The rumen primary and secondary metabolite content is intimately related to its community of bacteria, protozoa, fungi, archaea and bacteriophages, ingested feed and the host. Despite the myriad of interactions and novel compounds to be discovered, few studies have explored the rumen metabolome. Here, we present the first study using ultra-high performance liquid chromatography tandem mass-spectrometry and Molecular Networking approach, and various extraction methods on the cell-free rumen fluid of a non-lactating Holstein cow. Putative molecules were annotated based on accurate fragmentation matching the Global Natural Products Social Molecular Networking library, public spectral libraries, or annotated manually. The combination of five extraction methods resulted on 1,882 molecular features observed. Liquid-liquid extraction resulted on the highest molecular features abundance, 1,166 . Sixty-seven compounds were annotated using Global Natural Products Social Molecular Networking library and public libraries, such as hydrocinnamic and azelaic acid, and monensin. Only 3.56% of molecular features (67) observed had positive match with available libraries, which shows the potential of the rumen as reservoir of novel compounds. The use of untargeted metabolomics in this study provided a snapshot of the rumen fluid metabolome. The complexity of the rumen will remain long unknown, but the use of new tools should be encouraged to foster advances on the rumen metabolome. The symbiotic relationship between the host and the microbiota results on the transformation of human non-edible feedstuff into high-quality products, such as meat and milk. The degradation of feed material in the rumen is a dynamic and continuous process orchestrated by a myriad of microbial enzymes, and this process is affected by the input and output of feed and water, ruminal pH and motility, and others2. Exploring the rumen microbial processes, interactions and functionality is key to improve feed efficiency, decrease methane emission, and to discover novel enzymes and antimicrobials5.The rumen is a complex anaerobic environment enriched in bacteria, protozoa, fungi, archaea and bacteriophages6. However, there is little information on the chemical composition of secondary microbial metabolites and the expressed proteins in the rumen. The first study to employ different analytical methods, including mass spectrometry (MS), to explore the rumen metabolome was performed by Saleem et al.7 The rumen fluid of 8 dairy cows fed different amounts of barley were evaluated and a total of 93 molecular features were observed. To further characterize the rumen fluid metabolome, Saleem et al.8 identified and quantified routinely detected metabolites such as amino acids, carbohydrates, organic acids and others. A total of 246 molecular features were identified, of which 116 were derivatized and extracted using a commercial kit and analyzed using direct flow injection tandem MS. Recently, Artegoitia et al.9 analyzed rumen fluid collected after slaughter of beef cattle using liquid chromatography-mass spectrometry (LC-MS) to explore potential metabolite markers related to average daily gain. Thirty-three metabolites were reported to be associated with differences on average daily gain.Recent studies using next-generation sequencing have provided evidence on the abundance of microorganisms and genes present in the rumen11. Thus, molecular families can be clustered within groups on a chemical map, providing the possibility to comprehensively interpret large metabolomics datasets.Metabolome investigations require a comprehensive approach to represent the molecular features present in a sample. The association between extraction methods, analytical techniques and annotation tools are essential to cover metabolites from various chemical classes, especially for complex samples. The recent developed annotation tool Molecular Networking (MN) based in MS allows users to visually and structurally evaluate related metabolites with similar fragmentation patternsKa. Also, we present the first study using MS-based MN approach to explore the rumen fluid metabolome.In this study, Liquid-Liquid Extraction (LLE), Solid Phase Extraction (SPE) and 3 variations of the Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) extraction method were used to provide a snapshot of the rumen fluid metabolome of a non-lactating dairy cow. Different extraction methods aimed to extract metabolites varying in chemical properties, such as polarity and p2O), magnesium sulphate , sodium sulphate , sodium chloride (NaCl) and acetic acid were obtained from Synth . Primary-secondary amine (PSA) and silica-based sorbent with C18 bonding (C18) were obtained from Supelco . Solid-phase extraction cartridges were obtained from Applied Separations  and filled using C18 phase  and Cyano phase . Ethyl acetate was obtained from Dinâmica . Water with 0.1% formic acid , formic acid (≥96% purity) and ammonium hydroxide solution  were obtained from Sigma-Aldrich . Methanol and acetonitrile of UHPLC grade were purchased from J.T. Baker . Ultra-pure water was produced using an EMD Millipore Direct-Q™ 3 system . Mixed cellulose esters membrane filter (47 mm diameter with 0.22 and 0.45 µm pore size) and glass vacuum filter holder for 47 mm disc filters were obtained from Merck Millipore .Sodium acetate  was used. All animal care and experimental procedures were conducted under the surveillance of the Animal Care and Use Committee of the Universidade Estadual de Maringá, Brazil (protocol no. 9013160518) and met the guidelines of the National Council for the Control of Animal Experimentation (CONCEA). Diet was offered for 21 days and consisted, on a DM basis, of 60% corn silage, 24% of corn grain, 10% of wheat bran, 4.8% of soybean meal, and 1.2% of mineral mixture (which contained 480 mg/kg of monensin). The cow was fed at 07 h 00 and 15 h 00 for g for 5 min at 4 °C, the pellet was discarded, and the supernatant was subsequently centrifuged at 13,000 g for 30 min at 4 °C. The cell-free supernatant was then filtered using 0.45 µm pore size membranes, and subsequently using 0.22 µm pore size membranes, both using a Millipore filtering system. Aliquots were stored at −20 °C for one day until extraction procedures were performed.At day 21, rumen content was sampled (300 mL) through a ruminal cannula 10 min before the morning feeding. The rumen content was filtered through 4 layers of cheesecloth into a glass amber container on ice. The pH of the rumen fluid was determined immediately after sampling using a pH meter . Filtered rumen fluid was centrifuged within 30 minutes at 1,000 One aliquot of cell-free rumen fluid (10 mL) was mixed with 10 mL of ethyl acetate and 1.0 g of NaCl for the LLE  was loaded into C18 cartridges and collected for analysis (SPE-1B). The cartridge was washed with 10 mL H2O, followed by elution with 6 mL acetonitrile (SPE-1A). C18 cartridges were activated with 5 mL of methanol followed by conditioning with 10 mL H2O. Homogenized cell-free rumen fluid (6 mL) was loaded into C18 cartridges and collected for analysis (SPE-2B). The cartridge was washed with 10 mL H2O, followed by elution with 6 mL methanol (SPE-2A). CN cartridges were activated with 5 mL of acetonitrile followed by conditioning with 10 mL H2O. Homogenized cell-free rumen fluid (6 mL) was loaded into CN cartridges and collected for analysis (SPE-3B). The cartridge was washed with 10 mL H2O, followed by elution with 6 mL acetonitrile (SPE-3A). CN cartridges were activated with 5 mL of methanol followed by conditioning with 10 mL H2O. Homogenized cell-free rumen fluid (6 mL) was loaded into CN cartridges and collected for analysis (SPE-4B). The cartridge was washed with 10 mL H2O, followed by elution with 6 mL methanol (SPE-4A). The 8 SPE aliquots were concentrated under nitrogen flow before UHPLC-MS/MS analysis.Solid-phase extractions  was concentrated under nitrogen flow (OQ-1). An aliquot of 1 mL of the upper acetonitrile layer was mixed with 150 mg anhydrous MgSO4 and 25 mg PSA sorbent, vortexed for 30 s and centrifuged for 1 min at 6,000 g (OQ-2). The OQ-2 procedure was repeated, however 25 mg of C18 was used instead of PSA (OQ-3). Extracts were analyzed using UHPLC-MS/MS.Homogenized cell-free rumen fluid (10 mL) was mixed with an equivalent volume of acetonitrile and vortexed for 1 min for the QuEChERS procedure4 and 1.7 g NaOAc.3H2O, for the buffered QuEChERS procedure13  was concentrated under nitrogen flow (BQ-1). A 1 mL aliquot was mixed with 150 mg anhydrous MgSO4 and 50 mg sorbent PSA, vortexed for 20 s and centrifuged for 1 min at 6,000 g (BQ-2). The BQ-2 procedure was repeated, however 50 mg of C18 was used instead of PSA (BQ-3). Homogenized cell-free rumen fluid (10 mL) was also mixed with an equivalent volume of 1% acetic acid in methanol (v:v), 4 g anhydrous MgSO4 and 1.7 g NaOAc.3H2O. The solution was vortexed for 1 min and centrifuged for 3 min at 11,000 g. The supernatant was split in 2 aliquots of 1 mL for the cleanup step. A 1 mL aliquot was mixed with 150 mg anhydrous MgSO4 and 50 mg sorbent PSA, vortexed for 20 s and centrifuged for 1 min at 6,000 g (BQ-4). The BQ-4 procedure was repeated, however 50 mg of C18 was used instead of PSA (BQ-5). Extracts were analyzed using UHPLC-MS/MS.Homogenized cell-free rumen fluid (10 mL) was mixed with an equivalent volume of 1% acetic acid in acetonitrile (v:v), 4 g anhydrous MgSO2SO4 anhydrous according to Wang et al.14 with minor modifications . The bottom layer was mixed with 10 mL solution of 1% ammonium hydroxide in acetonitrile (v:v). The extract was vortexed for 2 min, centrifuged for 10 min at 6000 g and the upper layer was collected (basic phase). An aliquot of 5 mL from the acid and basic phases were mixed and the remainder aliquots were concentrated separately under nitrogen flow . The combined 10 mL aliquot was split in two aliquots. One aliquot was mixed with 0.5 g NaAc, 50 mg C18 and 75 mg PSA sorbent, vortexed for 2 min and centrifuged for 10 min at 6,000 g (ABQ-3). The other aliquot was mixed with 0.5 g NaAc and 50 mg C18, vortexed for 2 min and centrifuged for 10 min at 6,000 g (ABQ-4). Extracts were analyzed using UHPLC-MS/MS.Homogenized cell-free rumen fluid was mixed with 1% acetic acid in acetonitrile (v:v) and 6 g Na® CSHTM C18 packed with 135 Å pore, 1.7 µm particle size, 2.1 × 100 mm column , at a flow rate of 0.2 mL min−1. The gradient mixture of solvents A  and B  was as follow: 5% B 0–1 min, 50% B 1–5 min, 95% B 5–10 min, maintained at 95% B 10–16 min, 5% B 16–18 min, and maintained at 5% B 18–21 min at 40 °C. The capillary voltage was operated in positive and negative ionization modes, set at 4500 and 3000 V, respectively; with an end plate offset potential of −500 V. The dry gas parameters were set to 8 L min−1 at 180 °C with a nebulization gas pressure of 4 bar. Data were collected from m/z 50 to 1300 with an acquisition rate of 5 Hz, and the 4 ions of interest were selected by auto MS/MS scan fragmentation.Extracts were analyzed using an ultra-high performance liquid chromatograph  coupled to a hybrid quadrupole time-of-flight high resolution mass spectrometer  equipped with an electrospray ionization source. Chromatographic separation was performed with an Acquity UHPLC10 (Supplementary Method S1). The GNPS approach consists of comparing fragmentation spectra (MS/MS experiments) and grouping molecules with similar chemical structures. Each spectrum is represented as a node in the visual MN, and spectrum-to-spectrum alignments are represented as lines that connect nodes, evidencing ions that are correlated with each other. Communication between the nodes are related to similarities in fragmentation spectra between the ions, and similarity between fragmentations patterns are evaluated via vector relation. Structurally related molecules exhibit similar fragmentation patterns; therefore, molecular families tend to unite within groups in the MN, referred as clusters. In addition, it is possible to determine the difference of m/z between nodes, defining spectral proximity between all MS/MS spectra in a dataset. This tool innovates and facilitates the analysis of large datasets. It also permits the comparison of molecular features to the GNPS spectral library and all publicly available data. Molecular networking was visualized using Cytoscape15. Metabolites with positive match with the GNPS library had both parent and fragment ions manually compared with GNPS spectral library and publicly available data  to generate the MN, according to GNPS guidelinesThe combination of five extraction methods in the rumen fluid of a non-lactating Holstein cow resulted on 1,882 molecular features analyzed by UHPLC-MS/MS. A total of 67 compounds were identified using publicly available molecular matches . The LLE also provided the greatest amount of annotated molecular features: 57 of 67. Compounds such histamine and tyramine  and 174 exclusive molecular features . The SPE had 48 annotated molecular features. N-[2-(1H-Indol-3-yl)ethyl]acetamide, 9-octadecenamide, EL and 7b,9-dihydroxy-3-(hydroxymethyl)-1,1,6,8-tetramethyl-5-oxo-1,1a,1b,4,4a,5,7a,7b,8,9-decahydro-9aH-cyclopropabenzoazulen-9a-yl acetate were observed for all SPE methods. 5-aminopentanoic acid, nicotinic acid, methionine, xanthine, azelaic acid and other 10 molecular features were observed in the SPE-B aliquots, which are usually discarded when SPE is used  and had the lowest abundance of exclusive molecular features . A total of 34 molecular features extracted using the original QuEChERS were annotated. Acid-base QuEChERS and buffered QuEChERS extracted a total of 707 and 531 molecular features, respectively. This corresponded to 37.57 and 28.21% of total observed molecular features, respectively. Acid-base QuEChERS and buffered QuEChERS had more exclusive molecular features compared to original QuEChERS, 109  and 177 , respectively. A total of 50 and 45 identified molecular features were extracted by the acid-base QuEChERS and buffered QuEChERS method, respectively.m/z 297.243) and 12,13-DiHOME (m/z 315.253). Cluster B , decanedioic acid  and dodecanedioic acid . Cluster C was formed by phenylalanine , 3-indoleacetic acid  and hydrocinnamic acid [M + H]+; (m/z 151.075) , monensin B  and monensin methyl ester  Fig. . Cluster16. Furthermore, many of the rumen bacteria remain uncultured and uncharacterized, which is more troublesome for eukaryotes17. Thus, the use of LC-MS favors the exploration of the interactome, rather than the metagenome. This is actually an important feature as integrative omics tools are only becoming available. For example, the exploration of the metabolome can result on the discovery of novel compounds, such as antimicrobials18 or other bioactive molecules19. To date, the majority of rumen metabolome studies have used UHPLC-MS, gas chromatography-mass spectrometry and nuclear magnetic resonance, with the latter the most used due to reliability and absolute quantification20. However, UHPLC-MS has the advantage of having increased sensitivity and coverage. Thus, a single UHPLC-MS run can result on huge amount of data. For example, we obtained 432,391 MS spectra in this study. To overcome the difficulty to analyze this massive amount of data, the GNPS MN was used to comprehensively visualize the dataset and to prospect novel compounds. Another challenge of metabolomics studies relies on our ability to annotate unknown molecular features or their origins. Indeed, there is evidence that only 1.8% of spectra in untargeted metabolomics experiment can be annotated21. To overcome this issue, metabolites spectra can be compared based on parent and fragment ion similarities to publicly available molecular matches.The extraordinary activity of microorganisms is based on their remarkable metabolic diversity and genetic adaptability, which makes them an important source of genetic resources for biotechnological advancement and sustainable development. The success for biotechnological processes is directly related to the diversity of microorganisms and the molecules they produce as a result of primary and secondary metabolism as well as the conservation of the genetic resources they provide. The rumen fluid is a complex matrix composed by a myriad of microorganisms, which interact among themselves and the host, and degrade plant material  from various sources. The intra- and interspecies interaction are responsible for the synergistic effect on the production of volatile fatty acids and microbial protein in the rumen. Despite the ability of the ruminant to degrade cellulose being responsibility of the microbiota, only recently a reference microbial genome catalog of the rumen was published, and yet a large portion of microorganisms remains unknown22. Using targeted approaches and a combination of analytical methods on the rumen fluid of cows fed diets varying in levels of barley, Saleem et al.8 observed 246 metabolites including: phospholipids, inorganic ions, gases, amino acids, dicarboxylic acids, fatty acids, volatile fatty acids, glycerides, carbohydrates and cholesterol esters. Aiming to identify markers for the rumen function that could lead to differences in average daily gain, Artegoitia9 observed 1,429 molecular features using the metabolomics untargeted approach. However, a large number of animals was used in that study (n = 16).Recent studies exploring the rumen fluid metabolome have mainly focused on how the metabolites are influenced by different diets. For example, organic acids, amino acids, amines, sugars and nucleosides/nucleotides, which are the core of the rumen fluid, are affected when dairy cows are fed diets varying in levels of concentrate23. Moreover, due to the vast microbial interaction and dispute inside the rumen, the rumen is a pool for novel antimicrobials24. Furthermore, the GNPS MN is a collaborative platform that is improved from the spectra deposited by users. The rumen is still a poorly explored environment and deposit of rumen metabolome dataset is required.In our study, 1,882 molecular features were observed and 67 were identified, which was 3.56% of total. The sources of these uncharacterized molecular features may range from secreted molecules by ruminal microorganisms, plants, the host, and administered drugs. This offers a possibility to further explore the ruminant in its environment. For example, bacterial secondary metabolites can be used to identify antimicrobial resistance26. Alternatively, SPE methods are focused on low to medium polarity compounds using octadecylsilane (C18) and Cyano (CN) phase cartridges, respectively. To increase the abundance of extracted compounds, solvents with different elution strengths can be used. Lastly, QuEChERS was used as alternative extraction method for compounds of medium and low polarity, but with lower material and labor cost compared to SPE. QuEChERS extraction methods is based on an extraction step that partition with organic solvent, followed by a clean-up step with sorbents, which improves the selectivity to the method.Liquid-liquid extraction method resulted on the greatest abundance of molecular features: a total of 61.96%, and it also resulted on the greatest quantity of annotated compounds. Liquid-liquid extraction method extracts compounds with moderate polarity, and nonpolar or hydrophobic characteristics. The pH of the extraction can also be explored as it influences the solubility of acidic and basic compounds. Thus, pH modifiers can increase the abundance of dissociated compounds in the organic phase−; m/z 165.056), which was present in multiple extractions is metabolized from cinnamic acid27 and is produced by the anaerobic bacterium Clostridium xylanolyticum28. Another annotated compound was the EL, which has potential to improve milk stability, the immune system of the dairy cow, and to prevent cardiovascular diseases, osteoporosis and diabetes in human30.The molecular feature 3-(2-hydroxyphenyl) propanoic acid , 3-indoleacetic acid  and hydrocinnamic acid [M + H]+; (m/z 151.075) formed a cluster  and 12,13-DiHOME (m/z 315.253) had a match with the GNPS, which were also identified by Artegoitia9. These are bioactive lipids derived from several polyunsaturated fatty acids, such as linoleic acid. The epoxidation metabolites of linoleic acid produce the epoxyoctadecenoic acid  and the epoxide hydration product dihydroxyoctadecenoic acid, . The modulation of these products can be associated with processes of inflammation in dairy cattle34.Phenylalanine . By observing the molecular features in the monensin cluster and the fragmentation pattern, we could infer that the unannotated structures m/z 679.402 and 707.433 are molecules structurally similar to monensin. Mass differences of −14 Da and +14 Da with monensin suggested differences in a methyl group in both cases. Inspecting the fragmentation pattern and comparing to public available data suggests both molecular features are monensin B  and monensin methyl ester 36.Monensin was observed and had the +; m/z 203.127) and dodecanedioic acid , which formed a cluster in the MN. These molecular features were observed the rumen of both cows and sheep and can be derived from the microbial hydrogenation of non-conjugated dienoic acids37. Azelaic acid  is an organic compound observed in wheat, rice, rye and barley38 and can be used as carbon sources by the rumen microbiota. The proximity of the unannotated structure m/z 217.143 to decanedioic acid and dodecanedioic acid nodes, and the evaluation of the fragmentation pattern supported that the unannotated structure m/z 217.143 belonged to the dicarboxylic acids family. Mass differences of −14 Da with decanedioic and dodecanedioic acids suggested differences of CH2 group in both cases. The fragmentation pattern compared to public available data suggested the structure of undecanedioic acid . This characteristic mass difference was also observed between the unannotated structure m/z 175.096 and the azelaic acid. The loss of -14 Da and fragmentation pattern suggested that the molecule was the suberic acid .Another example is related to azelaic acid, decanedioic acid ([M + H]This investigation of the rumen fluid metabolome of a non-lactating Holstein cow using different extraction methods, liquid chromatography-mass spectrometry and Molecular Networking analysis resulted on 1,882 observed molecular features, which is the greatest abundance of molecular features reported to date. Several extraction methods resulted on various quantities of exclusive molecular features. The liquid-liquid extraction method had more exclusive and total molecular features. Only 67 molecular features observed had positive match with the Global Natural Products Social Molecular Networking database and public libraries, which corresponded to 3.56% total molecular features. Of those, amino acids, dicarboxylic acids, carboxylic acids, lactones, lignans, fatty acids derivatives and indole compounds were observed. Four clusters observed in the Molecular Networking were presented and two clusters were manually explored so that novel compounds could be identified. This highlights the use of ultra-high performance liquid chromatography tandem mass-spectrometry and Molecular Networking to explore the potential of the rumen as a reservoir for novel compounds.Supplementary Material


Reliable estimates of the current and future disease burden of antimicrobial resistance (AMR) are essential to combat the global drug-resistant infection crisis , 2. Yet,Major, recent efforts to estimate AMR burden include the Global Burden of Disease (GBD) 2016 study , the RevMore accurately measuring AMR burden requires a fully described scientific measurement framework . The gloThe accuracy of AMR burden estimates depends on the quality and availability of input data . PresentThe development of GLASS is a step in the right direction, but it is at an early stage . GLASS lNecessarily stringent privacy concerns further limit data sharing. Linked data that matches clinical bacteria to patient mortality outcomes, patient information and comorbid conditions, are essential to measure the excess mortality risk of resistant infections – and they are virtually non-existent. In the absence of comprehensive and diverse datasets from various regions and income-bases, GBD estimates rely heavily on modeling, which increases uncertainty. This is particularly concerning in low-income regions, where surveillance data is almost non-existent, access to essential antimicrobials is limited, and substandard antibiotics are widely available , 3.High-income nations face their own challenges. An evaluation of Australia’s AMR response highlighted the need for a more robust animal surveillance system, and better One Health integration . AlthougDespite its shortcomings, Australia’s model of integrating AMR surveillance into system-wide risk-mitigation strategies may offer valuable lessons. The national Antimicrobial Use and Resistance in Australia network has identified alarming trends in resistance and antimicrobial use (AMU) . SurveilAMU surveillance initiatives in Australian tertiary and aged care facilities have facilitated the inclusion of antimicrobial stewardship standards within national medication safety and quality frameworks . OveruseBetter surveillance infrastructure requires ongoing funding and support from key donors and policymakers. The UK’s £265 million Fleming Fund for surveillance initiatives in low and middle-income countries is a welcome display of global leadership . LikewisRegional capacity-building must be part of a broad, coordinated international response. Governing bodies must assist countries to build AMR surveillance systems, while supporting the development and funding of national action plans in fragile states – currently, only one-fifth of national action plans are properly funded . To effeWithout a more concerted and collaborative global response, the disastrous forecast made in the Review on AMR of 10 million annual deaths from resistant infections by 2050 might eventuate. Initiatives that enhance monitoring capacity for resistant infections, and bolster the accuracy of AMR burden estimates, will provide essential information to guide policy and prevent a calamity of this scale.


Opioids are powerful addictive agents that alter dopaminergic influenceon reward signaling in medium spiny neurons (MSNs) of the nucleus accumbens.Repeated opioid exposure triggers adaptive changes, shifting reward valuation tothe allostatic state underlying tolerance. However, the cellular substrates andmolecular logic underlying such allostatic changes are not well understood.Here, we report that the plasticity of dopamine-induced cyclic AMP (cAMP)signaling in MSNs serves as a cellular substrate for drug-induced allostaticadjustments. By recording cAMP responses to optically evoked dopamine in brainslices from mice subjected to various opioid exposure paradigms, we defineprofound neuronal-type-specific adaptations. We find that opioid exposure pivotsthe initial hyper-responsiveness of D1-MSNs toward D2-MSN dominance asdependence escalates. Presynaptic dopamine transporters and postsynapticphosphodiesterases critically enable cell-specific adjustments of cAMP thatcontrol the balance between opponent D1-MSN and D2-MSN channels. We propose aquantitative model of opioid-induced allostatic adjustments in cAMP signalstrength that balances circuit activity. Muntean et al. examine how opioid exposure influences cyclic AMP (cAMP)responses to dopamine in striatal medium spiny neurons (MSNs). They describeallostatic adaptations in the processing of dopaminergic signals by D1-MSN andD2-MSN populations as opioid administration progresses from acute exposure tochronic use, and they define molecular elements contributing to the process.  The anatomical connectome of the mammalian brain provides the infrastructurefor synaptic communication, which underlies functional neuronal circuits instructingaction selection. A remarkable feature of neural systems is the boundless adaptableplasticity in response to input stimuli, allowing response adjustments based onexperience while maintaining homeostatic integrity . This prThe basal ganglia contain two prominent dopaminergic inputs: dopamine neuronsfrom the midbrain substantia nigra pars compacta (SNc) and ventral tegmental area(VTA) project to the striatal caudate putamen (CPu) and nucleus accumbens (NAc),respectively . DopaminThe NAc is composed of ~95% medium spiny neurons (MSNs), which are thekey element in processing dopamine signals. Two major subpopulations of MSNs featuredistinct transcriptional landscapes, most prominently delineated by differentialexpressions of either the dopamine D1 receptor (D1-MSNs) or the dopamine D2 receptor(D2-MSNs) . These pThe major physiological and direct cellular consequence of dopamine action onboth MSN populations is the positive and negative influence on cyclic AMP (cAMP)production mediated by the D1 and D2 dopamine receptors, respectively . There iin vivotool offered by the cAMP-encoded reporter (CAMPER) mice  mice  to interCAMPER mouse model, which has been calibrated to reportabsolute cAMP values with nanomolar resolution  and acute opioid exposure . In addiWe proceeded to analyze signal integration properties of the circuit bydelivering trains of stimulation. Consistent with the reversal of the elementalresponse properties of D1-MSNs toward their drug-naive state, we found thatresponses from chronically treated mice also partially regained their ability tosum the response into a tetanic wave at 3-min ISIs . HoweverWe further examined how chronic opioid exposure changes the responseproperties relative to the drug-naive state, when animals are withdrawn from thedrug exposure for 24 h prior to experimentation, modeling an abstinent state. In thisThe profound influence of opioids on dopaminergic signaling in MSNsprompted an investigation of molecular determinants that shape responseproperties underlying plastic changes. Dopamine is released in the striatum as adiffuse volume transmitter , with re3-isobutyl-1-methylxanthine(IBMX), increased the baseline cAMP level in both MSN subpopulations in adose-dependent manner , a family of enzymes that metabolize cAMP to delimitsignal propagation . Bathapt manner . After ft manner  and 3G. t manner  and3H. Having established the roles of both the DAT and PDEs in shapingdopaminergic signaling, we next sought to determine their contributions to theadaptations in response parameters triggered by opioid exposure. For thesestudies, we chose to use the acute morphine protocol to minimize compounding theinfluence of multi-drug interactions, which could trigger neuroplastic eventsassociated with chronic paradigms . As withWe next applied a similar strategy to dissect the contribution ofpostsynaptic elements in opioid influence by blocking the PDE with IBMX . We founTo better understand the implications of observed neuron-type-specificalterations on the processing of dopaminergic inputs, we next modeled changes instrength of phasic dopamine release by varying the intensity of the stimulation,while collecting MSN responses over the wide range of the VTA firing frequencies. AnalysiWe next interrogated the contributions of the DAT and PDEs tomorphine-induced adaptations of frequency tuning and response sensitivity.First, we compared elemental responses evoked by varying frequencies recorded inslices from saline-(naive) and acute morphine-exposed mice following the DATblockade –6C. In dUsing similar experimental logic, we next interrogated the contributionof PDEs to morphine-induced changes in frequency tuning of the sensitivity todopaminergic inputs. In drug-naive  D1-MSNs, blockade of PDEssignificantly increased sensitivity to dopamine; however, this effect was notobserved in mice exposed to acute morphine –6I. AgaiOur experiments revealed the extensive impact of opioid exposure onmultiple parameters of MSN responses in processing dopaminergic inputs shaped byboth pre- and postsynaptic mechanisms. In order to better understand theunderlying logic of these changes, we modeled the multivariate data by applyinga principle component analysis on correlations to reduce the dimensionalitybetween core signaling features and visualize their impact. We consideredchanges in key response parameters including the basal cAMP set point, responseamplitudes, signaling duration, and oscillatory rebound in defining clustersacross morphine treatment paradigms .Most notably, this analysis revealed that both types of MSNs, undernaive conditions, exhibit broadly dispersed clusters that likely underscoretheir ability to integrate an adaptive range of neuromodulation and maintaindiversity in their responsiveness. Experimental opioid paradigms werecharacterized by distinct patterns that shared partial overlap with naiveneurons, but also clustered in a unique manner. In both MSN subtypes, prolongedopioid exposure in the abstinence and tolerance paradigms sharply shiftedprinciple components in the opposite direction of neurons in mice subjected tothe initial acute morphine exposure. The D1-MSN profile from the abstinence andtolerance paradigms concentrated into zones with mutual overlap, yet maintainedareas of exclusivity, supporting our observations of overall signalingcompression in these neurons .InteresZ score, combining key responseparameters that defined clusters in the principle component analysis  in influencing dopamine response dynamics andadaptations induced by opioid exposure in a cell-specific manner. PDEs arerecognized for their role in striatal pathophysiology  and haveCollectively, our observations suggest that both presynaptic mechanismsorchestrated by the DAT and postsynaptic properties delineated by PDEs areessential factors that distinctly shape opioid plasticity in the mesolimbiccircuit by inducing temporal cell-specific adaptations to bias activity betweenMSN subtypes. As critical elements in the processing of neurotransmitterresponses, both the DAT and PDEs act to selectively adjust the discreteparameters of cAMP signaling to provide both cell-autonomous mechanisms,contributing toward programming diverse allostatic states. Taken together, thisprompts a model where opioid exposure is time stamped by distinct profiles ofalterations in the processing of dopaminergic signals by striatal neurons, withpatterns of bidirectional cAMP modulation serving as substrates for calculatingallostatic shifts in reward valuation. We hope this model can provide a usefulframework for the exploration of molecular players, circuit mechanisms, andneuromodulatory logic and guide future studies on adaptive neural control ofbehavior, such as parsing the plastic features of other abusive drugs  or affective disorders , that are beyond the scopeof this study.kirill@scripps.edu). This study did not generate new uniquereagents.Further information and requests for reagents and resources may bedirected to, and will be fulfilled by, the Lead Contact, Kirill A. Martemyanov through Hamilton syringes. Viral particles wereinfused at 150 nl/min  into the VTA . Sufficient diffusion of viral particles was achieved byallowing the syringes to remain in place for five additional minutes afterthe infusion was completed.Adult mice were subjected to isoflurane anesthesia for bilateralstereotaxic delivery of AAV5-hSyn-hChR2(H134R)-mCherry , MgCl2 (1), NaHCO3 (25),NaH2PO4 (1.25), Glucose (25), Kynurenic acid (1).Slices were then transferred to gassed ACSF absent kynurenic acid with 2 mMCaCl2 for 30 min prior to performing experiments. Recordingswere made in a chamber perfused with gassed ACSF, void of kynurenic acid, at2 mL/min. Dopamine-mediated responses were isolated by inclusion ofpicrotoxin (100 mM), DNQX (10 μM), CGP55845 (300 nM), and scopolaminehydrobromide (200 nM) in the ACSF as described  wereanesthetized with isoflurane followed by decapitation and rapid excision ofthe whole brain. In order to maximize preservation of mesolimbic circuitconnections, sagittal sections (300 μm) were cut between15–20° respective to the midline  on a vibescribed .CAMPER mouse brain slices by imaging FRET changes fromNAc MSNs anatomically identified in the ventral region of the striatumapproximately 5 mm rostral to midbrain mCherry fluorescence (from VTA hChRinfusion). Sub-NAc geography between core and shell was not distinguished.Utilizing a Leica TCS SP8 MP confocal microscope, excitation of FRET donor(mTurquoise) was achieved with a Ti:sapphire laser (Coherent) tuned to 850nm whereupon simultaneous donor  and acceptor bandpass emission XYZ image stacks were collectedthrough a 25X objective lens at 10 s intervals. Neuronal cell bodies weredefined as regions of interest and raw fluorescence intensity from bothchannels was used to calculate FRET using ImageJ software. Δ FRETvalues were converted to cAMP concentrations by interpolation from astandard calibration curve. Channelrhodopsin stimulation in the VTA wasachieved with 0.3 mW of 470 nm of light delivered from a 400 μm corefiber optic cannula from a fiber-coupled LED (Thorlabs). Facilitation ofoptical stimulation trains (20 flashes of light each 2 ms in duration) wereconfigured with a Pulse Train Generator (Prizmatix). DAT blockade wasachieved by bath application of cocaine hydrochloride (10 μM). PDEblockade was achieved by bath application of 3-Isobutyl-1-methylxanthine(IBMX) at concentrations indicated in the text.As previously described , intraceStatistical analysis was performed using Prism GraphPad software whereStudent’s t test and ANOVA were used for pairwise comparisons with theuse of asterisks to indicate statistical significance . At leastthree biological replicates were performed for each experiment with data fromall samples included. Graphs report mean values with the presence of error barsto denote standard error of the mean. Principle component analysis oncorrelations was performed using JMP 14 software.The datasets supporting the current study are available from the LeadContact on request.1


The manipulation of molecule-electrode interaction is essential for the fabrication of molecular devices and determines the connectivity from electrodes to molecular components. Although the connectivity of molecular devices could be controlled by molecular design to place anchor groups in different positions of molecule backbones, the reversible switching of such connectivities remains challenging. Here, we develop an electric-field-induced strategy to switch the connectivity of single-molecule junctions reversibly, leading to the manipulation of different connectivities in the same molecular backbone. Our results offer a new concept of single-molecule manipulation and provide a feasible strategy to regulate molecule-electrode interaction.  •in-situ switch the connectivity of single-molecule junctionsA strategy to •A concept to manipulate the molecule-electrode interaction•A molecular switch triggered by the varying of electric field•Experiments were combined with calculations to probe the switching mechanism Molecular Electrochemistry; Quantum Electronics; Electronic Materials The one-dimensional (1D) conductance histograms of M1 (blue) and M1-H (red) are constructed from ∼2000 of such traces. As shown in G0 represents the formation of gold atomic point contact , suggesting the formation of two types of junction geometries, with about two orders of magnitude conductance difference.Protonated pyridinium on of M1 A, which echnique  in the s contact , and the contact . We find0−5.8G0 B, which 0−5.8G0 . DiffereM1-H  conductance-displacement histogram of M1-H E demonst0−6.0G0 C. The siium ring B, which ium ring  and S17.ium ring , the in-idiniums A, which se added .M1L-H formed by the protonation of M1L, leading to a 1.3 nm difference between two possible connectivities , as shown in M1-H is gradually suppressed and almost completely suppressed in the bias of 0.40 V. Meanwhile, the formation of high-conductance junctions in M1-H becomes more and more favorable with the increasing of bias. We quantitatively characterize the junction formation probability for both the low- and high-conductance junctions of M1-H in different bias , to characterize the single-molecule conductance of ic field . We alsoic field D also vaic field E, suggesic field , the absM1-H, we carried density functional theory (DFT) calculation to study the different binding geometries between M1 and M1-H. We find that the dipole moment of M1-H is eight times larger than M1 , as shown in M1-H is the in-backbone connectivity (θ = 0) with a parallel orientation to EEF, whereas M1 does not show explicit dependency to θ. The calculation result is consistent with the bias-dependent junction formation probability, in which the in-backbone connectivity of M1-H becomes more and more dominant in higher bias (M1 and M1-H, the electrostatic potential distributions of M1-H shows significantly high positive charge distribution around the pyridinium ring (M1-H would be another factor in facilitating the formation of high-conductance junctions in M1-H.To investigate the connectivity switching mechanism in  than M1 A, attribM1 (M1-H . The modher bias A. Besideium ring B, so thaM1-H is localized at the pyridinium ring, which is distinct to M1 with its LUMO delocalized around the molecular skeleton. The localized LUMO of M1-H weakens the back-donating bonding from gold to sulfur, leading to weaker Au-S bond, which is confirmed by DFT calculation (−1 in M1 to 234 cm−1 in M1-H (M1-H reduces the competition to form the end-to-end connectivity between two sulfurs and makes the formation of the in-backbone connectivity more favorable. Thus, we think both the electric field and the weakened Au-S bonds contribute to the formation of high-conductance junctions in M1-H.We also find that the formation of pyridinium has a distinct effect on their frontier orbitals. As shown in culation E and sur in M1-H  (Kocharo in M1-H . The weameta-connectivity to shorter para-connectivity, we manipulate the charge-transport distances, which significantly enhance the conductance difference between two connectivities. The mechanism of the switching is further investigated by experiments and DFT calculation, revealing that the protonation-enhanced dipole moments have significant interaction with the electric field, which favors the formation of in-backbone para-connectivity. Our studies suggest that the interplay between the dipole moment of molecules and EEF will lead to a reversible connectivity switching strategy, which would provide a new concept to manipulate the molecule-electrode interaction and be promising for constructing new conceptual molecular devices.In conclusion, we have developed an electric-field-induced strategy for reversible switching the connectivities of single-molecule junctions. Through the switching from longer M1-H may also lead to the switching of quantum interference in the charge transport through the single-molecule junctions. For instance, the changes from meta-connection to para-connection may switch the patterns of quantum interference from destructive to constructive states and also offer a new opportunity for interference-based molecular devices. However, the understanding of quantum interference patterns needs further investigations, which are challenging to be accomplished at the current stage.The switching from the end-to-end connection to the in-backbone connection of All methods can be found in the accompanying


Zea mays as having a “closed organization” that differs from Arabidopsis with the development of xylem conforming to predictable rules. We speculated that root apical meristem organization in a wild subspecies of Z. mays (a teosinte) would differ from a domestic sweetcorn cultivar (‘Honey Bantam’). Careful comparison could contribute to understanding how evolutionary processes and the domestication of maize have affected root development. Root tips of seedlings were prepared and sectioned for light microscopy. Most sections were treated with RNase before staining to increase contrast between the walls and cytoplasm. Longitudinal and serial transverse sections were analyzed using computer imaging to determine the position and timing of key xylem developmental events. Metaxylem development in mexicana teosinte differed from sweetcorn only in that the numbers of late-maturing metaxylem vessels in the latter are typically two-fold greater and the number of cells in the transverse section of procambium were greater in the latter, but parenchymatous cell sizes were not statistically different. Promeristems of both were nearly identical in size and organization, but did not operate quite as previously described. Mitotic activity was rare in the quiescent centers, but occasionally a synchronized pulse of mitoses was observed there. Our reinterpretation of histogen theory and procambium development should be useful for future detailed studies of regulation of development, and perhaps its evolution, in this species.Classical histology describes the histological organization in  Z. mays). This common name is now understood to have been traditionally applied by indigenous people to three non-maize species  and three subspecies of Z. mays , all of which have older natural histories than domesticated cultivars of Z. mays and are more similar to each other structurally than they are to domesticated maize. The inclusion of the latter three into species Z. mays was done by virtue of the “reproductive criterion”  as well as by morphological, geographical, isozyme, and DNA RFLP analysis criteria [Maize is believed to have been domesticated beginning about 9000 years ago from one of several species of large grasses, commonly known as “teosinte”, native to what is now central Mexico ,2. “TeosZ. mays ssp. parviglumis) had on gametophyte and embryo development has been done, and significant shifts in timing and patterning were revealed that account for the larger size of the caryopses of maize [Z. mays subspecies, notably with proportionally larger stele and xylem vessel cross-sectional areas in domesticated maize [Z. mays could similarly contribute to our understanding of how evolutionary processes and domestication might have affected developmental patterns in the primary roots of domesticated maize compared to Z. mays teosintes. A study of this kind in roots has only been done for an unusual family of dicotyledons: Podestemaceae, a family that has rather distinctive root morphology [Z. mays are particularly well suited to precise anatomical analysis because their closed apical organization and predictable histogenesis make measurements more manageable and meaningful . Furthe[Z. mays .Zea mays ‘Kelvedon 33′), and he reported that the first four metaxylem files “differentiated simultaneously” and that metaxylem differentiation was correlated with quiescent center size.Over the last 150 years much work has been invested in evaluating and understanding the structure and patterns of histogenesis in root apical and primary meristem zones (RAMs). Our general understanding of RAM structure and behavior was well reviewed by Jiang and Feldman  and HeimThe cellular organization of the apical meristems of primary roots of flowering plants has been described as being “closed” or “open” ,10,11,12Arabidopsis thaliana, which is a dicotyledonous plant in the Brassicaceae [A. thaliana can be detected in the plerome because cells that divide to form cell files that will become tracheary elements are slightly larger than the flanking cells that produce differentiating phloem cell types. The outermost cells of the plerome produce a uniseriate pericycle by anticlinal divisions transverse to the root axis. Some aspects of the molecular genetic regulation of primary root vascular development have recently been revealed in the Arabidopsis system [A. thaliana, which has very narrow roots, four cells occupy the center of the periblem, and these, by virtue of their mitotic inactivity, constitute the quiescent center (QC) [The best-characterized example of a closed system is that of sicaceae ,17,18. Wsicaceae  describes system ,21,22,23ter (QC) , which iter (QC) ,7,24.Arabidopsis. In grass root apices the uniseriate root cap-producing calyptrogen is usually quite distinct and the protoderm and ground meristem are produced by a single dermatogen-periblem histogen layer immediately basipetal to it. As in Arabidopsis, the procambium that becomes the pericycle, xylem, and phloem of the stele is reportedly produced by the uniseriate plerome layer [Triticum) [Avena sativa) [Hordeum sativum) [Oryza sativa) [Zea mays) [Plants in the Poaceae (grasses) typically have root apical meristems with closed organization that also have defined histogen layers, though the organization differs slightly from me layer ,26. Careriticum) , oats (A sativa) , barley sativum) ,29,30, rea mays) ,32,33,34Arabidopsis root system is not typical of most plant species. Furthermore, errors of interpretation due to misunderstood terminology or due to oversimplification and/or extrapolation from model systems such as Arabidopsis that are uncharacteristically simple by comparison to most plant species can lead to confusion, for example, in the otherwise excellent review by Miyashima et al. [Arabidopsis thaliana continue to be undertaken so that as increasingly effective and versatile molecular tools are developed the histological topology of other plant systems shall have been mapped in detail and such new strategies can be used with precision. This has been recently done well for roots of a range of dicotyledonous plants [Z. mays during the artificial selection typical of domestication.Unfortunately, the numerical precision and predictability of the a et al. . It is is plants . Here weZ. mays subspecies (mexicana) and a modern, highly-derived commercial cultivar (Z. mays ‘Honey Bantam’); and (2) to evaluate the anatomical differences that that may contribute to our understanding of the factors that cause dimensional differences apparent in their primary root tips. Preliminary data led us to believe that some earlier analyses of maize closed root apical organization that relied heavily on longitudinal sections and coarser levels of resolution due to the methods prevalent at the time missed some important details of the development process in this species. This ultimately led us to reassess the way histogens have been defined and the way they behave in Zea mays RAMs.The objectives of the present study were (1) to use a new approach to slide preparation that more clearly reveals structural patterns at the tissue and cell level to enhance precision thin-sectioning and high magnification analysis of RAM structure, procambium development, and late-maturing metaxylem vessel initiation in an undomesticated We found that treating tissue sections with RNase to remove cytoplasmic RNA before staining with toluidine blue O, which heavily stains cytoplasmic RNA, simplified evaluation of cell wall patterns and mitotic figure recognition by making cell walls and chromatin in nuclei more clearly visible compare . This enZ. mays ssp. mexicana teosinte had the closed anatomical organization typical of most grasses and were very similar to that of sweetcorn  were mainly limited to cells of the dermatogen-periblem complex, the plerome, and two somewhat less organized layers of cells basipetal to the plerome that contained procambial (vascular) initials and gave rise to all vascular cell types except pericycle. These four cell layers were centrally located at the tip of the root A,C, and Z. mays types, which is noteworthy especially considering the significant differences in the ultimate dimensions of their steles  or two-to-three (sweetcorn) rings of additional cells, and then the margin initials . There wr steles . Immaturr steles A,C. In tr steles B. Accordr steles C.The remainder of the procambium was produced by proliferation of cells from the two somewhat irregularly organized layers of vascular initial cells immediately basipetal to the plerome A,C. We dt = −4.72, df = 3.71, p = 0.011, n = 3), indicated that a larger number rather than a larger cell size of this cell type primarily accounted for the larger diameters of sweetcorn steles compared to those of teosinte  divisions that increase the number of cells in the vicinity of and between developing vessels . It was Feldman , that in the RCJ ,7. The c the RCJ  for iden the RCJ  added th vessels F–H. This vessels . Neverth vessels . We wereZ. mays.From an evolutionary perspective it is noteworthy that the organization of the promeristem, specifically the size and organization of the histogens in teosinte and sweetcorn , Table 1Allium cepa [Z. mays cv. ‘Golden Bantam’, it is understandable that mitoses would be frequently missed if they are synchronized, as we observed.Our most novel finding, however, was that on the relatively rare occasions that mitotic activity was found in the QC it was found with many mitotic figures in the same histogen layer C. In othium cepa ,38, but ium cepa  and Jensium cepa  that themexicana teosinte differed from sweetcorn only in that the numbers of late-maturing metaxylem vessels in the latter are typically two-fold greater and the number of cells in transverse section of procambium were greater in the latter, but parenchymatous cell sizes were the same. This suggests that over evolutionary time either the frequency of mitotic activity or its temporal persistence within the parenchymatous tissue of the procambium (or both) increased during domestication perhaps as a result of artificial selection. Perhaps this additional activity stimulated additional LMX initials to form. Verification of this hypothesis by future studies, especially at the molecular level, would be useful for comparing monocots with dicot systems in which such information has begun to be revealed. Primary root promeristems of both taxa were nearly identical in size and organization, but they did not operate quite as previously described. Mitotic activity was rare in the quiescent centers, as previously reported, but occasionally a synchronized pulse of mitoses was observed there. Our quantitative analysis and reinterpretation of histogen theory and procambium development should be useful for future detailed studies of the regulation of development, and perhaps its evolution, in this species.Metaxylem development in Zea mays L. ssp. mexicana from Snow Brand Seed Co. Ltd., Sapporo, Japan) and sweetcorn  were surface sterilized in 10% (v/v) household bleach. 20–25 seeds each were sown under axenic conditions in 1 L beakers filled with moistened vermiculite (375 mL water/L vermiculite) that had been covered with foil and autoclaved. The beakers were then placed in constant 25 °C in a continuously dark growth chamber for 3 or 4 d . The 1 d difference was required because teosinte radicles typically required 1 d longer to emerge than those of sweetcorn. Consistently at noon one day after emergence, all primary roots that were 0.5–1.0 cm long were then collected for fixation and sectioning. The relative developmental time we chose for this study was early but was determined to be after the hiatus of cell division during radicle emergence and after the reestablishment of the QC [Cultivation methods were modified after Gladish and Niki . TeosintFor conventionally prepared specimens, procedures for LM were modified from Niki et al. . Root tiAt neutral pH toluidine blue O principally stains carbohydrates and nucleic acids. Buffering the stain solution at pH 7.0 or lower prevents toluidine blue O from staining proteins in the cytosol to any significant degree. RNA is abundant in the cytosols of most meristematic cells, most of which lack vacuoles. Removing RNA by RNase treatment therefore makes nuclei, mitotic chromosomes, and cell walls more distinct after pH7 toluidine blue O staining by minimizing the staining of cytosolic contents. Therefore, some RNase-treated serial transverse and longitudinal sections were prepared for high resolution and high contrast LM micrographs as follows. Sections prepared as above in Technovit 7100™ resin and mounted on glass slides were treated with ribonuclease A  at 20 µg/100 µL 0.05M phosphate buffer solution (pH 7.2). The slides were kept in an incubation box at 100% humidity at 37 °C for 4 h. The slides were then stained, viewed, and photographed as described above.A total of 55 primary root tips of teosinte seedlings were serially sectioned transversely and 16 were serially sectioned longitudinally. A total of 19 seedling primary roots of the ‘Honey Bantam’ maize cultivar were serially sectioned transversely and 18 were serially sectioned longitudinally. All were examined microscopically after being stained by one of the two staining protocols. Of the transversely sectioned primary root tips of teosinte and sweetcorn seven of each type were selected at random for RNase treatment and subsequent detailed measurements. These constituted the physical “library” from which we sampled for the various comparisons reported here.Digital photomicrographs of transverse sections cut 97.5–100.5 µm from the RCJ and prepared as described above and photographed at the same magnification from three roots each of teosinte and sweetcorn were prepared as follows for quantitation. With Photoshop CS6, all cells from and including the pericycle outwards were erased. The resulting images were “thresholded” at the same setting, and all cell contents that remained were erased, leaving only cell walls. The images were inverted, leaving only the cytoplasmic areas uniformly black. Vascular conducting cells  were identified from the original image and erased, which left only parenchymatous cells. With NIH Image 1.63, pixel sizes were calibrated to a photomicrograph of a stage micrometer taken at the same magnification, and the sectional areas of the cytoplasms were computed. Boxplots of area data for each section were generated, mean cell area 95% confidence intervals were computed and compared by one-way ANOVA, and mean vascular parenchymatous cell numbers and LMX cross-sectional area proportions of the procambium from three teosinte and three sweetcorn root transverse sections were compared using Student’s t-test with Minitab 16 .


Kaposi’s sarcoma-associated herpesvirus (KSHV) is the infectious etiologic agentassociated with Kaposi’s sarcoma (KS), primary effusion lymphoma, and multicentricCastleman disease. It has been shown that high KSHV prevalence and high incidence ofboth classic KS and AIDSassociated KS are found mostly among people of Uygurethnicity in Xinjiang, while people of Han ethnicity in Xinjiang have a higher KSHVseroprevalence than those of other Han populations in mainland China. However, it isstill unclear why there is such geographical and population variation in KSHVdistribution in China. In this work, we focused on the populations in the Kashgarregion and Urumqi area, where a total of 1294 research subjects were randomlyselected to investigate the potential correlation between KSHV prevalence anddifferent ethnicities in endemic areas of Xinjiang, and to determine risk factorsthat may affect KSHV infection rates or KS incidence. We identified a highseroprevalence of KSHV and high peripheral blood DNA infection in the general Uygurand Han populations in both Urumqi and Kashgar regions of Xinjiang, and determinedthat advancing age, low education level, and stationary population status affectKSHV infection rates. Further, KSHV-positive Uygur participants were shown to havehigher prevalence of neutralizing antibodies and neutralizing antibody titers thanKSHV-positive Han participants. Supplementary material is available for this article at 10.1007/s12250-017-4049-9 and is accessible for authorized users. Prevalence of Kaposi’s sarcoma-associatedherpesvirus in Uygur and Han populations from the Urumqi andKashgar regions of Xinjiang, China


Vibrio vulnificus can cause severe necrotic lesions within a short time. Recently, it has been reported that the numbers of wound infection cases in healthy hosts are increasing, for which surgical procedures are essential in many instances to eliminate the pathogen owing to its rapid proliferation. However, the mechanisms by which V. vulnificus can achieve wound infection in healthy hosts have not been elucidated. Here, we advance a systematic understanding of V. vulnificus wound infection through genome-wide identification of the relevant genes. Signature-tagged mutagenesis (STM) has been developed to identify functions required for the establishment of infection including colonization, rapid proliferation, and pathogenicity. Previously, STM had been regarded to be unsuitable for negative selection to detect the virulence genes of V. vulnificus owing to the low colonization and proliferation ability of this pathogen in the intestinal tract and systemic circulation. Alternatively, we successfully identified the virulence genes by applying STM to a murine model of wound infection. We examined a total of 5418 independent transposon insertion mutants by signature-tagged transposon mutagenesis and detected 71 clones as attenuated mutants consequent to disruption of genes by the insertion of a transposon. This is the first report demonstrating that the pathogenicity of V. vulnificus during wound infection is highly dependent on its characteristics: flagellar-based motility, siderophore-mediated iron acquisition system, capsular polysaccharide, lipopolysaccharide, and rapid chromosome partitioning. In particular, these functions during the wound infection process and are indispensable for proliferation in healthy hosts. Our results may thus allow the potential development of new strategies and reagents to control the proliferation of V. vulnificus and prevent human infections. Vibrio vulnificus is a Gram-negative halophilic bacterium and opportunistic human pathogen that causes primary septicemia and wound infections , in vivo-induced antigen technology (IVIAT), and signature-tagged mutagenesis (STM) have been used to identify virulence factors of V. vulnificus that are functionally expressed in vivo, focusing on primary septicemia  toxin is known to be indispensable for pathogenicity and colonization  for the animal experiments. The mice were housed in a controlled environment with a 12:12-h light-dark cycle and were fed rat chow MF  and tap water. Ambient temperature during the study was maintained at about 23°C.All animal studies were carried out in strict accordance with the Guidelines for Animal Experimentation of the Japanese Association for Laboratory Animal Science. The animal experimentation protocol was approved by the President of Kitasato University based on the judgment of the Institutional Animal Care and Use Committee of Kitasato University .Vibrio vulnificus CMCP6 is a clinical isolate from a male septicemic patient at the Chonnam National University Hospital, South Korea (1 and Kyoto Encyclopedia of Genes and Genomes (KEGG)2. Escherichia coli BW19795 with signature-tagged mini-Tn5Km2 in pUT were used for conjugation of transposons to V. vulnificus. ΔpomA is a non-motile strain used as a control strain for the swarming assay (V. vulnificus E4 is unencapsulated strain (translucent colony morphology) and was used as a control strain for colony morphology. This strain was isolated from seafood in Florida, and kindly provided by Dr. Shinichi Miyoshi   .E. coli BW19795 with signature-tagged mini-Tn5Km2 in pUT were combined with V. vulnificus on a nitrocellulose Hybond C membrane  for conjugation, placed onto an M9 agar plate, and incubated at 25°C (V. vulnificus were grown in Luria-Bertani (LB) medium containing 100 mg/ml of kanamycin for 12 h at 37°C in 96 well plate separately. We checked each bacterial growth by measuring optical density at 600 nm (OD600) with a microplate reader , and the mutants were pooled, washed with LB medium without any anti-biotics, and used as an input pool. Mice were subcutaneously inoculated with 106 CFU of the input pool into right caudal thighs  and fixed with CL-1000 Ultraviolet Crosslinkers . DNA probes were amplified by polymerase chain reaction (PCR) with Dig-labeled primers: Dig 1 (5′-Dig-CAT GGT ACC CAT TCT AAC-3′) and Dig 2 (5′-Dig-TAC CTA CAA CCT CAA GCT-3′). PCR was performed in a 50-μl reaction mix containing 25 μl of 2 × PCR Buffer for KOD FX Neo, 5 μl of 2 mM dNTPs, 2 μl of primer mix , 0.5 μl KOD FX Neo polymerase , 1 μl DNA template (about 200 ng genomic DNA), and distilled water. Thermal cycling conditions were as follows: (i) 5 min at 94°C; and (ii) 25 cycles of 15 s at 94°C, 45 s at 59°C, and 10 s at 68°C. The hybridization processes were performed in a hybridization oven  .V. vulnificus genome: Arb1 (5′-GGC CAC GCG TCG ACT AGT CAN NNN NNN NNN GAT AT-3′), Arb3 (5′-GGC CAC GCG TCG ACT AGT CAN NNN NNN NNN TTC AA-3′), Arb4 (5′-GGC CAC GCG TCG ACT AGT CAN NNN NNN NNN CCA CG-3′), Arb5 (5′-GGC CAC GCG TCG ACT AGT CAN NNN NNN NNN ACT GA-3′), Arb6 (5′-GGC CAC GCG TCG ACT AGT CAN NNN NNN NNN ACG CG-3′), Arb7 (5′-GGC CAC GCG TCG ACT AGT CAN NNN NNN NNN TGG CA-3′), and Arb for 2nd (5′-GGC CAC GCG TCG ACT AGT CA-3′). Thermal cycling conditions of first round were as follows: (i) 5 min at 95°C; (ii) 6 cycles of 20 s at 95°C, 20 s at 30°C, and 2 min at 72°C; (iii) 30 cycles of 20 s at 95°C, 20 s at 45°C, and 2 min at 72°C; and (iv) 5 min at 72°C. PCR was performed in a 50-μl reaction mix containing 5 μl of 10 × PCR Buffer for Paq5000, 2 μl of 2 mM dNTPs, 2 μl of primer mix , 0.5 μl Paq5000 polymerase , 1 μl DNA template (about 200 ng genomic DNA), and distilled water. Thermal cycling conditions of second round were as follows: (i) 30 s at 95°C; (ii) 30 cycles of 20 s at 95°C, 30 s at 55°C, and 4 min at 68°C; and (iii) 5 min at 68°C. PCR was performed in a 50-μl reaction mix containing 5 μl of 10 × PCR Buffer for Taq DNA polymerase with ThermoPol, 5 μl of 2 mM dNTPs, 2 μl of primer mix , 0.5 μl Taq DNA polymerase with ThermoPol , 1 μl the PCR product of the first round, and distilled water. The DNA sequences of the PCR products were determined by using the Fasmac sequencing service  and used to search for sequence homologies in the KEGG.The transposon inserted sequence was determined via arbitrarily primed PCR using specific primers targeting the transposon: Rev consensus2 I-out 1st (5′-CCA TGG GTA AGA TTG GTT CGA A-3′), consensus1 I-out 1st (5′-GGT ACC TAC AAC CTC AAG CT-3′), and Rev consensus1 I-out for 2nd (5′-AGC TTG GTT AGA ATG GGT ACC-3′), and random primers targeting the pomA, approximately 1000 bp of each 5′ and 3′ of flanking regions of PomA encoding gene were amplified by PCR with the primers: pomA Up Fw (5′-GGG GTG ACG CCA AAG TAT ATG GTG AAT GCG AGC GTT TG-3′), pomA Up Rev (5′-TCT TAA GTT TGC TTC CCT CAT GCT ATT TTC CGA TTT ACC GC-3′), pomA Down Fw (5′-AAC GGG AGG AAA TAA TCA CGG GAG TAT GTG ATG GAT GAC G-3′), pomA Down Rev (5′-CTT AAC GGC TGA CAT GGG GAC GAG CAT TTC TGC TCA TC-3′), and pomA Flag Rev (5′-TTA TTT CCT CCC GTT TTA GAT TAC AAG GAT GAC GAC GAT AAG ATC GTT GAT ATC GAG GGC AC-3′). The amplified DNA were cloned into the suicide vector pYAK1, which contains the chloramphenicol resistant gene and sacB gene conferring sensitivity to sucrose (To delete a 762-bp DNA fragment of  sucrose .cheY was amplified using specific primers: cheY up Fw (5′-CTG CAG TGA ATG TGA GCC TCG AAC TC-3′) and cheY down Rev (5′-GGA TCC GCA TTG AGA AGA TCC CTG TC-3′). It cloned into the pGEM-T Easy vector . To delete a 381-bp DNA fragment of cheY, the plasmid was amplified by inverse PCR using specific primers: Inv Fw (5′-TTA GGG CCC CAA AAT TGC CTC CAC TGA AT-3′) and Inv Rev (5′-GCC GTG CAC TAA ACC TCG TTT GAA GAT TA-3′), treated with DpnI to digest methylated parental plasmids, and self-ligated. The plasmid was digested with ApaI and ApaLI and then cloned into the suicide vector pYAK1.A 2859-bp DNA fragment including pomAKO and pYAK1-cheYKO, were introduced into Escherichia coli BW19795. The transformants were combined with V. vulnificus on a nitrocellulose Hybond C membrane  for conjugation, placed onto an M9 agar plate, and incubated at 25°C  and cheY Rev (5′-CTC GAG TTA TAA ACG TTC AAA AAT TTT ATC TAG-3′). It cloned into pACYC184 by Infusion cloning reactions . The complementing plasmid pACYC184-cheY was introduced into V. vulnificus via electroporation. After inoculation onto LB plate containing 10 μg/ml chloramphenicol and incubation overnight at 37°C for selection, the strain retaining the plasmid was named pcheY. The cheY gene could be expressed from the cat promotor on this plasmid (The  plasmid .Vibrio vulnificus CMCP6 parent strain (WT) were grown in LB medium, and each signature-tagged transposon insertion mutants were grown in LB medium containing 100 μg/ml of kanamycin at 37°C. Overnight cultures (100 μl) were inoculated into 2 ml of fresh medium and incubated for 2 h. Log-phase bacteria were inoculated onto LB plates containing 0.3% agar and incubated for 12 h at 37°C.WT and E4 were grown in LB medium, and each signature-tagged transposon insertion mutants were grown in LB medium containing 100 μg/ml of kanamycin at 37°C. Overnight cultures (5 μl) were inoculated onto LB plate and incubated for 12 h at 37°C. The opacity of the colonies was examined.cheY were grown in LB medium containing 50 mg/ml of rifampicin, and pcheY was grown in LB medium containing 50 mg/ml of rifampicin and 10 μg/ml chloramphenicol with agitation (163 rpm) at 37°C. Overnight cultures (100 μl) were inoculated into 2 ml of fresh medium and incubated for 2 h. Bacteria were harvested, washed with PBS (pH 7.2) containing 0.1% gelatin, and resuspended in fresh medium. Then, 106 CFU/mouse were subcutaneously inoculated into right caudal thighs. Infected mice were sacrificed at 6 h post-infection. The collected muscular tissue was suspended in PBS containing 0.1% gelatin, homogenized for 5 s with a lab mixer IKA EUROSTAR digital , and centrifuged at 800 rpm for 5 min. The supernatant was plated at 10-fold serial dilutions in duplicate on LB agar containing 50 μg/ml rifampicin and incubated for 12 h at 37°C. V. vulnificus colonies were counted, and the number of CFU/g of muscle tissue was determined as a bacterial burden in muscle tissue.WT andΔ3 and their putative function: cellular component, metabolism, regulation, and uncertain function proteins , C-ring (fliM), hook (flgK), filament cap (fliD), and motors (pomA and motX)  and flagellar hook-length control (fliK) were responsible for flagellin export  . Genes fn export . All strn export . Genes fn export . Our finThe genes involved in metabolism included those responsible for the utilization of carbohydrate and iron Figure . GlycosyV. vulnificus requires an iron acquisition system for their proliferation in healthy hosts.In iron metabolism, the genes for VuuA and IutA were detected in this study Table . These ain vitro but were detected by the STM method  in the tissue compared with WT, and the bacterial burden was recovered by complementation with cheY (Figure V. vulnificus proliferation at the local site of the wound infection.In order to sense and respond to various environmental conditions, the two-component system, which regulates the direction of flagellar rotation based on sensing of chemoattractants or chemorepellents in bacterial chemotaxis , along wed Table . To deteV. vulnificus, such as MARTX, VVH, VvpE, the and iron acquisition system, are located on chromosome II (We classified and selected 71 clones from among 5418 independent transposon insertion mutants. Although genes involved in the known virulence factors of osome II , our detPseudomonas aeruginosa indicate that the FimT, FimV, and CpaE-like protein are involved in Tfp assembly (P. aeruginosa is essential for twitching motility, however, that of V. vulnificus is expected to be functionally distinct (V. vulnificus infection (V. vulnificus, especially those isolated from wound infection sites, presented higher numbers of pilus fibers per cell than environmentally isolated strains. V. vulnificus exhibited decreased adhesion to cells, biofilm formation, and lethality in iron dextran-treated mice. Together with these data, the results of the present study first showed the essential genes for and importance of pili in the wound infection, suggesting the possibility that Tfp of V. vulnificus may be involved in the adherence to cells and biofilm formation in soft tissues at the wound infection site (Figure Type IV pili are known to play a role in cell adhesion, twitching motility, and the uptake of foreign genes . Studiesassembly . Tfp of distinct . Pili confection . Gander e Figure .V. vulnificus proliferation in a local wound infection site (The flagellum is structurally composed of the basal body, hook, and filament . The basion site . These fion site .in vivo as well as in vitro and may not able to spread in soft tissues which have complex and intricate structures. It must have resulted in a non-sufficient proliferation by ΔcheY at the local wound infection site and not be detected from spleen as a STM output (Figure V. vulnificus to spread in soft tissue and proliferate at the site of wound infection. Notably, the chemotaxis of V. vulnificus associated with tissue tropism or invasion of the systemic circulation has not previously been investigated. However, although approximately 40 genes encoding MCPs exist on V. vulnificus chromosomes, these were not detected in the present study. Identification of the chemoattractants underlying bacterial spread in soft tissue and transition to the systemic circulation will therefore likely be helpful to prevent the development of sepsis.Bacteria change swimming direction by tumbling consequent to switching the direction of flagellar rotation from counterclockwise to clockwise  and movet Figure . TogetheSalmonella enterica serovar Typhi, Vibrio cholerae, and E. coli, secrete siderophore during intestinal tract infection (V. vulnificus also secretes siderophore in primary septicemia through the digestion of raw seafood (V. vulnificus infection (V. vulnificus infection. Unlike in septicemia, in soft-tissue infection, one a single study has reported that P. aeruginosa secretes siderophore, in a murine model of surgical site infection (V. vulnificus during wound infection in healthy hosts (Figure V. vulnificus is enhanced under in vitro iron-restricted conditions (Several pathogens, such as nfection . It is t seafood . Notablynfection . Unlike nfection . These os Figure . It is cnditions .Vibrionaceae bacteria such as V. vulnificus, V. cholerae, and Vibrio parahaemolyticus have two differently sized chromosomes that each contain an origin for replication (V. vulnificus in the wound infection. In particular, several studies on E. coli have shown that MukB acts as a homodimer, condenses DNA, and facilitates chromosome segregation (lication . This chregation .murG mutant by STM. In P. aeruginosa and E. coli, MurG plays a key role in the biosynthesis of the peptidoglycan layer to form a glycosidic bond between N-acetylmuramyl pentapeptide and GlcNAc for bacterial cell walls (We also detected a ll walls . The bacll walls .wbpP gene encoding GlcNAc C4 epimerase is CPS-deficient. Thus, GlcNAc metabolism is essential for the synthesis of CPS. CPS is considered essential for in vivo proliferation, as clinically isolated strains typically produce CPS unlike environmentally isolated strains (V. vulnificus is essential for the evasion of phagocytosis by macrophages as well as for serum resistance (V. vulnificus to proliferate in the systemic circulation (V. vulnificus serve as inducers of tumor necrosis factor-alpha (V. vulnificus contribute to the initial establishment and fatal outcomes in both primary septicemia and wound infection (Figures  strains . It has sistance . In addisistance . Synthessistance . In turnsistance . It was culation . Howeverculation . These rculation . In addior-alpha , which h Figures .V. vulnificus during wound infection highly depends on its characteristics; motility, iron-acquisition, CPS, LPS, and two circular chromosomes. In addition, we identified novel factor: chemotaxis, which likely enables V. vulnificus to spread toward a suitable environment, rapidly proliferate especially at the local infection site, and aggravate wound infection within a short period. These factors comprise attractive potential candidates for the development of antibiotics or vaccines to control infection.Our findings demonstrate that the pathogenicity of KY and TKas contributed to the conception and design of the study. KY performed the majority of the experiments and analyzed the data. MM and TKad assisted STM. KY wrote the manuscript. SU analyzed and supervised this study. All authors contributed to manuscript revision and read and approved the submitted version.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.


Genomics and particularly mutational profiling have deepened our understanding of cancer pathology and laid the foundation for precision therapies as a major field in modern oncology. Comprehensive genetic profiling has even led to proposals to replace the current histology-based WHO tumor typing with molecular classes based on the rationale that similar molecular alterations have similar clinical relevance across cancers -3. HowevOne way to assess the functional impact of genetic alterations in cancer is to study their relationship with corresponding (phospho-)proteomic profiles. Using publicly available proteogenomic profiling data across major cancer types, we propose a computational approach that systematically evaluates if and to what extent genetic aberrations observed in tumors are associated with distinct proteomic profiles . Our anaTo achieve this, we further propose a combined experimental and computational systems proteogenomics approach that allows for a reduction of the mutational complexity and facilitates the identification of functionally relevant molecular aberrations that can be exploited to overcome resistance against targeted (mono-)therapy in individual patients . It combIn summary, the ability to interpret and assess the functional and clinical relevance of the increasingly comprehensive mutational profiling data accruing not only in research but also in clinical cancer diagnostics is reaching its limits when relying only on pre-clinical experimental data and knowledge on biological pathways. Moreover, the combinatorial complexity of potential druggable target combinations is incompatible with testing in classical clinical trials. As a solution, combining genomic with proteomic profiling and classical computational analysis as well as advanced machine learning techniques can contribute to identifying functionally and clinically relevant molecular alterations which in the future may complement molecular diagnostics for precision oncology.


There is a debate as to whether topic structures in Chinese involve A’-movement or result from base-generation of the topic in the left periphery. If Chinese topicalization was derived by movement, under the assumptions of Friedmann et al.’s Relativized Minimality (Lingua 119:67–88,  The topic structure has been widely discussed in the linguistic literature and authors have critically discussed whether it is derived by movement or not . Not only ya, but also a, me, ne and ba can be used as topic markers  or a class of entities, but other syntactic categories can also constitute the topic. Generally, the comment is a clause, but not obligatorily. The topic marker can be null as in (1a), or overt like a/ya are almost equal to a simple pause and their function is simply to signal a topic, without any additional meaning. Ba often follows an aforementioned event and ma often follows an aforementioned object, while ne sometimes signals a contrast between the marked topic and another topic or a juxtaposition of two topics , which, together with (1), illustrate the variation encountered in topic structures.Li xiansheng ‘Mr. Li’ is related to an empty element in the comment clause, the topic in (2a) is resumed by an overt pronoun ta ‘him’ in the comment clause. Second, double noun constructions (‘double-subject sentences’ in Li and Thompson changjinglu ‘giraffe’ and bozi ‘neck’ in (2b) are involved in a part-whole relation, or the first NP shuiguo ‘fruit’ and the last NP yingtao ‘cherry’ in (2c) have an inclusive relation. Third, the comment as a whole may be a predicate related to the topic as in (2d), without any indication of a gap in the comment. Fourth, the topic can also be an adverbial phrase as in (2e), where the adverbial yijiuliuba nian ba yue ershi’er ri ‘(on) August 22, 1968’ is derived from the position after the subject wo ‘I’ as argued by Xu and Langendoen  in which the topic ngendoen .Two contrasting analyses of Chinese topic structures have been proposed in the literature . There is no gap in any of these sentences. An ‘aboutness’ relation between the comment clause and the topic, rather than a gap-antecedent relation, exists. These ‘gapless’ sentences have been noted by a number of linguists Li Si ‘Li Si’, is extracted from inside a complex NP. If a resumptive pronoun is inserted in the gap position, the sentence becomes acceptable. This indicates that the ungrammaticality of (4) is not due to semantic or pragmatic anomaly, but because the topic element cannot be extracted from the complex NP island , the topic, pro in Chinese, limited to the subject position of finite clauses in Chinese. Consider (6)  and the grammaticality of (5)? Huang , 1989 prZhang San  or to a previously mentioned discourse topic such as John . In (6b), the null object can only refer to a previously introduced discourse topic . Huang claimed that the null subject in (6a) is a null pronominal, since an overt pronoun such as ta ‘him’ in the same position functions in the same way, while the null object in (6b) cannot be a null pronominal, because the overt pronoun in the same position can either refer to the matrix subject Zhang San or to a discourse topic. Accordingly, the null object is better analyzed as a variable, which is A’-bound by an empty operator and cannot be A-bound by the matrix subject (see 6c). The comparison between (6a) and (6b) led Huang to conclude that there is a subject/object asymmetry with respect to empty categories in Chinese , the null subject may refer to the matrix subject nt Huang : 552.pro, which allows base-generation. In (4), the closest antecedent of the empty category is the subject of the comment clause, wo ‘I’, not the topic Li Si ‘Li Si’. Accordingly, the sentence receives the reading ‘As for Li Si, I like my own voice of singing’ which is not comprehensible, as the topic is not related to the comment. Thus, either with a trace or with a pro, the sentence (4) is ill-formed. In turn, consider (5), in which the empty category is coindexed with the closest compatible antecedent and turns out to be coreferential with the topic Li Si ‘Li Si’. Thus, the linking between the pro and the topic is created without violating any principle of grammar and the sentence (5) is grammatical.Let us go back to (4) and (5). In both examples, as Huang et al.  noted, tHowever, the subject/object asymmetry is questionable. One classical example is given in (7) from Xu : 78.(7)Xxiaotou ‘thief’, but within an appropriate context it can refer to the discourse topic John. It seems that the syntactic restrictions can be overridden by semantic and pragmatic plausibility. Thus, the distribution of the empty pronoun is not limited to the subject position of finite clauses as Huang .ya). The topic is Li xiansheng ‘Mr. Li’ and the subject is wo ‘I’. The topic occurs in sentence-initial position and is related to an empty element in the comment clause. However, in (8b), the topic and the subject are identical. With a topic marker such as ya, the sentence receives the topic interpretation (Interpretation 1); without the topic marker, the sentence can be regarded as a canonical SVO sentence (Interpretation 2) or as a topicalization sentence with the subject being the topic given the proper context (Interpretation 1). Consider (9).In the literature, not only (8a) but also (8b) have been regarded as topic structures (Li and Thompson Li xiansheng ‘Mr. Li’ has been mentioned in the previous discourse (Speaker A) and is being discussed again (Speaker B). It can be followed by an optional topic marker such as ya, and the remaining clause renshi wo ‘know me’ is about this specific person. Given that the phrase satisfies the conditions for being the topic, we consider the sentence as a topic structure.In the sentence by speaker B, To examine whether Chinese children distinguish topic and subject at the early stages, Chien and Lust  tested 9baobao ‘baby’) much more frequently than the first NP (i.e. baobao ‘baby’) in the coordinate construction (25.74 vs. 2.38%). They were more likely to omit the first NP (i.e. Xiaohua ‘Xiaohua’) than the second NP (i.e. Xiaohua ‘Xiaohua’) in the control-construction (48.59 vs. 3.36%). Based on the fact that children were able to reduce redundant topics, Chien and Lust suggested that young children were sensitive to the topic-subject distinction to some degree. However, the findings were confounded by problems with the experimental materials used, e.g., in (10b) instead of treating the first Xiaohua ‘Xiaohua’ as the topic, Xiaohua jiejie can be interpreted as the subject of (10b) with the genitive maker de omitted (i.e. Xiaohua de jiejie ‘Xiaohua’s older sister’).The results of the experiment showed that children dropped the second NP X…How do you wonder who could solve this problem?). Second, when the intervener’s featural specification is included in the target’s featural specification as in (12b), the structure (e.g. Which problem do you wonder how to solve?) is ruled in by the RM principle . Finally, when the featural specification of the intervener and that of the target are disjoint as in (12c), the structure (e.g. What do you think John solved?) is ruled in by RM.Based on RM expressed in terms of features Rizzi , three dwh-questions by agrammatic Broca’s aphasics  leads to ungrammaticality and disjunction of feature specification as in (12c) results in grammaticality, but indeed some differences between the adult and the child system arise concerning (12b). According to Friedmann et al. , the aduIn the context of these findings, now we focus our attention on object topicalization (with OSV order) as in (13a) and subject topicalization (with SVO order) as (13b). As discussed above, with the appropriate context, the two structures constitute a minimal pair of topicalization sentences.If topicalization involves A’-movement, RM applies to the structure, and in that case the sentences in (13) have the structure in (14) in which ‘D NP’ stands for a nominal expression. The first ‘D NP’ is the topic phrase, which is attracted to the left peripheral position by the bundle of features , where ‘TOP’ designates the topic feature expressed overtly or covertly by the topic marker, and ‘NP’ designates the nominal feature that the topic also bears. We adopt the feature [+ NP] as in the RM framework, where it is understood as a categorial feature, an assumption that may be problematic. Therefore, we suggest that this feature might be a referential feature.In (14a), the subject of the comment also bears a [+ NP] feature and intervenes between the topic and its copy. According to the RM approach, in the configuration (14a), a RM violation may be detected by children because they may not consider the featural specification of the topic and that of the subject distinct. In order to consider them distinct, a subset relation has to be computed, but (young) children are assumed to have limited computational resources which would prevent them from making the computation. In contrast, no problem arises in SVO topicalization sentences as illustrated in (14b), because there is no intervener between the topic and its copy. In summary, we expect children to display difficulties in comprehending OSV topicalization sentences compared with SVO topicalization sentences, if topicalized structures are derived by A’-movement.pro as in (15)  to 10 . The results showed that the mean of sentences with an overt topic marker were numerically lower than those with covert topic marker , but no significant difference between them was found (t (23) = 1.66, p > .05). By carrying out two experiments, we can make a clear-cut distinction between overt topic markers and covert topic markers.In our study, we aimed at evaluating the two hypotheses above. We did this through two experiments on the acquisition of Chinese subject and object topicalizations, one with the presence of an overt topic marker (Experiment 1) and another in the absence of an overt topic marker (Experiment 2). In Experiment 1, an overt topic marker ause Chu  and in ESixty-six Mandarin-speaking children aged from 3:0 to 6:11 participated in this experiment. They were divided into four age groups: the 3-year-old group , the 4-year-old group , the 5-year-old group  and the 6-year-old group . They lived in Zhejiang, China and were developing normally. An additional adult group  served as control.ya, was used. We used 8 transitive verbs: bang ‘help’, da ‘hit’, gai ‘cover’, hua ‘draw’, kan ‘look at’, tui ‘push’, yao ‘bite’ and zhui ‘chase’.The stimuli consisted of 8 OSV topicalization sentences like (17a) and 8 SVO topicalization sentences like (17b). An overt topic marker, Zhe-ge nanhai, zai kan shu ‘As for this boy, (he) is reading books’), and that did not include an overt topic marker. See the complete list in the Appendix.All of the experimental sentences were semantically reversible, and the noun phrases were animate. In addition, there were 8 filler sentences including verbs that were not semantically reversible  is hitting the cat’, and the participant was asked to point to the picture matching the sentence.To make the use of topicalization felicitous, we created an appropriate context. The experimenter asked participants to describe the picture first, e.g., to name the characters, Participants were tested individually. Each participant was asked to look at the experimental pictures on an iPad screen. After providing an appropriate context for topicalization, the experimenter presented the target sentence and the participant pointed to a picture (out of two). Two practice items were presented to ensure that participants understood the task.The experiment yielded a total of 1056 responses from children and 160 from adults . Half of the responses corresponded to OSV topicalizations and the other half to SVO topicalizations. Table We analyzed the data using the lme4 package in the R environment  = 7.03, p < .01; Wald Z = − 2.97, p < .01). We further compared the two structures in each age group, and found that only the 3-year-old group and the 4-year-old group performed significantly better in SVO topicalization sentences than in OSV topicalization sentences (χ2 (1) = 4.54, p < .05; Wald Z = − 2.27, p < .05, and χ2 (1) = 4.38, p < .05; Wald Z = − 2.25, p < .05, respectively), suggesting that there is a subject/object asymmetry on the comprehension of Chinese topicalizations in 3- and 4-year-old children.First of all, sentence type yielded a significant effect (χ2 (3) = 51.51, p < .001). By changing the reference categories, we compared each age group with the other age groups. Table Second, age yielded a significant effect , the 4-year-old group , the 5-year-old group  and the 6-year-old group . As in Experiment 1, all the children lived in Zhejiang, China and were developing normally. An additional adult group  served as control.The materials used in Experiment 2 were identical to those used in Experiment 1 except that an overt topic marker was not used in the experimental sentences, as illustrated in (18a) for OSV topicalization sentences and in (18b) for SVO topicalization sentences.To iterate, we aimed at making a clear-cut distinction between overt topic markers and covert topic markers, so we did not use the intonational break in Experiment 2. As in Experiment 1, we provided an appropriate context to make the use of topicalization felicitous. Given that the initial phrase of the sentence is a topic, both structures regardless of word order (OSV vs. SVO) are topicalizations.The experiment yielded a total of 1280 responses from children and 160 from adults. Half corresponded to the comprehension of OSV topicalizations and the other half to SVO topicalizations. As in Experiment 1, we excluded the analysis of filler sentences, as all were answered correctly. Table 2 (1) = 2.71, p = .10; Wald Z = − 1.70, p = .09). Neither within the 3-year-old group, nor within the 4-year-old group did we find any significant difference between the two sentence types (χ2 (1) = 2.11, p > .05; Wald Z = − 1.55, p > .05, and χ2 (1) = 1.03, p > .05; Wald Z = − 1.02, p > .05, respectively). The results indicate that comprehension of topicalization sentences with the OSV order and those with the SVO order did not differ.First of all, the sentence type factor did not predict the comprehension ability (χ2 (3) = 38.03, p < .001). As is evident in the output of the analysis  = 6.05, p < .05; Wald Z = 2.57, p < .05). We contrasted the difference for each age group, and found that the significant difference was evident in the 3-year-old group (χ2 (1) = 14.57, p < .001; Wald Z = 4.16, p < .001) and in the 4-year-old group (χ2 (1) = 9.29, p < .01; Wald Z = 3.29, p < .01).We further compared the results of the two experiments  was significantly worse than that of Chinese topicalization without an overt topic marker (Experiment 2).ya are not just optional elements.As it is evident in Experiment 1, children comprehended significantly better subject topicalizations (with SVO order) than object topicalizations (with OSV order) when the topic marker is overt in the structure. Specifically, there is a subject/object asymmetry on the acquisition of Chinese topicalization with an overt topic marker at 3 and 4 years of age . This contrasts with children’s performance on topicalizations without an overt topic marker in Experiment 2: although the accurate rates of subject topicalizations were numerically higher than object topicalizations , no difference between the two structures reached significance in any age group. Moreover, we noticed that the accuracy rate in Experiment 2 was much higher than in Experiment 1, specifically at age three and four. These findings indicate that children treat the structure with an overt topic marker and that without an overt topic maker differently in the early stage of development, and certainly suggest that topic markers such as Two analyses are offered in the literature on topic structures: the movement analysis and the base-generation analysis. They make different predictions for acquisition when combined with the RM approach, as we discussed before. According to the RM principle, a local relation between X and Y cannot hold if Z has the same feature specification as X and acts as a possible candidate for the same relation, as in (19a). If topicalization involves A’-movement, RM applies as in (19b). Since we assume that (young) children have trouble computing the subset relation, they may not consider the topic and the subject distinct and may consider (19b) a RM violation. In this case, we predict that object topicalization would be harder than subject topicalization. On the other hand, if topicalization does not involve A’-movement and the topic is base-generated in the left periphery, RM does not apply and no asymmetry between subject and object topicalizations is expected.The observed subject/object asymmetry in Experiment 1 is consistent with the predictions of the movement analysis, as children at 3 and 4 years of age did show great difficulty in object topicalization. The lack of asymmetry in Experiment 2 seems to support the base-generation analysis, as children do not seem to have trouble establishing the relation between the topic and the empty category in the comment clause, in spite of the presence of the subject. However, one may question this explanation on different grounds, which we consider below.The girl kissed the boy and went to the beach in which the subject of the first conjunct is the subject of the second conjunct) and object relative clauses (The girl that the boy kissed). The former structure presumably involves no syntactic movement, while the latter involves A’-movement. They tested Hebrew and European Portuguese speaking children aged 3–5 and found that, in both languages, children’s difficulty in understanding the coordination sentences with crossing dependencies resembled the difficulty in object relative clauses. The authors ascribed the difficulty of the coordination sentences with crossing dependencies to the effects of linear intervention, as in this structure the intervener (the boy) linearly precedes the null subject of the second conjunct, without c-commanding it. Accordingly, the authors concluded that children’s difficulty with crossing dependencies is not restricted to A’-movement phenomena. In the present study, regardless of whether the topic marker is overt, in object topicalization sentences, the subject of the comment intervenes at least linearly between the topic and the object position, while in subject topicalization sentences there is no such a linear intervener. If we generalize Friedmann and Costa’s . This means that both structures must be derived by movement.However, it is generally agreed that topicalization does not involve movement at least in the case of ‘gapless’ topic structures , at age three, was quite differently from that of subject topicalization without an overt topic marker (88%), and was more similar to that of object topicalization without an overt topic marker (76%). The same pattern holds for the 4-year-old group. This fact suggest that children are not ignoring the topic marker, but they do not master it fully at age three and four. Firstly, as mentioned earlier, the discourse pragmatic roles of each topic marker are different , but, when the topic marker is overt, their comprehension accuracy decreased for both subject topicalizations and object topicalizations (Experiment 1). Critically, these contrasting findings indicate that topic markers such as