that both variables seem appropriate to determine the relative load in the seated chest press exercise in older adults. Nevertheless, it is important to point out that peak velocity is generally considered to be more suitable for measuring ballistic movements , as it captures the fastest point during the concentric portion –45. On tThe current study demonstrated that older men presented higher movement velocity values than older women in almost all relative loads in the seated chest press, except for 95 and 100% 1RM. Previous research with physically active young adults corroborates these results demonstrating that men present higher lifting velocity values than women in almost all relative loads in the bench press, except for heavier loads (~80–100% 1RM) , 24, 25.-1 difference) [-1 difference) than strength-trained older women for the same relative loads [In the present study, older women reached, on average, lower mean velocities than those observed in a previous study with strength-trained older women (~0.07 m·sference) . These dve loads . Althougve loads , suggestve loads .-1) than physically active young adult women (~0.07 m·s-1) and men (~0.08 m·s-1) [In line with previous research , these r8 m·s-1) , 24, 25.8 m·s-1) , 46. The8 m·s-1) –49.The estimated velocity values presented in the current study for each relative load in older women and men can have high practical relevance in clinical, training, and research contexts. The first point is that the velocity values can be used as a reference for prescribing relative loads during a resistance training program, assuming that the repetition velocity will be recorded in all sessions. This way of prescribing the relative load has the advantage of knowing in real-time whether the participant is training according to the programmed relative load and adjusting the absolute load if large deviations from the target velocity are observed , 50, 51.This study presents several limitations that need to be addressed. First, a larger sample size would allow generalizing the proposed sex-specific regression equations to other older individuals. Second, although there were no significant differences between older women and men in age (a 4-year difference), recruiting a sample with a minimal age difference would perhaps allow for more accurate results regarding the differences between sexes in movement velocity for each relative load. Third, comparing the velocities obtained in the resistance machine versus a different one  would be necessary to understand the transferability of the results and the accuracy of the regression equations when using different machines. Finally, comparing the load-velocity relationship in the horizontal leg press and seated chest press in both sexes would allow a direct analysis of the differences in the movement velocity in all relative loads in both exercises.The current research demonstrates that it is possible to accurately determine the load-velocity relationship in the seated chest press exercise in older adults, a widely used exercise in geriatric research and fitness facilities. Therefore, the provided velocity values associated with each relative load and the proposed sex-specific equations may be useful for clinicians, coaches, and researchers to implement velocity-monitored resistance training in older adults and analyze the changes in the load-velocity profile in the seated chest press throughout its course.S1 Data(XLSX)Click here for additional data file.


Clostridium butyricum, Akkermansia muciniphila, Faecalibacterium prausnitzii, and Bacteroides fragilis, for the remediation of neurodegenerative diseases are discussed.Increasing clinical and preclinical evidence implicates gut microbiome (GM) dysbiosis as a key susceptibility factor for neurodegenerative disorders, including Alzheimer’s disease (AD) and Parkinson’s disease (PD). In recent years, neurodegenerative diseases have been viewed as being driven not solely by defects in the brain, and the role of GM in modulating central nervous system function via the gut–brain axis has attracted considerable interest. Encouraged by current GM research, the development of new probiotics may lead to tangible impacts on the treatment of neurodegenerative disorders. This review summarizes current understandings of GM composition and characteristics associated with neurodegenerative diseases and research demonstrations of key molecules from the GM that affect neurodegeneration. Furthermore, applications of new probiotics, such as  Age-related neurodegenerative diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD), are chronic and progressive neurological disorders with a range of causes and clinical presentations ,2. The pIn this communication, we review recent progress in the understanding of gut dysbiosis and neurodegeneration, with particular emphasis on the potential application of new probiotics for AD and PD therapies.Neurodegenerative diseases are relatively common and are progressive, and devastating neurological disorders. As the global population ages, the incidence and prevalence of neurodegenerative diseases have risen rapidly in the past two decades. AD is the most prevalent neurodegenerative disease and the leading cause of dementia worldwide. Histopathologically, AD is characterized by β-amyloid (Aβ)-containing extracellular plaques and tau-containing intracellular neurofibrillary tangles . The preIn general, Aβ peptides begin to accumulate approximately 10–30 years before the onset of dementia, which occurs in the early stages of AD and is detectable in the basal temporal and medial frontal regions . The earFirmicutes phylum  and Bacteroidetes phylum (including the Rikenellaceae family and Prevotellaceae family). At the genus level, fecal samples from AD patients have been found to show increased abundances of Ruminococcus . β-. β-RickeSalmonella and Escherichia/Shigella), modulates the release of proinflammatory cytokines, and participates in antigen presentation. In intestinal epithelial cells, autophagy enhances the tight junction barrier function owing to the reduced permeability of ions and small molecules due to lysosomal degradation of claudin-2 . InLactobacillus plantarum PS128 alleviates nigral dopaminergic neuronal death and motor deficits in a PD mouse model . A. ABacterGastrointestinal dysfunction serves as a prodromal symptom preceding the clinical manifestations of neurodegenerative diseases. Currently, there is growing interest in new probiotics as potential therapeutic agents. The safety and tolerability of these novel probiotics need to be validated in both animal models and human trials in order to develop personalized applications. Understanding the mechanisms by which probiotics colonize in the gut could lead to the development of “personalized” bacterial therapies. Another challenge is storage due to the strict anaerobic conditions required during microbial collection and freeze drying. Even so, the current state of new probiotics remains largely promising in the context of neurodegenerative diseases for the purpose of slowing down or preventing neurodegeneration, as well as developing effective therapeutic interventions.


Drosophila melanogaster, the widespread inversion polymorphism In(3R)Payne underpins latitudinal clines in fitness traits on multiple continents. Here, we use single-individual whole-genome sequencing, transcriptomics, and published sequencing data to study the population genomics of this inversion on four continents: in its ancestral African range and in derived populations in Europe, North America, and Australia. Our results confirm that this inversion originated in sub-Saharan Africa and subsequently became cosmopolitan; we observe marked monophyletic divergence of inverted and noninverted karyotypes, with some substructure among inverted chromosomes between continents. Despite divergent evolution of this inversion since its out-of-Africa migration, derived non-African populations exhibit similar patterns of long-range linkage disequilibrium between the inversion breakpoints and major peaks of divergence in its center, consistent with balancing selection and suggesting that the inversion harbors alleles that are maintained by selection on several continents. Using RNA-sequencing, we identify overlap between inversion-linked single-nucleotide polymorphisms and loci that are differentially expressed between inverted and noninverted chromosomes. Expression levels are higher for inverted chromosomes at low temperature, suggesting loss of buffering or compensatory plasticity and consistent with higher inversion frequency in warm climates. Our results suggest that this ancestrally tropical balanced polymorphism spread around the world and became latitudinally assorted along similar but independent climatic gradients, always being frequent in subtropical/tropical areas but rare or absent in temperate climates.Since the pioneering work of Dobzhansky in the 1930s and 1940s, many chromosomal inversions have been identified, but how they contribute to adaptation remains poorly understood. In  Chromosomal inversions are structural mutations that cause the gene order of a chromosomal segment to be reversed , 1921. BDrosophila pseudoobscura Payne, an 8-Mb large paracentric inversion that spans roughly one-third of the right arm of the third chromosome  and whose frequency varies latitudinally on several continents, most prominently along the North American and Australian east coasts  in subtropical and tropical climates but rare or absent in high-latitude, temperate areas around the world , did thi3R Payne inversion polymorphism on four continents: in its ancestral range in Africa and in derived populations in Europe, North America, and Australia. First, we seek to elucidate the adaptive genetic basis of this inversion by combining new phased sequencing data for3R Payne inverted and standard karyotypes isolated from North American populations in Florida and Maine with published sequencing data from the African ancestral range as well as from Europe and Australia. We use these data to investigate patterns of phylogeography, nucleotide variability, linkage disequilibrium (LD), karyotypic divergence, and allele sharing across populations. Second, to identify potential targets of selection spanned by the inversion, we combine FST outlier analyses with transcriptomic analysis of karyotypes from a derived Florida population; because 3R Payne has been implicated in climate adaptation, we performed RNA-sequencing (RNA-seq) across two developmental temperatures.Here, we address these fundamental questions by investigating the evolutionary genomics of the We discuss our results in the light of theoretical predictions about expected patterns of variation and divergence of inversions  and balaIn(3R)Payne captured adaptive alleles in the ancestral African range that predate the origin of the inversion. Yet, contrary to the abovementioned corollary, we find relatively weak differentiation among inverted chromosomes across continents. These results indicate that the adaptive allelic content of the inversion might be ancestral and shared among populations: Selection appears to have favored the spatial assortment of this ancestral polymorphism on multiple continents in a parallel fashion, resulting in qualitatively identical latitudinal clines and mediating “global” (species-wide) adaptation.Consistent with either model, our results suggest that 3R Payne is a cosmopolitan adaptive polymorphism  that have captured an adaptive haplotype, we might expect major peaks of divergence between inverted and standard chromosomes in the center of the inversion, which are due to the interplay of homogenizing flux and divergent selection opposing recombination (In(3R)Payne in North American samples , North America (n = 3), and Australia (n = 2) (fig. 1A and In(3R)Payne and other inversions using several African populations and single populations from Europe (France) and North America . Based on the average number of pairwise nucleotide differences per site . In contrast to neighbor-joining trees, Neighbor-Net allows one to represent conflicting signals in the data, for example, due to recombination.To study the phylogeography of B and 3R Payne arose in sub-Saharan Africa (In(3R)Payne P; see Materials and Methods): here, the network structure mainly reflects geography, not 3R Payne karyotype (fig. 1C and In(3R)Payne on genetic variation might go well beyond its breakpoints .In addition to the dominant signal of monophyletic divergence between inverted and standard karyotypes, we also found a weaker signal of geographical substructure within the inverted and standard clusters of chromosomes, indicating some divergence within karyotypes among continents . On the other hand, consistent with equilibrium (long-term) balancing selection, π was higher for African standard chromosomes inside as compared with outside the inverted region .In both Africa and derived populations,  pattern  online. ants see ; this alπ for non-African 3R chromosomes may be due to the interplay of sufficient time for gene flux having homogenized variation between karyotypes , selection, and the out-of-Africa bottleneck. The fact that levels of variation in derived populations are very similar between standard and inverted chromosomes could imply that a substantial number of individuals carrying 3R Payne has migrated out of Africa during the range expansion.The absence of clear differences in D to test for departures of the site frequency spectrum from an equilibrium standard neutral model (D = 0), positive values of D indicate an excess of intermediate frequency variants and might be consistent with a bottleneck or balancing selection; by contrast, negative values of D indicate an excess of rare variants which may result from a recent population expansion, from purifying selection, or from the affected genomic region having recovered variation after a selective sweep Payne represents a balanced polymorphism, including our analyses of nucleotide variability above. For example, consistent with some form of balancing selection, In(3R)Payne segregates at intermediate frequencies in subtropical/tropical populations around the world: For example, the inversion attains an average frequency of ∼45% in subtropical southeastern North America and ∼60% in tropical Australian populations Payne under crowded larval conditions, giving further credence to a scenario of balancing selection.The fact that different low-latitude populations exhibit different intermediate inversion frequencies is consistent with epistatic coadaptation: Under such a model, there exist multiple equilibria and quasi-equilibria for the frequency of the inversion, and the frequency which it will ultimately attain will depend on the history, the initial conditions of the population, and/or the local environment . AlthougIn(3R)Payne can locally reach near fixation or fixation in some Australian populations Payne segregates at intermediate frequencies in the majority of low-latitude populations around the world and that fixation of the inversion is rare  LD among markers without reference to karyotype; 2) LD between a marker and inverted versus standard arrangements; and 3) LD between markers within inverted or within standard chromosomes. Because inversions strongly reduce the products of recombination in heterozygous state, heterokaryotypes (or pools of inverted and standard chromosomes) should exhibit increased LD as compared with homokaryotypes (aspect 2); for sufficiently old inversions that evolve neutrally, we might expect that LD decreases toward the center of the inversion due to gene flux between the karyotypes, even though such a pattern is difficult to distinguish from direct positive selection at the breakpoints . Within 3R Payne inversion and marker loci has been previously studied by In(3R)Payne in Australian samples using RAD-sequencing data. Here, we sought to use phased genomic data to compare patterns of LD in the region spanned by 3R Payne in African, European, North American, and Australian samples with single-nucleotide resolution (LD between the solution  online.r2) within the region spanned by In(3R)Payne and a slower decay of LD with physical distance (A). A plausible explanation is that this pattern is due to drift within the two semiisolated subpopulations of inverted versus standard chromosomes, with inverted chromosomes exhibiting both lower recombination and lower eN. The pattern of decay was similar for inverted chromosomes across continents, except for the Portuguese sample, perhaps due to the rather small number of sampled chromosomes and the overall lower frequency of 3R Payne in Europe  and Australia (Queensland) closely resembled that of inverted chromosomes (A), probably reflecting bottlenecks in the derived samples. The patterns for the derived populations were generally less clear than those for the Zambian population, presumably due to the out-of-Africa bottleneck.As compared with standard chromosomes, inverted arrangements showed significantly higher levels of short-range LD , we observed strong long-range LD within the inverted region, involving SNPs several million base pairs away from each other and suggesting that major associations among loci are driven by heterokaryotypes not homokaryotypes. These patterns were similar for the other continents, with major LD between but not within karyotypes Payne and between these loci and the inversion itself, including marked associations in the center of the inversion. In the African sample, we also observed LD between the breakpoints and center regions of elevated LD, yet these central clusters of high LD were much less prominent than those in the derived populations . Given that In(3R)Payne is at least ∼129,000 years old and globally quite frequent (eN is large (∼106), gene flux should have had ample opportunity to break down strong LD associated with this inversion. Our data are thus consistent with the selective maintenance of the center peaks inside the inversion. On the other hand, D. melanogaster has undergone an expansion from southern-central Africa and a major out-of-Africa bottleneck, began to spread from the Middle East into Europe ∼1,800 years ago, and colonized North America and Australia ∼100–150 years ago (In(3R)Payne.How do our data compare with these predictions? Assuming a gene flux rate nversion , the timears ago : Demogra3R Payne karyotype, we used FST, a normalized measure of pairwise allele frequency differentiation  and when comparing low-latitude inverted with high-latitude standard arrangements Payne.First, we observed marked divergence in the region spanned by the inversion between inverted and standard chromosomes on all four continents (effect of “K”), with pronounced peaks in the breakpoint regions  between the “endpoints” of the clines on different continents (In(3R)Payne in North America and Europe Payne is underscored by strong correlations between levels of FST with respect to karyotype across continents, including Africa  that is common to derived populations in Europe, North America, and Australia, with alleles in this peak being in strong LD with the breakpoints , assuming an equilibrium balanced polymorphism under an island model of population structure . The pattern in the Zambian sample might thus be compatible with 3R Payne representing a long-term balanced polymorphism  local adaptation but having arisen prior to the out-of-Africa migration, we failed to observe elevated divergence within the inversion body among inverted chromosomes from different continents  online.FST ≥ 0.9 between inverted and standard chromosomes in the North American sample from Florida, among African  standard and inverted chromosomes , possibly indicating that they represent ancestral alleles that have been captured by the inversion. The major enrichment of “inversion-specific” alleles among African inverted relative to standard chromosomes  might also be consistent with the inversion having captured these alleles before the out-of-Africa expansion. If so, this would speak against a scenario whereby the inversion spread to some appreciable frequency by drift and then gained adaptive variants via influx from the subpopulation of standard chromosomes by recombination or through new mutations, with the inversion driven to high frequency by hitchhiking.This analysis revealed that alleles that are highly “inversion-specific” outside of Africa are polymorphic among omosomes A, possibA by using highly inversion-specific alleles (FST ≥ 0.9) as defined based on the Zambian population  also revealed major frequency differentiation between African inverted and standard alleles in derived populations, consistent with the notion that African alleles underpin the divergence of In(3R)Payne karyotypes in derived populations  in the African sample with a frequency difference of ≥50% between standard and inverted chromosomes: The resulting pattern delineates the breakpoints clearly, indicating that divergence in the African sample is driven by suppressed recombination at the breakpoints. Also, note the two “mini-peak” regions away from the breakpoints  where flux is expected to be much higher than at the breakpoints: The locations of these mini-peaks correspond well with those of the major central peaks seen in European, North American, and (for the second peak region) Australian samples . Overall, we observed significant sharing of candidate genes and SNPs across continents (A and B)—these loci might thus underlie the shared pattern of karyotypic divergence across continents  and to complement our genomic analyses, we analyzed RNA-seq data from adult female whole-body transcriptomes of different In(3R)Payne karyotypes. Our data set consisted of nine biological replicates each for Florida inverted (FI), Florida standard (FS), and Maine standard (MS) homokaryotypes , with each group reared at two developmental temperatures (18 °C and 25 °C) prior to RNA extraction . Because the 3R Payne inversion is involved in climate adaptation  showed significant DE in response to temperature  and 0.45% in response to the effect of karyotype plus geography    online.P > 0.05; see SuperExactTest; P < 0.05; In(3R)Payne makes a major contribution to latitudinal differentiation in gene expression patterns.Interestingly, we failed to identify any DE in response to the effects of geography alone t, In(3R)Mo, and In(3R)K; see In(3R)Payne karyotype affected DE across the entire genome . Despite these genome-wide transcriptional effects, DEGs were enriched within the region spanned by the inversion . By contrast, we failed to find enrichment for effects of temperature . Beyond DEG involved in regulating expression, GO analysis revealed that the inversion polymorphism also affects the expression of genes involved in growth, development, and reproduction (fig. 8C), as might be expected given the multifarious effects of 3R Payne on fitness traits such as body size, survival upon starvation, cold shock survival, and lifespan . Given that 3R Payne affects DE inside the inversion body as well as across the entire genome, variants in the breakpoints cannot fully account for the transcriptional effects of the inversion. These results agree well with the conjecture that inversions such as In(3R)Payne affect gene expression as a consequence of linked allelic variation maintained by selection for suppressed recombination  tests . This analysis identified a core set of 86 overlapping genes (see top right corner of fig. 9A) which are all located within In(3R)Payne or in close proximity to it  tests  applied gly DEGs A. This a3R with FST ≥ 0.9 between inverted and standard arrangements in Florida and 317 transcriptomic candidates with significant DE between karyotypes at 18 °C (FIFS18). Comparison of these two groups of candidates revealed a significant overlap, comprising 74 genes .We subsequently focused on 470 candidate SNPs on In(3R)Payne-associated candidate loci, based on our genomic, transcriptomic, and overlap analyses, is provided in In(3R)Payne Payne . These c3R)Payne . Our datD. melanogaster, In(3R)Payne, on four continents: in its ancestral African range and in derived populations in Europe, North America, and Australia. Based on our population genomic and transcriptomic analyses, we offer the following conclusions and conjectures:Here, we have sought to refine our understanding of the adaptive nature of a common cosmopolitan chromosomal inversion polymorphism in 3R Payne polymorphism is monophyletic, consistent with a single mutational origin in Africa at least ∼129 kya  significant sharing among continents of loci that are strongly differentiated between the karyotypes, both in the breakpoint regions and the center of the inversion, and 2) an absence of pronounced genetic divergence among inverted chromosomes from different continents. Analyses of inversion-specific alleles that are nearly or completely fixed in non-African populations within the African population sample suggest that the inversion has captured adaptive alleles in its ancestral range prior to the inversion having migrated out of Africa and become cosmopolitan.3R Payne appears to have spread out of its ancestral tropical range and then become assorted by spatially varying selection in a parallel manner, causing the formation of similar clines on several continents  balancing selection maintaining the inversion polymorphism cf. , but thentinents . This scntinents ; this continents .3R Payne is involved in parallel or “global” Payne frequency and altitude in African populations. Because 3R Payne does typically not fix under warm conditions but appears to be selected against under cool conditions, it is an intriguing possibility that the loci captured by the inversion provide some form of balancing  selection independent of temperature yet happen to render it less tolerant to cool temperatures.Our results and previous work cf.  suggest  habitat . Similar3R Payne is susceptible to cool conditions  reveal pronounced effects of inversion karyotype on gene expression that depend on developmental temperature: Expression levels are higher for inverted chromosomes at low temperature, perhaps due to a loss of buffering or compensatory plasticity cf.  and cons3R Payne inversion has pervasive genome-wide effects on gene expression, consistent with trans-acting regulatory effects. Functional effects of this inversion are thus unlikely explained by lesions at the breakpoints alone. Together with analyses of divergence and LD, our results support the idea that 3R Payne maintains nonrandom associations among adaptive loci t, In(2R)NS, In(3L)P, In(3R)C, In(3R)Mo, and In(3R)Payne; see SMB6; TM6B; Bloomington Drosophila Stock Center [BDSC] stock #5687) in an ebony (e1) mutant background. F1 offspring, which were heterozygous for the wild-type chromosome and the balancer, were visually selected based on the dominant tubby (Tb1) marker mutant phenotype and backcrossed to the balancer strain to amplify the wild-type chromosomes. Using PCR markers (In(3R)Payne  segregated independently of the visual marker of the third chromosome balancer . Since we had not consistently selected for both visual markers during the isolation process, we could not rule out that the wild-type second chromosomes might occasionally have recombined with those of the lab strain. We therefore excluded sequencing data from all chromosomal arms other than 3L and 3R from downstream genomic analyses.To investigate karyotype-specific patterns of genetic variation and differentiation between karyotypes we established isofemale lines from populations in North America  see  online.  markers , we deteDrosophila Population Genomics Project [DPGP]; We generated single-individual (phased) sequencing data using a subset of North American lines from Florida and Maine see  online tm Tris–Cl, 100 mm EDTA, 1% SDS, and 1 mg/ml Proteinase K) for 30 min at 56 °C and 30 min at 70 °C. The lysate was treated with RNAse A  at 37 °C for 30 min prior to adding 39 μl of 8 m potassium acetate, followed by another incubation step for 30 min on ice to precipitate protein. After centrifugation at 14,000 × g for 15 min, we mixed the supernatant with one volume of phenol–chloroform–isoamyl alcohol (ratio 25:24:1). The aqueous phase was further washed with 0.75 volume of chloroform prior to precipitation of DNA by adding 3 volumes of ice cold 100% ethanol. After incubation at 4 °C for 2 h, followed by centrifugation at 14,000 × g for 15 min, we washed the pellet with 70% ethanol, dried it at room temperature, and then resuspended the DNA in 50 μl of TE buffer. Genomic DNA (gDNA) libraries of each sample were sheared using a Covaris instrument  and prepared for paired-end sequencing at the Lausanne Genomic Technologies Facility (GTF), using the Illumina TruSeq Nano Library preparation kit . Samples were sequenced on a HiSeq 2000 Illumina Sequencer to 100-bp paired-end reads.For each of the DNA libraries, we jointly homogenized whole tissue by bead beating  and incubated the homogenate in lysis buffer (100 mIn(3R)Payne is involved in climate adaptation Payne. To do so, we performed RNA-seq assays on isochromosomal lines carrying either the inverted or standard arrangement from Florida, where the inversion is polymorphic, and on lines carrying the standard arrangement from Maine, where the inversion is absent , including scripts, is available here: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and then trimmed and filtered with cutadapt  and common prokaryotic and eukaryotic symbionts of Drosophila, including Saccharomyces cerevisiae (GCF_000146045.2), Wolbachia pipientis (NC_002978.6), Pseudomonas entomophila (NC_008027.1), Commensalibacter intestine (NZ_AGFR00000000.1), Acetobacter pomorum (NZ_AEUP00000000.1), Gluconobacter morbifer (NZ_AGQV00000000.1), Providencia burhodogranariea (NZ_AKKL00000000.1), Providencia alcalifaciens (NZ_AKKM01000049.1), Providencia rettgeri (NZ_AJSB00000000.1), Enterococcus faecalis (NC_004668.1), Lactobacillus brevis (NC_008497.1), and Lactobacillus plantarum (NC_004567.2), to avoid paralogous mapping. We filtered mapped reads for a mapping quality ≥ 20 and used Picard  to remove duplicate reads and realigned sequences flanking insertions and deletions (indels) with the Genome Analysis Toolkit, GATK Payne and randomly selected an equal number of strains with standard arrangement third chromosomes. Consensus sequence files were downloaded from the Drosophila Genome Nexus (DGN) website (http://www.johnpool.net/genomes.html), filtered for polymorphic sites and merged into a single VCF file using custom-made software. Genomic coordinates from D. melanogaster reference v.5 were converted to v.6.African data. The African strains were collected in Siavonga (Zambia) and sequenced as haploid embryos to obtain fully phased sequences; they were bioinformatically karyotyped for various inversions as part of the DPGP resource see , 2016. WIn(3R)Payne, we randomly picked an equal number of strains with standard arrangement on the third chromosome and obtained the genomic data from Dryad (http://doi.org/10.5061/dryad.403b2). In addition to this European low-latitude sample from Portugal, we integrated phased sequencing data from 14 noninverted strains from a high-latitude population in Umeå (Sweden) into our analyses, which were sequenced as haploid embryos  and Yering Station (temperate Victoria) (18 standard lines) had been screened by the authors for 3R Payne using PCR markers (https://doi.org/10.5061/dryad.5q0m8) and converted genomic coordinates from D. melanogaster reference v.5 to v.6 prior to downstream analyses. Note that this data set does not include genomic information for the first 12 million bp on chromosome arm 3R.Australian data. Sequence data for the Australian continent were obtained from  markers . We obtaIn(3R)Payne in Australia that deviate from observations based on single-individual sequencing data of African flies (In(3R)Payne. Yet, these authors found a pattern of strong, highly localized divergence between flies from Queensland and temperate flies from Victoria (where the inversion is very rare or absent), irrespective of In(3R)Payne karyotype. To explore why the patterns in the Australia data might differ from those observed in Africa and North America, we compared the 55 Australian libraries  to high-confidence sequencing data from 42 isogenic lines from Siavonga  which had previously been characterized for the presence or absence of In(3R)Payne (In(3R)Payne karyotype and/or their geographic origin. Because 3R Payne is of monophyletic African origin (In(3R)Payne (see an flies  and poolan flies . In cont3R)Payne , 2016. On origin  online, ayne see  to bioinayne see  online. D. melanogaster strains from Africa, Europe, North America, and Australia were analyzed with respect to In(3R)Payne based on a compilation of sequencing data from the abovementioned sources (see In(3R)Payne, we analyzed 3,766 SNPs located within the breakpoints of In(3R)Payne. Second, we reconstructed genome-wide patterns of phylogenetic relationships among these samples independent of chromosomal inversions by focusing on 4,849 SNPs that were randomly drawn from the left and right arms of the third chromosome in 200-kb distance from the breakpoints of In(3L)P and In(3R)Payne. Using custom-made software, we combined and converted allelic data from all lines to the NEXUS file format and calculated unrooted phylogenetic networks based on the Neighbor-Net inference method  to obtain SNP-wise estimates of nucleotide diversity π and Tajima's D in samples with phased sequencing data. Because vcftools provides window-wise estimates based on total window size but does not account for positions in a given window that do not fulfill the same quality criteria as the polymorphic sites, we obtained average values in 100-kb nonoverlapping windows using custom-made software. We first generated mask files where positions for which >50% of individuals did not fulfill heuristic quality criteria  were flagged with a “0,” whereas all other positions that passed were flagged with a “1.” We then calculated window-wise averages of π and Tajima's D separately for inverted versus standard chromosomes using the information in the mask files for population samples with phased sequencing data. To test for differences in genetic variation with respect to 1) geography, 2) karyotype, and 3) genomic region, we analyzed samples from Siavonga , Póvoa de Varzim , Homestead , and Innisfail . We considered all window-wise averages of π and Tajima's D between positions 3R:16,432,209 and 3R:24,744,010 as being located “inside” In(3R)Payne. To define a representative “control” region “outside” of In(3R)Payne, we choose a random sample of equal size, composed of average estimates of π from 3L and 3R located outside the interval ranging from 3R:14,232,209 to 3R:26,744,010. To account for potential long-range effects of In(3R)Payne, we extended the actual length of the inversion by 2 Mb on both ends. Using R, we performed a three-way analysis of variance (ANOVA) of the form iy = O + K + G + O ´ K + O ´ G + G ´ K + O ´ G ´ K + iɛ, where iy is the continuous dependent variable π or Tajima's D in the ith sample, O denotes the categorical factor “origin” with four levels , K represents the factor “karyotype” with two levels , and G stands for the factor “genomic region” with two levels (inside vs. outside of the inversion), followed by all possible interactions, and where ɛ represents the error term. Based on the coefficients estimated from this model, we calculated planned contrasts using the R package emmeans to test for significant differences in genetic variation between karyotypes inside the inverted region. To search for a potential signature of balancing selection , we also estimated Tajima's D for pooled samples of inverted and standard chromosomes  did not make a qualitative difference.We quantified genetic variation using the software packages omes see  online. r2) Payne using custom-made software as described in r2 statistic can be affected by large variance due to rare alleles, and because this might confound analyses of LD patterns (In(3R)Payne and restricted analyses to pairwise r2 among SNPs within 100-kb distance. Following the approach in We estimated LD within and among karyotypes from low-latitude populations in Siavonga , Póvoa de Varzim , Homestead , and Innisfail  for which phased sequencing data were available. Squared allele frequency correlations (r2)  were calpatterns , we restr2 with physical distance. Here, E(r2) is the expected value of r2, n represents the sample size, and C is the product of the population-scaled recombination rate (ρ = 4erN) and the distance in base pairs, which we estimated using nonlinear regression in R. For each population sample, we employed the R function nls which fits a model based on nonlinear least squares across both karyotypes. Subsequently, we fitted the same model but additionally accounted for karyotype as a grouping factor using the function nlsList in the R package nlme (In(3R)Payne karyotype, we tested for differences in the goodness of fit of the two nested models by analysis of deviance, using the function anova_nlslist in the R package nlshelper . We first investigated how In(3R)Payne affects genetic differentiation within and among populations. We focused on population samples from the endpoints of latitudinal gradients in Europe, North America, and Australia for which the low-latitude populations harbored In(3R)Payne at appreciable frequencies and for which the inversion was absent in high-latitude populations. Our LD analyses mentioned above revealed elevated LD inside inverted karyotypes, indicating that adjacent SNPs do not evolve independently. We thus compared average FST in 100-kb nonoverlapping windows within the inversion breakpoints (“inside”) to a similarly sized “outside” set of average FST values that were randomly chosen from the third chromosome in 2-Mb distance from the breakpoints of In(3L)P and In(3R)Payne, as defined above. For the regions defined as “inside” and “outside” the inversion and for each continent separately, we tested for differences in average pairwise FST values using the following comparisons as input data: 1) samples from the same low-latitude population with different karyotypes , 2) samples with standard arrangement from different populations at the endpoints of a given continental latitudinal gradient , and 3) samples with different karyotypes from different geographical populations within the same continent . To analyze these data, we used one-way ANOVA of the form iy = C + iɛ, where iy represents pairwise FST in the ith genomic window, C is the categorical factor “pairwise comparison” with three levels , and ɛ represents the error term. To determine which of the three levels of C differ from each other, we performed Tukey's HSD (honestly significant difference) post hoc tests. The abovementioned between-karyotype FST estimates . Finally, we also examined whether FST was elevated within the breakpoints of 3R Payne when comparing inverted chromosomes between continents. We focused on SNPs shared across populations and calculated FST in 100-kb nonoverlapping windows in all pairwise combinations across inverted chromosomes from Zambia, Portugal, Florida (USA), and Queensland  using vcftools (v.0.1.16). FST values were plotted against their genomic positions for all comparisons in R using the ggplot2 package.To quantify the amount of genetic differentiation between samples for single SNPs and for averages in 100-kb nonoverlapping windows, we estimated pairwise sing KST —these anIn(3R)Payne, we focused again on the samples from Siavonga , Póvoa de Varzim , Homestead , and Innisfail  and isolated SNPs positions that exhibited FST ≥ 0.9 between In(3R)Payne inverted and standard individuals within a given population; we considered all genes as candidate loci if at least one candidate SNP with FST ≥ 0.9 was located inside or within 2-kb proximity of the 3′ and 5′ ends of a given gene, since these regions harbor regulatory elements , using the following parameters: -l 101 (average fragment length = 101 bp); -s 10 (average standard deviation of fragment length = 10); -b 100 (number of bootstrapped samples = 100); –rf-strand ; and –single (reads are single ended). We focused on gene-specific expression patterns and summed up all transcript-specific read counts for each gene using custom-made software following the approach in R package edgeR (3.20.9)  based on the geographic origin and karyotype of the samples  and the two rearing temperatures (18 °C and 25 °C). To account for potential line effects, we included replicate line identity as a random effect nested in karyotype. Using the voom function of limma, precision weights were calculated for log2-CPM-transformed read counts to account for the relationship between mean and variance in RNA-seq data when fitting linear models to expression data Payne was tested by creating contingency tables based on candidate and noncandidate genes located either inside or outside the region spanned by the breakpoints of In(3R)Payne and using FETs in R. We further compared our candidates to the candidates reported by SuperExactTests  , 2011. Lge limma . By emplion data . We usedge topgo . After cO-Module , we visuO-Module  and CytoO-Module . EnrichmactTests .3R Payne, we compared genomic and transcriptomic candidates. Genomic plus transcriptomic data were only available from North American populations . We tested for significant overlaps between FST-based and differentially expressed candidate genes using SuperExactTests (R package RRHO (P values, whereas genomic candidates were ranked based on average FST of the 10% top most highly differentiated SNPs located within 2-kb proximity of a given candidate gene. RRHO tests for significant overlaps between gene lists are based on hypergeometric tests calculated while sliding across all possible thresholds in the two ranked lists. Besides visual representation of changes of significance with decreasing rank, RRHO allows to define an “optimally” overlapping gene set. We used this “optimal” set to test for enrichment of GO categories using topGO in the R package limma.To refine our set of candidate loci associated with actTests ; as the age RRHO . RNA-seqmsad118_Supplementary_DataClick here for additional data file.


SMARCB1 (95%) or SMARCA4 (5%) genes [ASCL1, with additional sub-entities recently reported), TYR , and MYC (overexpressing MYC and HOXC clusters). These subgroups seem to be associated with distinct genetic and clinical features [Atypical Teratoid/Rhabdoid Tumors (AT/RT) are malignant pediatric tumors of the Central Nervous System (CNS) and are molecularly characterized by a biallelic alteration of the features  but theiSMARCB1-deficient) to evaluate the sensitivity/specificity of various combinations of immunohistochemical (IHC) markers to predict the molecular subgrouping. Using previously reported results and in-house datasets [https://www.molecularneuropathology.org/mnp/). Consistently, 42/51 were assigned to a subgroup of AT/RT with a calibrated score (≥ 0.9) . The nine remaining cases were classified as AT/RT-TYR (n = 4), AT/RT-MYC (n = 4), and control tissue (n = 1) but with a low calibrated score and were thus excluded from subsequent analyses. Their overall distribution within subgroups is depicted by the specificity of t-Distributed Stochastic Neighbor Embedding (t-SNE) analysis , ASCL1 was excluded from our analyses. Next, nineteen different combinations of five different antibodies were tested to evaluate the accuracy of AT/RT subgrouping using the IHC markers we selected ; AT/RT, SHH , AT/RT, TYR , CNS NB, FOXR2 , CNS tumor, BCOR ITD ; CTRL, HEMI ; ETMR, C19MC ; ETMR, DICER1 ; MB, non-WNT/ non-SHH ; MB, SHH ; MB, WNT . Cohort cases are designated by their numberSupplementary Material 1: Fig. S1. t-distributed stochastic neighbor embedding (t-SNE) analysis of the DNA methylation profiles of the 51 investigated tumors alongside selected reference samples of the DKFZ classifier (v12.5). Reference DNA methylation classes: AT/RT, Supplementary Material 2: Fig. S2. Additional immunohistochemical results.A: Comparison of results for molecular subtyping using nineteen different immunohistochemical panels . NEC: Not Elsewhere Classified. *designate the panels with the highest accuracy for subtyping. B: Discrepant cases with immunohistochemical findings (magnification x400). Black scale bars represent 50 μmSupplementary Material 3: Detailed data of the cohort and synthesis