With the integration and miniaturization of electronic devices, thermal management has become a crucial issue that strongly affects their performance, reliability, and lifetime. One of the current interests in polymer-based composites is thermal conductive composites that dissipate the thermal energy produced by electronic, optoelectronic, and photonic devices and systems. Ultrahigh thermal conductivity makes graphene the most promising filler for thermal conductive composites. This article reviews the mechanisms of thermal conduction, the recent advances, and the influencing factors on graphene-polymer composites (GPC). In the end, we also discuss the applications of GPC in thermal engineering. This article summarizes the research on graphene-polymer thermal conductive composites in recent years and provides guidance on the preparation of composites with high thermal conductivity. Thermal management has become a crucial issue in the modern electronics industry as electronic devices have become more integrated and miniaturized. The power required for some processor modules can reach 250 W in a high-performance computer, leading to heat loads as large as 1 kW in this system [−1 K−1 [Polymers have a lot of advantages, such as being lightweight, low cost, easy to process, and exhibiting good corrosion resistance. However, most polymers are heat insulators and have a thermal conductivity between 0.1 and 0.5 W m−1 K−1 , which i−1 K−1 . PhononsIn past years, a lot of works have studied thermal conductive polymer-based composites. Many different materials with high thermal conductivity have been used as fillers to improve the thermal conductivity of composites, such as boron nitride (BN) ,7,8,9, c−1 K−1, which was one of the highest thermal conductivities of currently known materials [Graphene has attracted great attention because of its unique two dimensional 2D) structure and novel properties, such as the zero-gap band structure, high electron mobility, and high thermal conductivity . Balandiaterials . AlthougD structuaterials ,4. In thIn this article, we review the advances in thermal conductivity of graphene-polymer composites in recent years. Special attention is given to the mechanism, the properties, and the influence factors of graphene-polymer composites. Additionally, we discuss the applications of thermal conductive graphene-polymer composites.−1 K−1 at room temperature, and the attribution from phonons is limited to 1–2% of the total [In solid materials, heat is carried by phonons and electrons . In metahe total . The thehe total . Howeverhe total . Figure he total . When onhe total ,31,32. Ihe total ,34,35,36Thermal conduction through a polymer is a complicated process, which is influenced by many parameters like crystallinity, temperature, orientation of the macromolecules, and so on ,38,39. PThe thermal conductive mechanism of graphene in polymers is more complex. In general, graphene has a very high specific surface area. When being added in a polymer, large numbers of interfaces are produced . These iIn recent years, more studies have been using graphene and its derived materials to prepare thermal conductive composite materials. The morphology of graphene in the polymer matrix significantly affects the thermal conductivity of the composites . In thisGraphene with a random orientation in the polymer matrix can be manufactured by many methods, such as solution mixing, melt mixing, and in-situ polymerization, etc. ,50,51,52There are a variety of specific orientations of graphene in the polymer matrix, such as orientation, segregated structure, 3D structure, and so on ,73,74,75There are many factors affecting the thermal conductivity of graphene-polymer composites, such as the defects on graphene, the orientation of graphene in the polymer, the graphene loading, and the surface modification, etc. ,4,5. In 3 W m−1 K−1 to ~4.0 × 102 W m−1 K−1 near room temperature as the density of defects changed from 2.0 × 1010 cm−2 to 1.8 × 1011 cm−2 [−1 K−1. This is because the high-temperature annealing heals defects and removes oxygen functional groups on graphene, thus reducing the phonon scattering centers [The characteristics of graphene have a great influence on the thermal conductivity of graphene-polymer composites ,91,92,93011 cm−2 . Xin et  centers . The mor centers . Kim et  centers . Kim et  centers .The loading of graphene exerts a significant effect on the electrical and thermal conductivity of the composites ,99,100. −1 K−1, which is higher than copper at room temperature. It is claimed that this dramatic enhancement is attributed to the rapid and effective heat-transfer path formed by orientated graphene [−1 K−1, 20% higher than that of GS/PDMS composite at the loading of 0.7 wt %. They believe that the unique interconnected structure of GF acts as an efficient thermal pathway in the polymer matrix [Many researchers believe that graphene with a specific orientation, like orientation and 3D structure, is much better than graphene with a random orientation when preparing thermal conductive composites ,88,89,90graphene . Zhao etIt is considered that the interface between graphene and the polymer plays an important role in thermal conductive composites. Since phonons are the main form of thermal conductance in graphene-polymer composites, bad coupling in vibration modes at the graphene-polymer interface will generate huge interfacial thermal resistance. Chemical bonding between graphene and the polymer can efficiently decrease the phonon scattering at the interface and reduce interfacial thermal resistance . Gao et Nowadays, with the improving demand in emerging industries, thermal conductive materials with novel properties are widely required ,108,109.2 and junction-to-ambient thermal resistance of less than 0.2 °C/W [−1 K−1. Employing graphene to prepare a thermal conductive material as TIM has been attracting a lot of attention.In the electronic industry, thermal management has been a serious challenge because of the miniaturization and functionalization of electronic devices. To control the temperature of all components in devices, an effective thermal conductive path must be used . Thermal−1 K−1, which is about 14 times that of the OA [The effort towards thermal energy storage has been intensified over the past years. Thermal conductivity is an important parameter in thermal energy storage materials, which significantly influences the rate of heat storage and extraction . Therefof the OA .−1 K−1. They think Lithium-ion batteries have become the major source of power for portable electronic devices. Separators are one of the major components of these batteries, and the improvement of thermal conductivity in separators is an option for long-lasting Li-ion battery fabrications [As batteries have become more powerful in recent years, thermal management has turned into a special issue in the battery system. When a battery is used at a high charging/discharging rate, the rate of heat generation may exceed the rate of heat dissipation. In this situation, the battery may be inefficient or even catch fire. A thermal management system is needed to maintain the battery pack at an optimum temperature. Khan et al. incorporated 8 wt % graphene nanoflake in polyacrylonitrile (PAN) fiber separators, and the thermal conductivity increased from 3.5 to 8.5 W mications . Hallaj ications .In this paper, we have reviewed the graphene-polymer thermal conductive material in recent years. The thermal conductive mechanisms in graphene, polymers, and their composites have been discussed. The recent advances on thermal conductivity of graphene-polymer composites have also been reviewed. Furthermore, we have discussed the factors influencing the thermal conductivity of graphene-polymer composites, such as the characteristics, the loading, the orientation of graphene, and the interface. Finally, the applications of thermal conductive graphene-polymer composites have been demonstrated. This review reveals the relationship between thermal conductive mechanisms and properties and also provides guidance on the preparation of composites with high thermal conductivity.


Mycobacterium tuberculosis is divided into several lineages, each with a different geographical distribution. M. tuberculosis lineage 1 (L1) is common in the high-burden areas in East Africa and Southeast Asia. Although the founder effect contributes significantly to the phylogeographic profile, co-evolution between the host and M. tuberculosis may also play a role. Here, we reported the genomic analysis of 480 L1 isolates from patients in northern Thailand. The studied bacterial population was genetically diverse, allowing the identification of a total of 18 sublineages distributed into three major clades. The majority of isolates belonged to L1.1 followed by L1.2.1 and L1.2.2. Comparison of the single nucleotide variant (SNV) phylogenetic tree and the clades defined by spoligotyping revealed some monophyletic clades representing EAI2_MNL, EAI2_NTM and EAI6_BGD1 spoligotypes. Our work demonstrates that ambiguity in spoligotype assignment could be partially resolved if the entire DR region is investigated. Using the information to map L1 diversity across Southeast Asia highlighted differences in the dominant strain-types in each individual country, despite extensive interactions between populations over time. This finding supported the hypothesis that there is co-evolution between the bacteria and the host, and have implications for tuberculosis disease control.Tuberculosis presents a global health challenge.  With tremendous efforts, some new drugs have recently been developed, but there has still been limited success in developing new effective tuberculosis vaccines. Thus a better understanding of the interactions between M. tuberculosis and its host is urgently needed.The burden of tuberculosis is high in developing countries in Asia, with 56% of all global cases found in five countries: India, Indonesia, the Philippines, Pakistan and ChinaM. tuberculosis lineages strongly associate with the geographic location, ethnicity and ages of the hosts2, which could be due to the founder effect or co-adaptation between the bacterial and host population, favouring their stable coexistence4. The latter hypothesis was supported by the sympatric association between bacterial lineages and patients’ origins5 which was weakened in HIV-infected patients6. The global population structure of M. tuberculosis lineage 4 further supported the co-evolution hypothesis7. Moreover, some candidate gene studies revealed an association with diseases caused by only some lineages8. Recently, genome-wide association studies (GWAS) successfully identified the HLA-DRB1*09:01 and CD53 genes associated with tuberculosis when the bacterial genotypes were considered10.13.Other aspects of genotype-phenotype associations have also been studied, particularly for lineages 2 and 4. The association varied between sublineages. The sublineages of the Beijing strains were associated with differential survival rates, virulence and responses to vaccine14 and almost completely congruent with the EAI (East African Indian) strains, defined by spoligotyping. The EAI strains were commonly reported in countries around the Indian Ocean16 and associated with higher ages of patients17. Pulmonary tuberculosis caused by L1 strains was more likely to be sputum negative and had a higher sputum conversion rate at two months after treatment18. L1 was less associated with drug resistance than L220. They were occasionally reported to associate with extrapulmonary tuberculosis21. In general, L1 isolates induced stronger cytokine responses and grew slower in macrophage culture23.Unfortunately, there have been few studies on the phenotypes of lineage 1 (L1). L1, as defined by SNP typing, is congruent with the Indo-oceanic strains, defined by LSP (large sequence polymorphisms)24. It is endemic in 11 of 30 high- burden countries. Some bacterial isolates, such as the “Asian human” type, from India and East Africa had distinct properties25 and were hypothesized to provide an explanation for the lack of efficacy of the BCG vaccine in the monumental clinical trial in Tamil Nadu, Southern India26. The described phenotypes might associate with L1 strains.Studies of L1 are essential for the End TB strategyet al. proposed classification based on a whole-genome single nucleotide variant (SNV) phylogenetic tree and provided a corresponding barcoding scheme27. While there have been many WGS studies of lineage 2 strains, studies of L1 strains are rather limited.Recently, Coll Comparing epidemiological findings between countries requires a reliable and high-resolution classification based on the bacterial genetic relationship. EAI isolates are classified by spoligotyping into “clades”, such as EAI1-EAI8. However, it is not clear whether the “clades” are monophyletic. There are “orphan” spoligotypes that are not yet assigned to any clades while assignment of some spoligotypes may be problematic. Correlation of the SNV phylogenetic tree to spoligotypes would provide better insights into the phylogeography of L1.M. tuberculosis L1 in Chiangrai, the northernmost province of Thailand, were sequenced and revealed significant diversity. Analysis of WGS was done to refine the SNV phylogenetics of L1, verify spoligotype clades and provide mapping between spoligotypes and the SNV phylogenetic tree. This finding provided a basis to map the distribution of predominant sublineages of L1 in various countries in Southeast Asia. Our results suggested an association between predominant sublineages and geography and ethnicity, despite a history of extensive interactions between various ethnic groups, supporting the hypothesis that there is some level of co-adaptation between M. tuberculosis L1 and human hosts.In this study, the genomes of 480 isolates of M. tuberculosis isolates, 480 were initially identified by LSP as belonging to the Indo-Oceanic family and had the 330 SNPs, previously reported to be specific to L127 (Supplementary Table Among 1174 successfully sequenced M. tuberculosis27. Its mean pairwise SNV distance (MPSD) in this study was 645.6 (range 0–965), while that of the globally reported L1 isolates was 7302, indicating high heterogeneity of L1 in Chiangrai. This finding might be related to the knowledge that Chiangrai is an ancient settlement originally with Austroasiatic language-speaking people and subsequently replaced by people who speak the Tai-Kadai language family29. It was historically controlled by several tribal kingdoms that resided now in Thailand, Myanmar and Lao. Chiangrai is home to several hill tribes and recent Chinese immigrants. Its modern roles as a transport hub and a major tourist attraction site lead to rapid urbanization and migration from throughout Thailand and other countries.A total of 41,157 SNVs were identified, with 22,491 SNVs, each present in only a single isolate. The total identified SNVs were more than in the previous reports  and 777777777777771 (SIT523) respectively. Neither pattern belongs to the EAI spoligotypes even though all four had the genetic markers of L1 (TbD1+ and RD239−). Isolates with SIT100 belonging to L1 have been previously reported31.The spoligotypes of 410 (85%) isolates were classified as EAI strains, while those of the other 66 isolates were not previously describedAnalysis of all 68 DVR segments revealed that all EAI isolates and unclassified isolates lacked DVR39-42, DVR44, known characteristics of EAI, and DVR48, which is not used for standard spoligotyping. This finding indicated that all unclassified isolates belonged to the EAI family. Some isolates had all the other 62 DVRs, indicating that the most recent common ancestor of the EAI group (EAI-MRCA) carried the deletion of only DVR39-42, 44 and 48.et al. studied 1601 globally collected WGS deposited in public databases, classifying 121 L1 isolates into five major groups, 1.1.1–1.1.3 and 1.2.1–1.2.227. The phylogenetic trees of the 480 isolates constructed by Bayesian inference  or the fixation indices greater than 0.33  of discrepancy. In seven cases, several DVRs were not identified computationally but they were detected by experimental spoligotyping, shown in Table The SNV phylogeny indicated that EAI2_MNL, EAI2_NTB and EAI6_BGD1 were monophyletic clades, corresponding exactly to L1.2.1.1, L1.2.1.2, and L1.1.3 respectively. Their DR regions were characterized by multiple deletions compared with the EAI_MRCA , typical for EAI5, belonging to 6 sublineages of L1.1.1  has only one more deleted DVR, DVR62, compared with SIT236. In this study, 14 of 20 isolates with SIT48, belonging to L1.2.2, L1.1.1.4 and L1.1.1.9, retained all the other unspoligotyped DVRs. As L1.2.2 separated from the other sublineages early in the evolution of L1 Fig. . Still, There were some sublineages associated with single DVR deletions such as L1.1.2.1-DVR18, L1.1.1.5-DVR18-21, L1.1.1.8-DVR53, and L1.1.1.6-DVR57-60. However, the associations were not completely specific and the single deletion event might allow for homoplasy, as in the case of DVR62 deletion.M. tuberculosis isolates from South and Southeast Asia usually have a single copy or low copy numbers of IS611032. Our study revealed that the isolates could belong to many sublineages except for L1.2.1 and L1.1.3  were usually found between studies performed in the same country, as expected, which might not be true if a large number of samples were intentionally selected from patients with specific forms of the disease, such as extrapulmonary or drug-resistant tuberculosis. There were numerous studies in India, where the percentages of L1 are known to be high in the southern and low in the northern part of the country. Nevertheless, the L1 spoligotype profiles were generally similar throughout the country, excluding eastern India.Correlations between studies in bordering countries were usually intermediate and higher than correlations between countries without common borders. Strong correlations were identified between Pakistan and Indian studies, particularly when homoplastic spoligotypes were excluded, conforming to the close historical relationship between the two countries. There were also strong correlations between the spoligotype profiles in Bangladesh and Myanmar (Supplementary Table Intriguing patterns of spoligotype profile correlations were observed in Southeast Asian countries. Countries in islands of Southeast Asia (ISEA), Indonesia and the Philippines, as well as Singapore and Taiwan are separated by seas but share similar L1 spoligotype profiles comprising predominantly EAI2_MNL (L1.2.1.1). In contrast, there was low similarity of spoligotype profiles between MSEA countries. In all cases, there were some levels of similarity of spoligotype profiles only between countries that shared common borders. The correlation coefficients between spoligotype profiles between countries without common borders were always low  isolates having SIT256, while the others had spoligotypes that could be derived from SIT256 by deletion. Both L1.2.1.2 and L1.1.1.8 had very low intragroup MPSDs of 106.4 and 174.4 and very high fixation indices of 0.61 and 0.63 respectively, indicating a close genetic relationship within each group. Together with the high prevalence, these results suggested high transmission activities of both sublineages.The two most common sublineages (L1.2.1.2 and L1.1.1.8) contributed to a third of L1 isolates in Chiangrai. Of the 94 members of L1.2.1.2, 83 (88%) had SIT89 or EAI2_NTB while the remaining 11 had new spoligotypes that could be derived from SIT89 by additional DVR deletions. The family was initially recognized by ISIn countries bordering Thailand, Cambodia, Malaysia and Myanmar, both SIT89 and SIT256 were reported but at much lower frequencies 39, should also belong to L1.2.1.1.EAI2_MNL or L1.2.1.1 was highly prevalent in the Philippines, accounting for more than 80% of all 40, while Cambodians speak the Khmer branch. The Thai language belongs to the Tai-Kadai family while Burmese belongs to the Tibeto-Burman family. The Malay language belongs to the Malayo-Polynesian branch of the Austronesian family. Each country also had a different predominant spoligotype belonging to different sublineages , while EAI2_MNL accounted for approximately 9%17, which might indicate a specific adaptation. In contrast, L1.2.1.1 was rare in Vietnam. This result is intriguing because the Cham people in the historical Champa Kingdom, in what is now southern Vietnam, spoke Austronesian. The kingdom lasted for several centuries and afforded ample interactions between Cham and Kinh, the major ethnic group in Vietnam. The rarity of L1.2.1.1 could be a consequence of the Champa Kingdom acquiring mainly Austronesian languages and culture rather than a large migration of genetic Austronesians42.The most common spoligotype in Vietnam was uniquely SIT139, a rare spoligotype classified as L1.1.1.1 in this study, accounting for 48% of Vietnamese L1 isolates43, which mostly belonged to L1.2.1.1, suggesting high transmissibility. Another study in Vietnam reported the clustering rate among EAI2_MNL (L1.2.1.1), an uncommon sublineage in that region, of only 18.3% while the clustering rate among EAI4_VNM, the predominant clade including SIT139, was 77.4%44. This result suggested that L1.2.1.1 and SIT139 spread differently in both populations, conforming to the co-adaptation hypothesis.Existing studies on the transmissibility of L1 supported the co-evolution hypothesis. A population-based study in Southern Taiwan revealed that the genotypic clustering rates of 60.8% among EAI strainsM. tuberculosis lineages at the population level across several studies. Although many recent studies provided genotypic details of the studied isolates, their phylogenetic relationships were usually not considered in the analysis45. Moreover, many studies used the phenotypes of the other strains in the same studied areas as the controls. The controls, therefore, varied between studies in both proportions of lineages and sublineages. Moreover, in most studies, information regarding host population genetic structures are usually not available.Robust phylogenetics provides a foundation for genotype-phenotype correlation studies. In general, it is difficult to compare phenotypes of In vitro phenotypic studies of M. tuberculosis typically involved a small number of strains and rarely had sublineage information, rendering comparison between studies difficult. A few recent studies, involving L1 isolates with sublineage information, included the autophagy study of EAI2_NTB (L1.2.1.2)46. A number of EAI isolates belonging to SIT1390 (L1.1.3.1)23 or EAI2_MNL (L1.2.1.1)47 were shown to induce high levels of inflammatory cytokines. Unfortunately, there are no known in vitro studies involving L1.1.1 or L1.1.2 isolates that merit further exploration.1, its predominant strain, SIT11 belonging to L1.1.2.2 , the Japan Anti-Tuberculosis Association (JATA), and the Ministry of Public Health, Thailand. The study recruited all newly-diagnosed culture positive tuberculosis patients with all nationality who were also willing to participate in a human genetic study from all pubic hospitals in Chiangrai. During the studied period, there were 15,805 new cases in Public Hospital Tuberculosis Registration System. Bacterial isolates were obtained from 7148 patients, of which both bacterial and host DNA were obtained from 1187 patients. 1174 M. tuberculosis isolates were successfully sequenced and 480 were identified to be Indo-Oceanic strains.The project was approved by the Ethical Committees of Chiangrai Prachanukroh Hospital, Chiangrai and the Thai Ministry of Public Health. Informed consent was obtained from all participants and/or their legal guardians. All methods were performed in accordance with the relevant guidelines and regulations.32. All the processes were performed in Class II biosafety cabinets.Bacterial samples from 1187 patients were successfully regrown in Lowenstein-Jensen medium in an appropriately contained clinical microbiology laboratory in Chiangrai using standard biosafety protocols and equipment. The bacteria were heat-killed and DNA was prepared as previously described51. The primer sequences are shown in Supplementary Table Lineages of the isolates in this study were initially identified by LSP using PCR primers specific to TbD1 , RD239 (for lineage 1), RD105 (for lineage 2), RD750 (for lineage 3) and 7 bp sequence at pks15/1 (for lineage 4)The 487 isolates were identified as Indo-Oceanic strains due the presence of the TbD1 and the absence of RD239.52. Spoligotyping investigates the presence or absence of 43 of the 68 DVRs. The results were coded into a binary pattern of 43 digits, which is typically converted to an octal code by the successive combination of three binary digits from the left, leaving the 15th code as still binary. M. tuberculosis L1 typically has EAI spoligotypes, i.e. the 10–12th octal code of 413. Some octal codes have been designated as spoligotype international type (SIT) and assigned to one of the various clades, such as EAI1, EAI2_MNL, and EAI2_NTB, among others. The SIT and clade assignment was performed by querying SITVITWEB 30. Spoligotypes were also predicted from whole-genome sequences using SpolPred software53.Spoligotyping was performed using a commercial kit  as previously describedM. tuberculosis samples were sequenced on the Illumina HiSeq. 2000 platform at the Wellcome Trust Sanger Institute, UK. The system produced paired-end reads in FastQ file format. We used Trimmomatic version 0.3654 to collect only the paired reads. Five isolates were not further analysed because the numbers of available reads were too small. The raw reads were filtered and discarded if their phred quality scores were lower than 20. The remaining reads were aligned by the BWA program55 and SAMtools56 using M. tuberculosis H37Rv (GenBank NC_000962.3) as the reference genome with alignment score more than 50, SAMtools base quality more than 23 and read depth more than 10. Two samples appeared to contain mixed nucleotide sequences and were also not further studied. The SNVs that were present in any drug-resistance gene, mobile genetic element, phage, PE/PPE region and non-homozygous SNVs were discarded. The remaining SNVs had been converted to an SNV-supermatrix using an in-house Python script before being used in the phylogenetic analysis. The sequencing data for the 480 samples used in this paper were submitted to the European Nucleotide Archive (ENA) of EMBL-EBI mirrored in the Sequence Read Archive (SRA) database. Actual read sequences can be queried and downloaded directly from the SRA database using the accession numbers listed in Supplementary Table SNVs of 480 Indo-Oceanic isolates were used for phylogenetic tree construction, with 24 isolates from the lineage 2–4 as outgroups. We analysed the generated supermatrix with two phylogenetic analytic methods, maximum likelihood (ML) and Bayesian inference (BI).57 via the SeaView program58. The core tree (starting tree) was generated using the BioNJ method. The analysis was run using GTR model, using the nearest neighbor interchange (NNI) method. The bootstrap analysis was calculated for branch support with 1,000 replications of pseudo-data.For ML analysis, we used PhyML54. The parameter “4by4” was used as the nucleotide substitution model. The BI analysis was performed with two simultaneous runs with four n-chain for each run. The analysis was run for approximately 10–20 million generations. The analysis was terminated when the average standard deviation of split frequencies was lower than 0.01. The first 25% of sample trees were discarded as burned-in. All trees were visualized using the FigTree program version 1.4.2. (http://tree.bio.ed.ac.uk/software/figtree/).For the BI methods, we used the MrBayes program57. The analysis was conducted at multiple levels of sublineage classification. The plots between the first three eigenvectors were examined.Principal Component Analysis was performed using Jalview 2.8.256. The fixation index (FST) was calculated based on the following formula (πbetween group − πwithin group)/πbetween group, where πwithin group is the average pairwise SNV distance within a group and πbetween group is the average pairwise SNV distance between all members in the group and all members not in the same group but in the same level of grouping. The statistical tests for the difference between the within-group averages and between-group averages were performed using Wilcoxon rank-sum test at α = 0.05 with Bonferroni correction multiple testing using R statistical package version 3.3.2.Pairwise SNV distances were calculated using MEGA5de novo using velvet software59. Analysis of the DR region in each genome was performed by carrying out a blastn search of the contig files containing the DR region. The identified contigs were mapped to areas around the position of 3119181–3123573 in the genome of H37Rv (GenBank NC_000962.3). The presence or absence of each of the 68 known direct variable region (DVR) sequences was determined and recorded as shown in Supplementary Table The sequence contigs of each isolate were constructed 6110 in the genome of each isolate was identified by ISMapper using the provided IS6110 sequence60 as the query and H37Rv as the reference genome with default parameters.The number of ISThe patient characteristics were described in descriptive statistics (as presented in Table Relevant research articles were identified by PubMed search using the keywords, spoligotype or genotype and tuberculosis and names of the countries in the ASEAN, South Asian and Pacific islands. The full papers were retrieved and examined for the presence of detailed descriptions of spoligotypes. Forty-one articles were identified with 6 having less than 20 EAI isolates in each study. The latter group was excluded. The list of retrieved papers is shown in Supplementary Table Supplementary InformationDataset for supplementary table S1Dataset for supplementary table S5Dataset for supplementary table S10


Language, humans’ most distinctive trait, still remains a ‘mystery’ for evolutionary theory. It is underpinned by a universal infrastructure—cooperative turn-taking—which has been suggested as an ancient mechanism bridging the existing gap between the articulate human species and their inarticulate primate cousins. However, we know remarkably little about turn-taking systems of non-human animals, and methodological confounds have often prevented meaningful cross-species comparisons. Thus, the extent to which cooperative turn-taking is uniquely human or represents a homologous and/or analogous trait is currently unknown. The present paper draws attention to this promising research avenue by providing an overview of the state of the art of turn-taking in four animal taxa—birds, mammals, insects and anurans. It concludes with a new comparative framework to spur more research into this research domain and to test which elements of the human turn-taking system are shared across species and taxa. It is cThe present paper will draw attention to this promising research avenue by providing an overview of the state of the art of turn-taking in non-human animal communication. As there has been a tremendous number of publications on temporally coordinated signalling—and we can only cite some of them—we have restricted our selection of citations to publications that either cite secondary literature or are exemplary for general phenomena. We will briefly define the predominant terms used, explain the main functional hypotheses proposed, and then discuss their implications for current findings in four animal taxa—birds, mammals, insects and anurans. We conclude that a systematic quantitative comparison of a representative range of turn-taking skills among a single set of human and non-human animal individuals is needed to test the hypotheses of Levinson & Holler ,12 and T2.The turn-taking system for conversation  applies In non-human animal studies, multiple and not always mutually exclusive terms have been used to describe coordinated—and not solely communicative—exchanges between interactants involving alternating turns . DiffereBox 1.Duetting. This term—sometimes also referred to as dialogue [pas de deux; [  dialogue ,22 or cadialogue —traditiodialogue . While sdialogue , others dialogue ,26. Somedialogue –29, whilde deux; ) and biode deux; .Chorusing. This term refers either to a cacophony of sounds or to the synchronous production of the same call type by more than two individuals [ividuals .Antiphonal singing/song. This term denotes a specialized form of duetting, in which one member of the pair starts a song that is then continued by the other member. The second member may complete the song or the members of the pair may take turns until completion [mpletion .Antiphonal calling. This term—sometimes also referred to as call-and-response—is defined as a minimum number of two individuals of any sex and/or age combination producing a vocalization in response to a preceding call [ing call . Some auing call .Turn-taking. This term was traditionally restricted to human spoken conversation but has recently been extended to other modalities and species. It denotes the orderly exchange of purely communicative signals or behaviours (e.g. peek-a-boo games in humans) between individuals characterized by principles for the coordination of turn transfer, which result in observable temporal regularities. The communicative signals delivered by turns can vary, as can the size and the order of turns, and techniques used to allocate turns to specific individuals [ividuals . Some scividuals .3.2—inspired by the extraordinary precision of antiphonal singing in tropical bird species—focused on mutual recognition, maintenance of contact between partners, as well as mutual stimulation, reassurance after disturbance and territory defence [Although temporal coordination in animal communication has attracted interest over several decades, no clear picture has yet emerged as to why individuals exchange signals. The explanations put forward generally have not distinguished between the function of signal exchange as such and the function of the exchange of specific signals , though the motivations for these may differ. The earliest hypotheses on the function of duets defence ,37. In t defence , followi defence ) based o defence ,30. In t defence  argued t defence ,40 sugge defence .Anura) and different orders of insects  suggest that the primary function of duets is to enable copulation by acting as mate-location devices [In stark contrast, relatively little research attention has focused on the function of duets in amphibians and insects, and antiphonal exchanges in monogamous and polygamous living societies. The few existing studies on amphibians  [Alauda gulgula) [Luscinia megarhynchos), perform ‘song matching’ (where the bird responds with the same song) or ‘vocal supplementing’  [Overall, birds use a large variety of different signals across species, which range from simple calls   to extengulgula) . Interacgulgula) , while ogulgula) , or engagulgula) . Some bial song) .Research on the time window of avian vocal interactions has focused predominantly on duetting, while investigations into the time window of call exchanges in non-duetting species are relatively rare . InformaTyto alba) [Sturnus vulgaris) [Analyses of time-specific relationships within vocal exchanges provided evidence that birds listen and respond to each other and show substantial flexibility in their temporal adjustments . For insto alba)  and Euroulgaris) . Nest-maulgaris) . This fiPoecile atricapillus) favour overlap, and European starlings prefer overlap avoidance [Lophortyx californicus) showed that males masking their females' identity prevented other unmated males from mating [In addition to avoidance of song overlap, birds have also been observed to adopt two additional roles: they either follow their temporal self-program—called ‘autonomous songsters'—or start their songs sometime before  a neighbour has finished singing—called ‘overlappers’ ,54. Thisvoidance –60. If ovoidance  or resulvoidance . For exam mating .Spreo bicolor; Cape glossy starling, Lamprotornis nitens: red-winged starling, Onychognathus morio; pale-winged starling, Onychognathus nabouroup) showed that the degree of territoriality highly influenced temporal relationships: the more communal the species, the more song overlap and choruses were observed during close-range interactions [In addition, temporal patterns of vocal interactions seem to be tightly linked to a species’ social structure . For exaractions .In sum, the use of distinct time windows in birds differs between but also within species ranging from overlap avoidance—representing a characteristic element of human turn-taking —to the s5.Research into turn-taking propensities of mammals is strongly biased towards non-human primates. Within the order of primates, studies have nearly exclusively been focusing on vocal exchanges of pair-bonded, and/or family living species e.g. ,48,63). ,63. 48,6(a)Lepilemur spp. [Tarsius spectrum [Callicebus cupreus [Callithrix jacchus [Cebuella pygmaea [Saimiri spp. [Cercopithecus campbelli [Theropithecus gelada [Hylobates spp. [Gorilla gorilla; Pan paniscus; Pan troglodytes; Pongo abelii [Indri indri; gibbons Hylobates spp.; Mentawi langurs Presbytis potenziani; Titi monkeys Callicebus cupreus [Saimiri sciureus) [Callithrix jacchus) [Some signs of turn-taking have been documented in all the major primate branches : prosimimur spp. ; Tarsiusspectrum ), New Wo cupreus ; Callith jacchus ; Cebuell pygmaea ; Saimiriiri spp. ), Old Woampbelli ; Theropis gelada ), smalletes spp. ,70) and o abelii ,64,71).  cupreus ,29), and cupreus ,63). The cupreus . In star cupreus ). Simila cupreus . Detaile cupreus . In addiciureus)  to 3000–jacchus)  but see jacchus) ). In addjacchus)  but see jacchus) ).Pan paniscus) and chimpanzees (Pan troglodytes) establish participation frameworks and adjacency pair-like sequences ; [Furthermore, spontaneous cooperative turn-taking has been observed in communicative gestural interactions of great apes in both captive and natural environments ,76. For (b)Outside of the primate order, vocal turn-taking has been studied in four distantly related mammalian groups, cetaceans, bats, elephants and mole rats.Delphinapterus leucas) [Tursiops truncatus) [Orcinus orca) [Eubalaena austrialis) [Physeter macrocephalus) [The most research attention has been devoted to cetaceans and provided evidence that vocal exchanges facilitating social interactions occur in a number of species including beluga whales ( leucas) , bottlenuncatus) , killer us orca) , southertrialis)  and sperephalus) . Bottlenephalus) ,82–84  ). Time wephalus) ,83,84. Oephalus) . Similarephalus) . Similarephalus) . They thephalus) ,81,87). ephalus) . Furtherephalus) , suggestDiaemus youngi) [Loxodonta africana) exchange vocalizations, such as low-frequency rumbles, to respond to calls from other females [Heterocephalus glaber) are one of the few eusocial mammal species, and use their most common vocalization type—the soft chirp—antiphonally with a latency of approximately 400 ms [Much less information is available regarding turn-taking in bats, elephants and mole rats. Bats engage in antiphonal calling between adults   or betwe youngi) ,88–92. T youngi) . Female  females ,94. A re females ,94, with females . Naked my 400 ms ,95.6.Eucoptura xanthenes) to relatively complex sequences involving females alternating their replies between the pulsed phrases of the male signal  [Ancistrus nigrovittata; 20–30 ms, speckled bush-cricket Leptophyes punctatissima) to even 850 ms in species relying on bioluminescent systems  calls from different males are spaced at intervals of 2–10 s and rarely overlap [Philautus leucorhinus) engage in vocal matching which appears to be based on the nature of the rival's call rather than simply being an example of vocal stereotypy [Bufo americanus) engage in synchronous, or near-synchronous, overlapping of calls [Xenopus laevis), for example, females may produce a ‘rapping’ vocalization in response to male calling which is then responded to with a male answer call [In anurans, turn-taking mainly takes place in the form of antiphonal advertisement calls by males to attract females and has been observed in most groups of frogs. Males producing vocalizations in close distance to each other (or in response to playbacks) will typically become temporally entrained such that overlap is avoided. Calls thus occur within defined time points after the completion of another call –103. For overlap . Males oereotypy . A smallof calls . And, whwer call .8.Overall, direct comparisons of turn-taking skills of non-human animals in relation to language origins  are highly constrained by lack of data, the application of different terms, methodological designs  and study environments . Furthermore, investigations have so far mainly been focusing on single call types  or songs  of species, limiting an in-depth understanding of the variability and underlying cognitive flexibility of turn-taking systems found in the animal kingdom.To date, the parameters tested across different taxa and species to infer the organization of non-human animal turn-taking have mainly concerned a single key element of full-blown human turn-taking—the time window. Hence, the temporal adaptation and alteration of signal production seems to be a basic element of sociality and communication in general, and may have been the first step in the evolution of turn-taking systems. However, it is currently virtually impossible to evaluate whether time windows across species and taxa are indeed similar phenomena or differ because different definitions, methodologies and signal types (differing in form and function) have been investigated.et al.’s [et al. [Most progress concerning an in-depth understanding of the degree of similarity between human and non-human communicative turn-taking systems has been made by studies taking into consideration Sacks et al.’s  systematet al.’s  and Fröh [et al.  investigper se to preceding signals, while in contrast species-specific time windows in monkey species are learnt [Future studies should push this approach even further by testing whether the most crucial hallmarks of human conversational turn-taking can be found in turn-taking systems of other animals: who should communicate, move or act and when should interactants do so . Such ane learnt , with ine learnt ,109–111.9.(A) Flexibility of turn-taking organization(B) Who is taking the next turn?(C) When do response turns occur?(D) What should the next turn do?The new framework enabling comparative, systematic, quantitative assessments of turn-taking abilities centres on four key elements characterizing human social action during conversation:The first element—flexibility of turn-taking organization (A)—refers to the phenomena of varying size and ordering of turns and intentionality involved in human turn-taking sequences . The eleThe second element—who is taking the next turn (B)—concerns who can or should produce the next signal and includes techniques for allocating turns to individuals or parties . Paramettime window or temporal relationship between an initiating turn and the response turn [The third element—when do response turns occur (C)—addresses the nse turn ,24. Sincnse turn  for ideaadjacency pairs [first pair parts , and second pair parts  [The fourth element—what should the next turn do? (D)—concerns one of the most fundamental structures in the organization of human conversation: cy pairs . An adjacy pairs  and expa grants) . This el grants) ,116.10.A major avenue of future research is to use the comparative turn-taking framework to characterize the turn-taking phenotype of a wide variety of primate species. This could be done through systematic testing of carefully chosen representatives of more than 50 genera of primates, which should then enable us to map out cladistically the evolution of primates' turn-taking skills and systems. Furthermore, recent findings on language competence and cognitive skills of members of the parrot and corvid family –119 haveSuch an unprecedented, systematic comparative approach will empower us to test whether cooperative turn-taking represents the most ancient infrastructure of the language system and has been the ‘small change’ that made a big difference in human history. This new field of comparative turn-taking will thus shed light on one of the ‘hardest’ problems in science  by testi


Q331K transgenic mice, we further unveil that TDP-43 downregulates Parkin mRNA, which involves an unidentified, intron-independent mechanism and requires the RNA-binding and the protein–protein interaction functions of TDP-43. Unlike Parkin, TDP-43 does not regulate PINK1 at an RNA level. Instead, excess of TDP-43 causes cytosolic accumulation of cleaved PINK1 due to impaired proteasomal activity, leading to compromised mitochondrial functions. Consistent with the alterations at the molecular and cellular levels, we show that transgenic upregulation of Parkin but downregulation of PINK1 suppresses TDP-43-induced degenerative phenotypes in a Drosophila model of ALS. Together, these findings highlight the challenge associated with the heterogeneity and complexity of ALS pathogenesis, while pointing to Parkin–PINK1 as a common pathway that may be differentially misregulated in TDP-43 proteinopathy.Parkin and PINK1 play an important role in mitochondrial quality control, whose malfunction may also be involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). Excessive TDP-43 accumulation is a pathological hallmark of ALS and is associated with Parkin protein reduction in spinal cord neurons from sporadic ALS patients. In this study, we reveal that Parkin and PINK1 are differentially misregulated in TDP-43 proteinopathy at RNA and protein levels. Using knock-in flies, mouse primary neurons, and TDP-43 SOD1, C9orf72, FUS, and TARDBP are reported to cause familial ALS that accounts for the remaining 10%2. Ubiquitin-positive cytoplasmic inclusions containing transactive response DNA-binding protein 43 kDa  in the brain and the spinal cord of patients are a main pathological hallmark of ALS3. Moreover, TDP-43-positive protein inclusions are found in a large spectrum of neurodegenerative disorders, including frontotemporal lobar degeneration, Alzheimer’s disease, dementia with Lewy bodies, polyglutamine diseases, and others8, which are collectively known as TDP-43 proteinopathy.Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by progressive motor neuron loss, leading to muscle weakness and wasting. ALS is incurable and the patients usually die within 2–5 years after diagnosis. The majority (>90%) of ALS cases are sporadic (sALS) with unknown causes, whereas mutations in genes such as 11. With the recent advancement of the next-generation sequencing, the RNA targets as well as the common principles of TDP-43-mediated RNA regulations are emerging15.In physiological conditions, TDP-43 protein is predominantly localized to the nucleus. It belongs to the heterogeneous ribonucleoprotein family and plays an important role in regulating gene transcription, RNA processing, transport, and stability, as well as the formation of stress granules. In disease conditions, ubiquitinated TDP-43 accumulates in the cytoplasm. The mislocalization and aberrant aggregation of TDP-43 cause dysfunction of various aspects of RNA metabolism as well as protein homeostasis, eventually leading to motor neuron degeneration Parkin is one of the reported targets of TDP-4313. Parkin is an E3 ubiquitin ligase involved in the clearance of damaged mitochondria via autophagy (termed “mitophagy”), which partners with PTEN-induced putative kinase 1 (PINK1) to execute the mitochondrial quality control function. PINK1 is a serine/threonine kinase, which after translation is continuously transported into mitochondria, cleaved, and released to the cytosol for proteasome-mediated degradation17. When mitochondria are damaged, PINK1 cannot be effectively cleaved and is subsequently anchored on the mitochondrial outer membrane, which in turn recruits Parkin to mitochondria and induces mitophagy18. Mutations in Parkin and PINK1 genes are linked to autosomal recessive early-onset Parkinson’s disease (PD)20.The long intron-containing pre-mRNA of22. Interestingly, in the spinal cord autopsy samples from sALS patients, neurons with TDP-43 protein inclusions have reduced Parkin protein levels12. In this study, we investigate whether Parkin and PINK1 are involved in TDP-43-induced neurodegeneration. We find that Parkin and PINK1 are differentially misregulated by TDP-43 at the RNA and protein levels. Consistently, genetic manipulations of Parkin or PINK1 exhibit the opposing modifying effects in a Drosophila model of TDP-43 proteinopathy. Collectively, we propose that distinct multilevel misregulations of Parkin and PINK1 contribute to the pathogenesis of TDP-43 proteinopathy.Increasing evidence points to mitochondrial dysfunction as a common pathogenic factor in ALSParkin pre-mRNA and TDP-43 loss of function (LOF) reduced Parkin mRNA levels in mouse brains13. Since TDP-43-induced neurodegeneration involves both LOF and gain-of-function (GOF) mechanisms23, in this study we investigated whether TDP-43 GOF affected Parkin and PINK1, starting with overexpressing human TDP-43 (hTDP-43) in mammalian systems. We examined the mRNA levels of Parkin and PINK1 in human 293T cells transfected with hTDP-43-HA and mouse primary neurons infected with lentivirus to express hTDP-43-HA . To overcome this problem, we utilized the CRISPR-based gene editing and the transgenic Cas9-gRNA system28 to generate an HA knock-in (KI) fly in which an HA-tag was inserted to the C terminus of the endogenous Drosophila Parkin (dParkin) gene  and two dParkin-stop codon guide RNA (gRNA) vectors (pBFv-U6.3-dParkin-stop gRNA-1 and gRNA-2). The HA donor and the dParkin-stop gRNA plasmids were mixed for micro-injection using a nanos-Cas9 founder line. The desired transformants were selected for subsequent balancing and background clearing, and were eventually established as a stable dParkin-(HA) KI line  KI flies to hTDP-43 flies and examined the dParkin-HA protein levels by western blotting. Compared to the control group (GMR driver only), expression of hTDP-43 in the fly eyes caused a significant reduction of the endogenous Parkin protein levels  constructed in the same expression plasmid was included as a control. The reverse transcription-PCR (RT-PCR) primers were designed to distinguish the Flag-tagged Parkin and V5-tagged PINK1 from the endogenously expressed Parkin and PINK1 mRNA in 293T cells (see Methods). As shown in Fig. Flag-Parkin were reduced by about 50% in TDP-43-overexpressing cells, whereas PINK1-V5 mRNA was not significantly changed , it was unlikely that the selective downregulation of plasmid-expressed, intron-free Parkin was due to a general effect of TDP-43 on transcription or the untranslated regions (UTRs) of the expression plasmid.We then examined whether TDP-43 could downregulate the mRNA levels of the plasmid-expressed, intron-free human ged Fig. . Consistged Fig. . In cont10. To understand what motif(s) are required and how TDP-43 regulates Parkin in the absence of any intron or gene-specific UTR, we generated the plasmids to express truncated TDP-43 protein: hTDP-43-HA-ΔRRM1, -ΔRRM2, and -ΔGRD  motifs involved in DNA and RNA binding, and a glycine-rich domain (GRD) domain at the C terminus that mediates protein–protein interaction with other members of the heterogeneous nuclear RNP protein familyGRD Fig. . We foun-43 Fig. . Furthervel Fig. . These rPINK1 mRNA or FL PINK1 protein levels (~64 kDa), there was a remarkable increase of cleaved PINK1 (~52 kDa29) in the western blots NK1 Fig.  stronglyNK1 Fig. . ConsideParkin pre-mRNA13 and intron-independent regulation of Parkin mRNA  of the 293T cells transfected with PINK1Δ1-104-V5 or vector. The basal mitochondrial respiration occurred similarly, whereas the spare respiratory capacity was significantly reduced in PINK1Δ1-104-V5 cells  driver59 to express hTDP-43 in adult fly neurons only (induced after eclosion of the adult fly). Expression of hTDP-43 in adult fly neurons induced an age-dependent decline of climbing capability  and might be recognized by PUM139. However, as the PUF recognition sequence usually locates within 3′UTRs, the exact mechanism of this enigmatic intron/UTR-independent regulation of Parkin mRNA is yet to be unraveled by additional research in the future.Furthermore, we find that downregulation of intron-free PINK1 mRNA or protein levels, we find that it selectively impedes PINK1 protein turnover and causes cleaved PINK1 to accumulate in the cytoplasm due to the impairment of proteasomal degradation , pcDNA-DEST47 PINK1 C-GFP (Addgene #1331655), and pcDNA3.1-TDP-43-Myc plasmid (a gift from H-Y Hu) by PCR using primers containing the Flag, V5, or HA-tag sequence. The PCR products were then subcloned into a pCAG vector56 using the XhoI/EcoRI sites. The pDsRed2-mito vector was from Clontech Laboratories, Inc.To generate the pCAG-Flag-Parkin, pCAG-PINK1-V5, and pCAG-hTDP-43-HA plasmids, DNA fragments encoding human Parkin, PINK1, and TDP-43 were amplified from YFP-PARK2 . The constructs were then subcloned into the pCAG expression vector as above. The PCR primers used were listed below:ΔRRM1-F1: 5′-ATGGCCATGGAGGCCGAATTCATGTCTGAATATATT-3′ΔRRM1-R1: 5′-CTCATCTTGGCTTTGGGATGTTTTCTGGACTGCTC-3′ΔRRM1-F2: 5′-CAAAGCCAAGATGAG-3′ΔRRM1-R2: 5′-GATGAGATGTTTTCTGGACTGCTC-3′ΔRRM2-F1: 5′-ATGGCCATGGAGGCCGAATTCATGTCTGAATATATT-3′ΔRRM2-R1: 5′-CTATTGCTATTGTGCTTAGGGCTTCTCAAAGGCTCATCTT-3′ΔRRM2-F2: 5′-CCTAAGCACAATAGC-3′ΔRRM2-R2: 5′-CATGTCTGGATCCCCGCGGCCGCCTACATTCCCCAGCCAGA-3′ΔGRD-F: 5′-ATGGCCATGGAGGCCGAATTCATGTCTGAATATATT-3′ΔGRD-R: 5′-CATGTCTGGATCCCCGCGGCCGCCTAACTTCTTTCTAACTGTCTATT-3′Δ1-104-V5 plasmid, the DNA fragments of truncated PINK1 were amplified from FL PINK1 by PCR and inserted into the pCAG vector as above. The PCR primers used were:To generate the pCAG-PINK1Δ1-104-F: 5′-CATCATTTTGGCAAAGAATTCCACCATGGGGCTAGGGCTGGGCCTC-3′PINK1Δ1-104-V5-R: 5′-GCTCCCCGGGGGTACCTCGAGTTACGTAGAATCGAGACCGAGGAGAGGGTTAGGGATAGGCTTACCCAGGGCTGCCCTCCATGAG-3′.PINK1All constructs were verified by sequencing to ensure the integrity of the cloned open reading frames.TM (Invitrogen) at 37 °C in 5% CO2. Transient transfection was performed using Lipofectamine™ 2000 (Invitrogen) in Opti-MEM (Invitrogen). Cells were transfected for 48 h before harvest. For proteasome inhibition assays, MG-132 was added into the medium 3 h before harvest at a final concentration of 50 nM.293T cells were cultured in Dulbecco’s modified Eagle medium (Invitrogen) supplemented with 10% (v/v) fetal bovine serum (BioWest) and GlutaMAXLentivirus was prepared according to the established protocols. Briefly, lentivirus packaging was performed by co-transfecting FHsynPW-TDP-43-HA with VSVG and delta 8.9 with the ratio of 1:1.5:2 into 293FT cells cultured in Opti-MEM I medium using PolyJet™ reagent (SignaGen). Culture supernatant was collected at 48 h after transfection and passed through a 0.45-μm filter. Viral particles were concentrated from culture supernatants by Lenti-X™ Concentrator (Clontech). Viral pellets used for neuronal infection were resuspended in Neurobasal medium (Invitrogen).Q331K mouse-derived culture, the cortical neurons of the transgenics or their littermates were isolated at E16–E18 and cultured as above. The RNA and protein levels of TDP-43Q331K neurons were examined at 15 DIV.Primary hippocampal neurons were isolated from C57BL/6 mouse hippocampus at embryonic day 14 (E14) and cultured in serum-free Neurobasal medium (Invitrogen) supplemented with 2% B27 (Life Technologies), GlutaMax, and penicillin-streptomycin (Invitrogen). At 14 days in vitro (DIV), neurons were infected with FHsynPW-TDP-43-HA for 5 days before extraction for RNA or protein. For TDP-43Q331K-myc transgenic mice24 were derived from the B6N.Cg-Tg (Prnp-TARDBP*Q331K) 103Dwc/J line  and maintained by breeding to the C57BL/6N background mice purchased from Vital River Laboratories Co., Ltd. All animal experiments were carried out in compliance with the institutional guidelines on the scientific use of living animals at Interdisciplinary Research Center on Biology and Chemistry (IRCBC) of the Chinese Academy of Sciences .The TDP-43Drosophila Stock Center: RNAi-mCherry ; UAS-lacZ ; elavGS (#43642); UAS-PINK1 (#51648); and UAS-Parkin (#51651). The following strains were obtained from the Tsinghua Fly Center: RNAi-PINK1 (#38262) and RNAi-Parkin (#38333). The transgenic fly strain of UAS-TDP-43 was generated by ΦC31 integrase-mediated, site-specific integration, and the attP landing site stock used was UAS-phi2b2a;VK5 (75B1).The following strains were obtained from the Bloomington elavGS driver,59 flies were raised on regular fly food supplemented with 80 μg/ml RU486 (TCI).Flies tested in this study were raised on standard cornmeal media and maintained at 25 °C and 60% relative humidity. For adult-onset, neuronal expression of the UAS or RNAi transgenes using the A 2xHA tag donor vector was generated from the pCR2-TOPO-3xp3-eGFP HR vector following the protocols on the flyCRISPR website. The 2xHA-stop codon-fused pBac 3′-terminal repeat fragments were created based on the pHD-2xHA-ScarlessDsRed vector by PCR reaction using the following primers: F1_5′-ACGTGCCAGACTATGCTTAACCCTAGAAAGATAATCATATTGTGACGTAC-3′, F2_5′-ATGTTCCAGATTACGCTGGATATCCATACGACGTGCCAGACTATGCTTAA-3′, F3_5′-gctagcctgacccgggGAAGACCTTACCCATACGATGTTCCAGATTACGCTGGA-3′ R_5′-aattagatcccctaggTAAAAGTTTTGTTACTTTATAGAAGAAATT-3′.NdeI/AvrII sites of pCR2-TOPO-3xp3-eGFP HR vector by In-Fusion HD Cloning Kit (Clontech) to get a transition vector. The pBac 5′-terminal repeat fragment was created from pHD-2xHA-ScarlessDsRed by PCR reaction using the following primers:A 338-bp PCR product was generated and cloned into the F_5′-cttatcgaatacgcgtGATATCTATAACAAGAAAATATATATATAA-3′ R_5′-ctcgagcgatcatatgCGTCTCCTTAACCCTAGAAAGATAGTCTGCGTAAAAT-3′.MluI/NdeI sites of the transition vector by In-Fusion HD Cloning Kit to get the pCR2-TOPO-2xHA-PBac-3xp3-eGFP vector.A 352-bp PCR product was generated and cloned into the dParkin flanking sequence without the stop codon was generated by PCR of the genomic DNA from the W1118 flies with the following primers:The L-arm of the F_ 5′-atccactagtgctagcGGATTTTGCGGCTCCATTGAATCTAATCAA-3′R_5′-aacatcgtatgggtaGCCGAACCAGTGAGCTCCCATGCAGTCGCG-3′.NheI/BbsI sites of pCR2-TOPO-2xHA-PBac-3xp3-eGFP by In-Fusion HD Cloning Kit to get a Parkin-2xHA transition vector. The R-arm of dParkin flanking sequence was generated similarly using the following primers:A 1032-bp PCR product was generated and cloned into the F_5′-tctttctagggttaaTCCTTCGTCATCGTTGTACGTCGGCCCGACGTGTCCTTTTTG-3′R_5′-tagatgcatgctcgagAGCGGCAAACCGAAAGGAATCTAGAACGTG-3′.BsmbI/XhoI sites of dParkin-2xHA transition vector by In-Fusion HD Cloning Kit to get the complete pCR2-TOPO-dParkin-2xHA-PBac-3xp3-eGFP HR donor vector.A 1149-bp PCR product was generated and cloned into the dParkin gene for CRISPR/Cas9 recognition were designed using the flyCRISPR Optimal Target Finder platform of flyCRISPR website. Two target sites close to the stop codon of the dParkin gene were found: gRNA-t1 site 5′-GAAGGATTAGCCGAACCAGTGGG-3′ and gRNA-t2 site 5′-TCGTTGTACGTCGGCCCGACGGG-3′. The target site fragments were generated by primer annealing and cloned into the BbsI sites of pBFv-U6.3 vector (modified from pBFv-U6.2) to get the pBFv-U6.3-dParkin-stop gRNA-t1 and gRNA-t2 plasmids.The target sites of dParkin-stop gRNA plasmids were mixed with the pCR2-TOPO-dParkin-2xHA-PBac-3xp3-eGFP HR donor vector for nanos-Cas9 founder line injection. Following green fluorescent protein (GFP)-mediated identification of successful KI flies, the GFP marker was removed through a single cross to PBac transposase. The embryo injection and selection of correct transformants were performed by BestGene Inc. and confirmed by PCR in the lab.The two pBFv-U6.3-actin or GAPDH were used as an internal control to normalize the mRNA levels of genes of interest. The qPCR primers used in this study are listed below:For quantitative PCR (qPCR), total RNA was isolated from fly heads, cell cultures, or mouse brain tissues using TRIzol (Invitrogen) according to the manufacturer’s instruction. After DNase (Promega) treatment to remove genomic DNA, the RT reactions were performed using All-in-One cDNA Synthesis SuperMix kit (Bimake). The cDNA was then used either for semiquantitative RT-PCR experiments by PCR amplification using the Taq Plus Master Mix (Vazyme) or real-time qPCR using the SYBR Green qPCR Master Mix (Bimake) with the QuantStudio™ 6 Flex Real-Time PCR system (Life Technologies). The mRNA levels of hβ-actin forward: 5′-GTTACAGGAAGTCCCTTGCCATCC-3′hβ-actin reverse: 5′-CACCTCCCCTGTGTGGACTTGGG-3′hPINK1 forward: 5′-CCCAAGCAACTAGCCCCTC-3′hPINK1 reverse: 5′-GGCAGCACATCAGGGTAGTC-3′hParkin forward: 5′-GTGTTTGTCAGGTTCAACTCCA-3′hParkin reverse: 5′-GAAAATCACACGCAACTGGTC-3′hTDP-43 forward: 5′-GGGAAATCTGGTGTATGTTGTCA-3′hTDP-43 reverse: 5′-GAAAATCACACGCAACTGGTC-3′PINK1-V5 forward: 5′-CAGACGTGAGACAGTTGGTG-3′PINK1-V5 reverse: 5′-GTAGAATCGAGACCGAGGAGAG-3′Flag-Parkin forward: 5′-CAAGGATGACGACGATAAGT-3′Flag-Parkin reverse: 5′-GCTGGAAGATGCTGGTGT-3′mGAPDH forward: 5′-CACCATCTTCCAGGAGCGAG-3′mGAPDH reverse: 5′-CCTTCTCCATGGTGGTGAAGAC-3′mPINK1 forward: 5′-CACACTGTTCCTCGTTATGAAGA-3′mPINK1 reverse: 5′-CTTGAGATCCCGATGGGCAAT-3′mParkin forward: 5′-TCTTCCAGTGTAACCACCGTC-3′mParkin reverse: 5′-TCTTCCAGTGTAACCACCGTC-3′dActin forward: 5′-GAGCGCGGTTACTCTTTCAC-3′dActin reverse: 5′-GCCATCTCCTGCTCAAAGTC-3′dPINK1 forward: 5′-GAGCAACAGCAGCATCAGAA-3′dPINK1 reverse: 5′-TGATGTTTGAATTCGCTGGA-3′dParkin forward: 5′-AGCGATGCCACGACAATAGAGC-3′dParkin reverse: 5′-GCGAAGGTTCCTCCTCCTCCAA-3′® 488 , and anti-chicken Alexa Fluor® 633 .The following antibodies were used for western blotting, immunoprecipitation, and immunofluorescence assays: mouse anti-FLAG ; mouse anti-HA ; rabbit anti-HA ; rabbit anti-TDP-43 ; rabbit anti-PINK1 ; mouse anti-Parkin ; mouse anti-V5 ; mouse anti-GAPDH ; rabbit anti-Tubulin ; mouse anti-Lamin C ; and chicken anti-MAP2 . Horseradish peroxidase-conjugated secondary antibodies: anti-mouse  and anti-rabbit . Fluorescent secondary antibodies: anti-mouse Cy5 , anti-rabbit Alexa Fluorg for 20 min at 4 °C. The resulting supernatant was used as the soluble fraction and the pellets containing insoluble fractions were dissolved in a 9 M urea buffer  after wash.Fly heads or cultured cells were lysed in 2% SDS lysis buffer  containing protease and phosphatase inhibitor cocktails (Roche). For separation of soluble and insoluble proteins, cells were lysed on ice using RIPA buffer  supplemented with protease and phosphatase inhibitors (Roche). Samples were sonicated and then centrifuged at 13 000 × Fresh brains of deep-anesthetized mice were rapidly excised and the motor cortices were isolated and collected in sterile 1.5 ml micro-centrifuge tubes. The samples were quickly plunged into liquid nitrogen and stored at −80 °C until testing. The brain tissues were then lysed and homogenized in ice-cold RIPA buffer supplemented with protease and phosphatase inhibitors (Roche).Equivalent amounts of lysates were resolved by electrophoresis through a 10% Bis-Tris SDS-polyacrylamide gel electrophoresis gel (Invitrogen) and probed with the primary and secondary antibodies listed above. Detection was performed using the High-sig ECL Western Blotting Substrate (Tanon). Images were captured using an Amersham Imager 600  and densitometry was measured using ImageQuant TL Software . The contrast and brightness were optimized equally using Adobe Photoshop CS6 (Adobe Systems Inc.). All experiments were normalized to GAPDH, tubulin, or actin levels as indicated in the blots and the values are plotted relative to the control  in across-assay comparison quantifications.15 followed by centrifuge at 3000 × g for 5 min at 4 °C. The supernatant was collected as the cytoplasmic fraction and the pellet was dissolved in tissue lysis buffer (Invitrogen) as the nuclear fraction.For nuclear–cytoplasmic fractionation, 20 flies per genotype were homogenized in the lysis buffer  by incubating for 5 min on ice as reported293T cells or primary neurons were grown on Nunc Chambered Coverglasses (Lab-Tek) and transfected with the indicated plasmids for 24 h. Thereafter, cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min at room temperature, permeabilized with 0.5% Triton X-100 in PBS for 15 min, and blocked with 3% goat serum in PBST (PBS + 0.1% Triton X-100) for 1 h at room temperature. The above primary and secondary antibodies in the blocking buffer were then incubated at 4 °C overnight or at room temperature for 1 h. After three washes with PBST, cells were mounted on glass slides using Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories).Fluorescent images were taken with Leica TCS SP8 confocal microscopy system using a ×63 oil objective . Co-localization of Parkin or PINK1 with mitochondria was evaluated by the line scanning analysis of LAS X and the protein puncta were counted using the “Analyze Particles” module of ImageJ. Images were assembled into figures using Adobe Photoshop CS6.g for 10 min at 4 °C. The proteasomal activity of the supernatant was determined by assaying the cleavage of a fluorogenic peptide substrate Suc-LLVY-AMC according to the manufacturer’s instruction. The fluorescence intensity was measured after the substrate peptide was incubated with the cell lysates at 37 °C at the end of the assay (60 min) using a microplate reader .Proteasomal activity was measured using a Proteasome Activity Assay kit . 293T cells were lysed in PBS with 0.5% NP-40 on ice for 10 min. The supernatant was obtained by centrifuge at 13 000 × 4 293T cells were seeded onto 96-well microplates pre-coated with poly-l-lysine. On the next day, the OCRs (pmol/min) of the cells in XF base medium containing 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose (Sigma-Aldrich) were assayed with the Seahorse CF Cell Mito Stress Test following sequential additions of 1 μM oligomycin, 0.5 μM Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone, and a combination of 0.5 μM antimycin A and 0.5 μM rotenone.Mitochondrial respiration functions were evaluated by measuring the OCRs using the Seahorse XFe96 Analyzer (Agilent) per the manufacturer’s instruction. Briefly, 24 h after transfection, 4 × 1058For the lifespan experiment, 20 flies/vial, 5–8 vials/group were tested. Flies were transferred to fresh fly food every 3 days and the number of dead flies of each vial was recorded. Flies lost through escape or accidental death were excluded from the final analysis. The median lifespan was calculated as the mean of the medians of each vial belonging to the same group, whereas the “50% survival” shown on the survival curves was derived from compilation of all vials of the group. For the climbing assay, 20 flies were transferred into an empty polystyrene vial and gently tapped down to the bottom of the vial. The number of flies that climbed over a distance of 3 cm within 10 s was recorded. The test was repeated three times for each vial and 5 vials of each genotype were tested.t-test at *p < 0.05, **p < 0.01, and ***p < 0.001. Error bars represent the standard error of the mean.Unless otherwise noted, statistical significance in this study is determined by unpaired, two-tailed Student’s Supporting Information


Background: Programmed death-ligand 1 (PD-L1) is expressed not only on some cancer cells, but also on the outer surface of placental syncytiotrophoblasts, which is assumed to induce maternal immune tolerance to fetal tissue via programmed death-1 (PD-1) receptors on T cells. Recently, levels of soluble forms of PD-L1 (sPD-L1) were reported to be higher in the serum of pregnant women (PW) than in non-pregnant women (non-PW). However, there have been no reports of the functional significance of PW's serum containing high sPD-L1 levels. Therefore, the aim of the present study was to clarify the role of sPD-L1 in the sera of PW as an immunosuppressive molecule by in vitro assays.Methods: As a post-hoc analysis of our previous cohort study, 330 pairs of serum from PW during the third trimester and cord blood (CB) from paired offspring without major complications were examined. Serum levels of sPD-L1 and sPD-1 were measured by ELISA. On mixed lymphocyte culture (MLC), 3H-thymidine uptakes in the presence of PW's, offspring's, or non-PW's serum were compared. Peripheral blood mononuclear cells (PBMCs) were cultured in the presence of PW's serum stimulated with PHA, and then cytokine levels were measured in supernatants by multiple cytokine analysis with or without anti-PD-L1blocking antibody.Results: The median sPD-L1 level was 8.3- and 6.9-fold higher in PW than in offspring and non-PW, respectively, whereas sPD-1 levels were lower in PW and offspring than in non-PW. On MLC, 3H-thymidine uptake in the presence of autoantigen was strongly reduced by co-culture with serum of both PW and offspring, compared with serum of non-PW. In contrast, uptake in the presence of alloantigen was moderately inhibited by PW's serum, whereas it was less suppressed by offspring's serum, compared with non-PW's serum. In the culture of PBMCs, tumor necrosis factor-α, interferon gamma, interleukin (IL)-2, and IL-4 levels were significantly higher in the presence of anti-PD-L1 blocking antibody than in culture not treated with antibody  or culture treated with isotype control antibody .Conclusion: The levels of sPD-L1 are elevated in PW's serum, which may, at least in part, suppress maternal immunity. Programmed death-ligand 1 (PD-L1) is expressed on some cancer cells to suppress anti-tumor immunity by interacting with the programmed death-1 (PD-1) receptor expressed on T cells , 3. By iin vitro assays.Both PD-L1 and PD-1 exist in membrane-bound form and induce local or peripheral immune tolerance by maintaining the quiescence of autoreactive T cells. Recently, soluble forms of PD-L1 and PD-1 (sPD-L1 and sPD-1), which are considered to be generated by proteolytic cleavage of the membrane-bound forms, have been detected in the serum of cancer patients , 9. ELISpost-hoc analysis, 330 pairs of PW and their offspring were randomly selected from our previous cohort study .Serum samples were collected from PW at 34 weeks of gestation. The offspring's serum (5–10 mL) was sampled from the placental side after placental delivery at birth. The serum samples were stored at −80°C prior to use. Serum levels of sPD-L1 and sPD-1 were measured by MO, using ELISA kits from Abcam  and RayBiotech , respectively, according to the manufacturers' protocols. Each sample was tested in triplicate for sPD-L1 and in duplicate for sPD-1, with the medians used for analysis. The lower detection limits for ELISA were 3.9 pg/mL for sPD-L1 and 20 pg/mL for sPD-1. The upper detection limits for ELISA were 1,300 pg/mL for sPD-L1 and 6,000 pg/mL for sPD-1.3H-thymidine uptake using a mixed lymphocyte culture (MLC) assay system at SRL Inc . Briefly, peripheral blood mononuclear cells (PBMCs) were obtained from three healthy male volunteers, named A, B, and C. For the MLC assay with autoantigen, fresh PBMCs were co-cultured with 13-Gy-irradiated PBMCs from the same donor in three patterns, i.e., fresh A—irradiated A, fresh B—irradiated B, and fresh C—irradiated C. For the MLC assay with alloantigen, fresh PBMCs were co-cultured with 13-Gy-irradiated PBMCs from different donors in four patterns, i.e., fresh A—irradiated B, fresh A—irradiated C, fresh B—irradiated A, and fresh C—irradiated A. These cells were cultured for 5 days with RPMI1640 and 20% of either a mixture of serum samples from PW, offspring, or non-PW, randomly selected from the cohort of this study. Each kind of serum was a mixture of at least 10 samples. Cells were pulsed with 3H-thymidine during the last 17.5 h of incubation and counted in a liquid scintillation counter . This MLC experiment was repeated for three sets using the same serum samples.Reactions of lymphocytes in the presence of either autoantigen or alloantigen were measured by 4 PBMCs per well in 96-well U-bottom plates were co-cultured with RPMI1640 and a mixture of serum samples derived from at least 10 PW randomly selected from the cohort of this study. The serum concentration was brought to 5%, and treated with 5 μg/ml of phytohemagglutinin (PHA)  for stimulation. Samples treated with anti-PD-L1 blocking antibody (5 μg/ml) , isotype control (5 μg/ml) , and samples without antibody were cultured according to the method of Andorsky et al.  was used for all analyses. Values of P < 0.05 were considered significant. These analyses were not corrected for multiple comparisons.Because the skewness test showed that sPD-L1 levels and sPD-1 levels were not normally distributed, their levels were compared among control non-PW, PW, and CB using the Kruskal-Wallis rank test (KW test). If the result of the KW test was significant, then the Mann-Whitney test was used to compare between two groups. Spearman's rank correlation, represented as rho, with linear regression was used to quantify the strengths of associations between two continuous variables: rho ≥0.4, strong; 0.4> rho ≥0.2, moderate; and rho <0.2, weak. The test for trends across ordered groups developed by Cuzick  was usedpost-hoc manner, 330 pairs of PW and offspring were analyzed in this study; the demographic and clinical data are shown in In a P = 0.0001) (P < 0.0001) and 6.9-fold higher in PW than in non-PW (P < 0.0001). The median level of sPD-L1 was lower in offspring than in non-PW (P = 0.003). Then, serum sPD-1 levels were compared among the three groups to find significant differences (KW test: P = 0.0006) (P = 0.0003), and 4.6-fold higher than in serum of offspring (P = 0.0001). However, the sPD-1 level was not significantly different between PW and offspring (P = 0.72). The median sPD-1/sPD-L1 ratio of non-PW was 41.6-fold higher than in PW (P < 0.0001) and 4.6-fold higher than in offspring (P = 0.001), and the ratio of offspring was 9.1-fold higher than in PW (P < 0.0001)  . The med 0.0006) . In the  0.0001) .P < 0.0001) (P < 0.0001)  . The sPD 0.0001) . In cont3H-thymidine uptake in the presence of autoantigen  and offspring , compared with that of non-PW. On the other hand, there was no difference in the uptake between sera from PW and offspring. In the presence of alloantigen, 3H-thymidine uptake was moderately reduced by co-culture with serum from PW , and not significantly reduced by co-culture with offspring's sera (P = 0.33), compared with that of non-PW.Level of oantigen  and allooantigen  were comP < 0.05). On the other hand, the levels of these cytokines did not show significant difference between samples without antibody and those treated with isotype antibody. There was a similar tendency for IL-6, but it was not significant (data not shown).In the presence of PW's sera, anti-PD-L1 blocking antibody increased the secretion of TNF-α , IFN-γ , IL-2 F, and IL-P = 0.0006) (P = 0.03) (P = 0.03), whereas there was no significant difference in sPD-L1 levels between non-smokers and past smokers.The levels of sPD-L1 were compared among non-smokers, past smokers, and current smokers among PW to find significant differences (KW test:  0.0006) . The sPD = 0.03) . In partOffspring anthropometric measurements at birth were compared with serum sPD-L1 levels in PW as both quartiles and continuous variables.P < 0.0001) and a moderate Spearman's rho  (The median body weight at birth in the highest sPD-L1 quartile (Q4) was 270 g heavier than in the lowest quartile (Q1). Thus, there was a significant trend for increased body weight with higher levels of sPD-L1  . On multP < 0.0001) and a weak Spearman's rho  (The median body height of the highest quartile (Q4) of sPD-L1 was 0.6 cm taller than that of the lowest quartile (Q1). There was thus a significant trend for increased body height with higher levels of sPD-L1  . On multP = 0.009) and a weak Spearman's rho  (The median head circumference of the highest quartile (Q4) of sPD-L1 was 0.4 cm longer than that of the lowest quartile (Q1). There was thus a significant trend for increased head circumference with higher levels of sPD-L1  . On multP < 0.0001) and a moderate Spearman's rho  (The median chest circumference of the highest quartile (Q4) of sPD-L1 was 0.5 cm longer than that of the lowest quartile (Q1). There was thus a significant trend for increased chest circumference with higher levels of sPD-L1  . On multin vitro experiments of cytokine production assays with PHA stimulation, where an attempt was made to block specific functions of sPD-L1 in PW's serum with anti-PD-L1 antibody, were added. These experiments showed that various cytokines were increased in the presence of anti-sPD-L1 blocking antibody, suggesting that the elevated sPD-L1 protein in PW's serum may be functional, able to suppress broad immune reactions, and may thus be considered to protect the placenta and fetus from maternal immunosurveillance, at least in part.In the present study, the median serum sPD-L1 level in PW during the third trimester was high, 8.3-fold higher than in offspring and 6.9-fold higher than in healthy age-matched non-PW controls. Although serum levels of sPD-L1 were reported to increase throughout gestation  when bloWith respect to clinical data, levels of sPD-L1 in PW showed a negative association with smoking. It has been well reported that maternal smoking impairs placental structure and function , 21. In There are several limitations of the present study. First, PW with major complications, such as gestational diabetes mellitus, pregnancy-induced hypertension, pre-eclampsia, preterm labor, or the need for emergent cesarean section, and who lacked high-risk fetal conditions, such as twins, intrauterine growth retardation, and congenital malformation, were excluded. Thus, associations between sPD-L1 levels of PW and these complications could not be examined. Instead, associations with smoking status and with fetal growth were examined to show that serum sPD-L1 levels can be a biomarker of placental function. Second, functional assays were added to support the hypothesis that sPD-L1 in addition to PD-L1 expressed on the surface of the placenta suppresses the maternal immune reaction to reject the placenta and fetus. It was demonstrated that the serum of PW showed stronger suppressive effects on autogenic and allogeneic immune reactions than serum of non-PW. Moreover, this suppressive effect of PW's sera was blocked using anti-PD-L1 antibody, as demonstrated by cytokine production assays. Third, MLC, which is used to evaluate the possibility of graft vs. host or host vs. graft disease in the field of bone marrow transplantation or organ transplantation, was used to examine immune reactions in the presence of PW's serum, considering the immunological paradox of pregnancy. However, there are many other types of functional assays. Fourth, since placental tissue was not collected in this cohort, for example, direct interactions between syncytiotrophoblasts and T cells could not be examined. Instead, the focus was not on membrane-bound PD-L1 molecules expressed on syncytiotrophoblasts, but on the soluble form of PD-L1 in the serum of PW. Fifth, sPD-L1 was measured at only one point in the third trimester during pregnancy. Sixth, placental PD-L1 protein expression was not measured by immunohistochemistry.3H-thymidine uptake in the presence of autoantigen was strongly reduced by co-culture with sera from both PW and offspring, compared with non-PW's serum, while uptake in the presence of alloantigen was moderately inhibited by sera from PW, but it was not significantly suppressed by offspring's serum, compared with non-PW's serum; (5) adding the anti-PD-L1 blocking antibody to sera from PW raised cytokine secretion (6); and, finally, sPD-L1 levels in PW and in non-PW were suppressed by smoking.In conclusion, this is the first study to measure both sPD-L1 and sPD-1 levels in the serum of PW, as well as paired offspring, and it showed that: (1) sPD-L1 levels were very high in the serum of PW in the third trimester, but low in paired offspring; (2) sPD-1 levels were lower in both PW and offspring than in non-PW; (3) there was a strong correlation of sPD-1 levels, as well as sPD-L1 levels, between PW and offspring; (4) on MLC, The novel finding of this study was that PW's serum may suppress both autogenic and allogeneic immune reactions, whereas offspring's serum may suppress mainly the autogenic immune reaction and only partly allogeneic immune reactions. It was further shown that anti-PD-L1 antibody impairs the immunosuppressive effects of PW's serum, and it was suggested that elevated sPD-L1 levels in PW's sera may be functional and play, at least in part, a role in suppressing the maternal immune reaction to alloantigen, i.e., placenta and fetus. Further research is needed to confirm this.MO and MU conceptualized and designed the study, drafted the initial manuscript, and reviewed and revised the manuscript. TK and HM collected and fixed clinical data and reviewed and revised the manuscript. MU performed experiments and analyzed data statistically. All authors approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


GS and GL) and selected tyrosine conformers in gas and aqueous phases obtained at M06-2X/6-31G(d) level are given. The data also include total energies of all complexes in the gas phase and the aqueous phase, binding energies, BSSE (basis set superposition error) correction, and BSSE-corrected binding energies in gas phase and solvation effect on the binding energies obtained at M06-2X/6-31G(d) level. Mulliken charges of tyrosine conformers in gas and aqueous phases, and the deformation energy for tyrosine and graphene in the gas phase complexes are provided. The values of the highest occupied molecular orbital (HOMO), the lowest unoccupied molecular orbital (LUMO) and HOMO-LUMO energy gaps for some of graphene-tyrosine complexes that were not reported in the article [1] are given. The data is related to the research article “Conformation dependence of tyrosine binding on the surface of graphene: Bent prefers over parallel orientation” [1].In this data article, M06-2X/6-31G(d) level optimized geometries of complexes of tyrosine conformers binding with graphene sheets are shown in top and side views with selected non-bonding distances. The images of frontier molecular orbitals from HOMO-15 to LUMO+15 of the complexes involving graphene with tyrosine conformers are presented and the isovalue is 0.003 au. For some complexes involving small graphene, the orbitals are from HOMO-5 to LUMO+5. The molecular orbitals highlighted with frames show obvious overlaps between the fragments. Total energies of small and large graphene ( Tyr1 conformer in GS/L-Tyr1-B and GS/L-Tyr1-B1 with selected non-bonding distances and binding energies. Tyr2 and Tyr3 conformers on graphene surfaces (GS and GL) obtained at M06-2X/6-31G(d) level and the selected non-bonding distances as well as the binding energies are provided. The complexes GS/L-Tyr2-D and GS/L-Tyr3-D showing the amino acids near the edges of graphene are depicted in GS-Tyr1-A, GS-Tyr4-A, GS-Tyr1-B, and GS-Tyr4-B. GS-Tyr1-A and GS-Tyr4-A, respectively. GS-Tyr1-B and GS-Tyr4-B, respectively. The pictures of selected FMOs from HOMO-15 to LUMO+15 showing obvious overlaps between the fragments for the complexes GL-Tyr1-A and GL-Tyr4-A, GL-Tyr1-B and GL-Tyr4-B are presented in Tyr in gas and aqueous phases, and the deformation energy for Tyr and graphene in the gas phase for selected complexes. Four selected conformers of tyrosine (Tyr) with two different sizes of the graphene sheets are shown in 2Selected conformers of tyrosine, two different sizes of graphene systems, and tyrosine-graphene complexes were fully optimized using M06-2X functional


Defect-free monolayers of graphene and hexagonal boron nitride are surprisingly permeable to thermal protons, despite being completely impenetrable to all gases. It remains untested whether small ions can permeate through the two-dimensional crystals. Here we show that mechanically exfoliated graphene and hexagonal boron nitride exhibit perfect Nernst selectivity such that only protons can permeate through, with no detectable flow of counterions. In the experiments, we use suspended monolayers that have few, if any, atomic-scale defects, as shown by gas permeation tests, and place them to separate reservoirs filled with hydrochloric acid solutions. Protons account for all the electrical current and chloride ions are blocked. This result corroborates the previous conclusion that thermal protons can pierce defect-free two-dimensional crystals. Besides the importance for theoretical developments, our results are also of interest for research on various separation technologies based on two-dimensional materials. Defect-free monolayers of graphene and hexagonal boron nitride are highly permeable to thermal protons, but are impenetrable to gases. Here the authors show that mechanically exfoliated crystals exhibit perfect proton selectivity, corroborating proton transport through the bulk without atomic-scale defects. As for the experiment, it was found that proton permeation through mechanically exfoliated crystals is thermally activated with energy barriers of ≈0.8 eV for graphene and ≈0.3 eV for monolayer hexagonal boron nitride (hBN)1. Further measurements using deuterons, nuclei of the hydrogen isotope deuterium, show that quantum oscillations raise the energy of incoming protons by 0.2 eV2. This correction yielded the total barriers of ≈0.5 eV for monolayer hBN and ≈1 eV for graphene. From a theoretical perspective, the latter value is notably lower  than that found in density-functional calculations for graphene7. To account for the difference, a recent theory suggests that graphene can be partially hydrogenated during the measurements, which makes its lattice slightly sparser; thus, making it more permeable to protons9. An alternative explanation put forward attributes the observed proton currents to atomic-scale lattice defects, including vacancies11. This was argued on the basis of ion-selectivity measurements using chemical-vapor-deposited (CVD) graphene11. Indeed, CVD graphene is known to possess a large density of atomic-scale defects that appear during growth14. Such defects are generally absent in mechanically exfoliated 2D crystals, which was proven conclusively in gas-leak experiments using the so-called nanoballoons17. Even a single angstrom-sized vacancy per micrometer-size area could be detected in those experiments17. Whereas it is plausible that vacancies and similar defects played a dominant role in experiments using CVD graphene11, extrapolation of those results to mechanically exfoliated 2D crystals is unjustifiable. To resolve the controversy, it is crucial to carry out similar ion-selectivity studies using mechanically exfoliated crystals with little or no defects15.Proton transport through two-dimensional (2D) crystals has recently been studied, both experimentally and theoretically1 that transport of thermal protons through high-quality graphene and hBN occurs through their bulk and does not involve vacancies and other atomic-scale defects.Here we report ion-selectivity measurements using mechanically exfoliated graphene and hBN monolayers. The crystals are found to be perfectly selective with respect to protons. The latter can permeate through the 2D membranes, whereas even such small ions as chlorine are blocked. The results support the previous conclusion18  as the proton-conducting medium1. Thicker crystals  exhibited no discernable conductance, again in agreement with the previous report1.The chips containing the individual 2D membranes . For concentrations above 1 mM, σ increased linearly with HCl concentration. At lower concentrations, the measured current was below our detection limit. The latter was determined by electrical leakage along surfaces of the liquid cell and was of the order of 1 pA as found using control devices with no holes in the SiN membranes19. In another control experiment, we used devices with the same SiN aperture but without a 2D crystal. They exhibited conductance at least ~1000 times larger than that for the devices with graphene or hBN crystals covering the aperture  and, by definition, they satisfy tH + tCl ≡ 1 and the inequality: 0 ≤ both tH and tCl ≤ 1. To find their values for our 2D membranes, we used the same setup as in the measurements discussed in Fig. + and Cl− ions towards equilibrium, from the high concentration (Ch) compartment to the low concentration (Cl) one. Therefore, the sign of the total ionic current at zero V indicates whether the majority carriers are protons (positive I) or chloride ions (negative). Figure I–V characteristics for monolayer hBN devices and concentration ratio ΔC ≡ Ch/Cl = 10. Independently of the absolute values of HCl concentrations, the zero V current was always positive, proving that the protons dominate ion transport through our membranes. The same behavior was found for graphene devices ln(ΔC = 10) ≈ − 58 meV at our measurement temperature of ∼20 °C and, therefore, the observation implies tH ≈ 1 or, equivalently, that all the ionic current through the membrane is due to proton transport. Within our experimental accuracy, the same perfect selectivity was also found for graphene 20.To corroborate the above result and obtain better statistics for the ion selectivity, we carried out similar measurements using different devices and several concentration ratios ranging from Δ 30 Fig. . For all11. The latter was reported to have σ ≈ 4 S cm−2 at 1 M HCl, in clear disagreement with our experiments for mechanically exfoliated graphene, where σ was nearly three orders of magnitude smaller. Furthermore, no current could be detected for 1 mM HCl concentration in our experiments; however, large current densities of ~10 mA cm−2 were reported in ref. 11 for CVD graphene membranes of the same area. The membrane potential reported for CVD graphene was also different, reaching only ∼8 mV for ΔC = 10, or ~7 times smaller than what we found for our devices. All this shows that the ion transport properties of exfoliated 2D crystals are radically different from those of CVD films where atomic-scale defects and, possibly, even macroscopic ones11 dominate ion transport. This conclusion is consistent with all the other evidence for intrinsic proton transport through 2D crystals, which was reported previously2.Finally, it is instructive to compare our results with those obtained previously in conceptually similar experiments but using CVD graphene19. This provides further support to the view that the activation barriers found for proton transport through high-quality graphene and hBN do not involve vacancies and other atomic-scale defects1, a conclusion important for further theory developments . Our results also have implications for the widely discussed use of atomically thin crystals as a novel platform for various separation technologies. In such technologies, selectivity is typically achieved by either perforating nanopores25 or exploiting those naturally occurring in CVD films27. The fast permeation of H+ through the 2D bulk is usually ignored but can be important for designing and optimizing the membranes’ properties.In conclusion, our experiments clearly demonstrate that mechanically exfoliated, defect-free 2D crystals allow only proton transport and block even small ions such as chlorine that has one of the smallest hydrated diametersDevice fabrication started by isolating atomically thin layers of graphene and hBN from bulk crystals. We used hBN crystals commercially supplied by HQ Graphene. The flake was first identified optically and then characterized using atomic force microscopy and Raman spectroscopy. Supplementary Fig. Supplementary Fig. I–V characteristics were measured, applying voltages between typically ±200 mV at sweep rates < 0.1 V min−1.Devices were clamped with O-rings to separate two reservoirs filled with HCl solutions and Ag/AgCl electrodes were placed inside each reservoir. The Supplementary Information