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1.

Background

Vocal sequences - utterances consisting of calls produced in close succession - are common phenomena in animal communication. While many studies have explored the adaptive benefits of producing such sequences, very little is known about how the costs and constraints involved in their production affect their form. Here, we investigated this issue in the chimpanzee (Pan troglodytes schweinfurthii) pant hoot, a long and structurally complex vocal sequence comprising four acoustically distinct phases – introduction, build-up, climax and let-down.

Results

We found that in each of these phases, and for the sequence as a whole, there was a negative relationship between the number of calls produced and their average duration. There was also a negative relationship between the total duration of some adjacent phases. Significant relationships between the fundamental frequency of calls and their number or duration were found for some phases of the sequence, but the direction of these relationships differed between particular phases.

Conclusions

These results indicate that there are trade-offs in terms of signal duration at two levels in pant-hoot production: between call number and duration, and between the relative durations of successive phases. These trade-offs are likely to reflect biomechanical constraints on vocal sequence production. Phase-specific trade-offs also appear to occur between fundamental frequency and call number or duration, potentially reflecting that different phases of the sequence are associated with distinct types of information, linked in different ways to call pitch. Overall, this study highlights the important role of costs and constraints in shaping the temporal and acoustic structure of animal vocal sequences.
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2.

Introduction

Human speech does not only communicate linguistic information but also paralinguistic features, e.g. information about the identity and the arousal state of the sender. Comparable morphological and physiological constraints on vocal production in mammals suggest the existence of commonalities encoding sender-identity and the arousal state of a sender across mammals. To explore this hypothesis and to investigate whether specific acoustic parameters encode for sender-identity while others encode for arousal, we studied infants of the domestic cat (Felis silvestris catus). Kittens are an excellent model for analysing vocal correlates of sender-identity and arousal. They strongly depend on the care of their mother. Thus, the acoustical conveyance of sender-identity and arousal may be important for their survival.

Results

We recorded calls of 18 kittens in an experimentally-induced separation paradigm, where kittens were spatially separated from their mother and siblings. In the Low arousal condition, infants were just separated without any manipulation. In the High arousal condition infants were handled by the experimenter. Multi-parametric sound analyses revealed that kitten isolation calls are individually distinct and differ between the Low and High arousal conditions. Our results suggested that source- and filter-related parameters are important for encoding sender-identity, whereas time-, source- and tonality-related parameters are important for encoding arousal.

Conclusion

Comparable findings in other mammalian lineages provide evidence for commonalities in non-verbal cues encoding sender-identity and arousal across mammals comparable to paralinguistic cues in humans. This favours the establishment of general concepts for voice recognition and emotions in humans and animals.
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3.

Background

Male sexually selected vocalisations generally contain both individuality and quality cues that are crucial in intra- as well as inter-sexual communication. As individuality is a fixed feature whereas male phenotypic quality changes with age, individuality and quality cues may be subjected to different selection pressures over time. Individuality (for example, morphology of the vocal apparatus) and quality (for example, body size and dominance status) can both affect the vocal production mechanism, inducing the same components of vocalisations to convey both kinds of information. In this case, do quality-related changes to the acoustic structure of calls induce a modification of vocal cues to identity from year to year? We investigated this question in fallow deer (Dama dama), in which some acoustic parameters of vocalisations (groans) code for both individuality and quality.

Results

We carried out a longitudinal analysis of groan individuality, examining the effects of age and dominance rank on the acoustic structure of groans of the same males recorded during consecutive years. We found both age- and rank-related changes to groans; the minimum values of the highest formant frequencies and the fundamental frequency increased with the age of males and they decreased when males became more dominant. Both age- and rank-related acoustic parameters contributed to individuality. Male quality changed with age, inducing a change in quality-related parameters and thus, a modification of vocal cues to male individuality between years.

Conclusions

The encoding of individuality and quality information in the same components of vocalisations induces a tradeoff between these two kinds of signals over time. Fallow deer vocalisations are honest signals of quality that are not fixed over time but are modified dynamically according to male quality. As they are more reliable cues to quality than to individuality, they may not be used by conspecifics to recognize a given male from one year to another, but potentially used by both sexes to assess male quality during each breeding season.
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4.

Background

Acoustic properties of vocalizations can vary with the internal state of the caller, and may serve as reliable indicators for a caller’s emotional state, for example to prevent conflicts. Thus, individuals may associate distinct characteristics in acoustic signals of conspecifics with specific social contexts, and adjust their behaviour accordingly to prevent escalation of conflicts. Common ravens (Corvus corax) crowd-forage with individuals of different age classes, sex, and rank, assemble at feeding sites, and engage in agonistic interactions of varying intensity. Attacked individuals frequently utter defensive calls in order to appease the aggressor. Here, we investigated if acoustic properties of defensive calls change with varying levels of aggression, and if bystanders respond to these changes.

Results

Individuals were more likely to utter defensive calls when the attack involved contact aggression, and when the attacker was higher in rank than the victim. Defensive calls produced during intense conflicts were longer and uttered at higher rates, and showed higher fundamental frequency- and amplitude-related measures than calls uttered during low-intensity aggression, indicating arousal-based changes in defensive calls. Playback experiments showed that ravens were more likely to react in response to defensive calls with higher fundamental frequency by orientating towards the speakers as compared to original calls and calls manipulated in duration.

Conclusions

Arousal-based changes are encoded in acoustic parameters of defensive calls in attacked ravens, and bystanders in the audience pay attention to the degree of arousal in attacked conspecifics. Our findings imply that common ravens can regulate conflicts with conspecifics by means of vocalizations, and are able to gather social knowledge from conspecific calls.
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5.

Background

Anecdotal and clinical theories purport that females are more responsive to smoking cues, yet few objective, neurophysiological examinations of these theories have been conducted. The current study examines the impact of sex on brain responses to smoking cues.

Methods

Fifty-one (31 males) cigarette-dependent sated smokers underwent pseudo- continuous arterial spin-labeled perfusion functional magnetic resonance imaging during exposure to visual smoking cues and non-smoking cues. Brain responses to smoking cues relative to non-smoking cues were examined within males and females separately and then compared between males and females. Cigarettes smoked per day was included in analyses as a covariate.

Results

Both males and females showed increased responses to smoking cues compared to non-smoking cues with males exhibiting increased medial orbitofrontal cortex and ventral striatum/ventral pallidum responses, and females showing increased medial orbitofrontal cortex responses. Direct comparisons between male and female brain responses revealed that males showed greater bilateral hippocampal/amygdala activation to smoking cues relative to non-smoking cues.

Conclusions

Males and females exhibit similar responses to smoking cues relative to non-smoking cues in a priori reward-related regions; however, direct comparisons between sexes indicate that smoking cues evoke greater bilateral hippocampal/amygdalar activation among males. Given the current literature on sex differences in smoking cue neural activity is sparse and incomplete, these results contribute to our knowledge of the neurobiological underpinnings of drug cue reactivity.
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6.

Background

Previous metabolomic studies have revealed that plasma metabolic signatures may predict epithelial ovarian cancer (EOC) recurrence. However, few studies have performed metabolic profiling of pre- and post-operative specimens to investigate EOC prognostic biomarkers.

Objective

The aims of our study were to compare the predictive performance of pre- and post-operative specimens and to create a better model for recurrence by combining biomarkers from both metabolic signatures.

Methods

Thirty-five paired plasma samples were collected from 35 EOC patients before and after surgery. The patients were followed-up until December, 2016 to obtain recurrence information. Metabolomics using rapid resolution liquid chromatography–mass spectrometry was performed to identify metabolic signatures related to EOC recurrence. The support vector machine model was employed to predict EOC recurrence using identified biomarkers.

Results

Global metabolomic profiles distinguished recurrent from non-recurrent EOC using both pre- and post-operative plasma. Ten common significant biomarkers, hydroxyphenyllactic acid, uric acid, creatinine, lysine, 3-(3,5-diiodo-4-hydroxyphenyl) lactate, phosphohydroxypyruvic acid, carnitine, coproporphyrinogen, l-beta-aspartyl-l-glutamic acid and 24,25-hydroxyvitamin D3, were identified as predictive biomarkers for EOC recurrence. The area under the receiver operating characteristic (AUC) values in pre- and post-operative plasma were 0.815 and 0.909, respectively; the AUC value after combining the two sets reached 0.964.

Conclusion

Plasma metabolomic analysis could be used to predict EOC recurrence. While post-operative biomarkers have a predictive advantage over pre-operative biomarkers, combining pre- and post-operative biomarkers showed the best predictive performance and has great potential for predicting recurrent EOC.
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7.

Background

Clinical statement alone is not enough to predict the progression of disease. Instead, the gene expression profiles have been widely used to forecast clinical outcomes. Many genes related to survival have been identified, and recently miRNA expression signatures predicting patient survival have been also investigated for several cancers. However, miRNAs and their target genes associated with clinical outcomes have remained largely unexplored.

Methods

Here, we demonstrate a survival analysis based on the regulatory relationships of miRNAs and their target genes. The patient survivals for the two major cancers, ovarian cancer and glioblastoma multiforme (GBM), are investigated through the integrated analysis of miRNA-mRNA interaction pairs.

Results

We found that there is a larger survival difference between two patient groups with an inversely correlated expression profile of miRNA and mRNA. It supports the idea that signatures of miRNAs and their targets related to cancer progression can be detected via this approach.

Conclusions

This integrated analysis can help to discover coordinated expression signatures of miRNAs and their target mRNAs that can be employed for therapeutics in human cancers.
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8.

Background

Evolutionary novelties often appear by conferring completely new functions to pre-existing structures or by innovating the mechanism through which a particular function is performed. Sound production plays a central role in the behavior of frogs, which use their calls to delimit territories and attract mates. Therefore, frogs have evolved complex vocal structures capable of producing a wide variety of advertising sounds. It is generally acknowledged that most frogs call by moving an air column from the lungs through the glottis with the remarkable exception of the family Pipidae, whose members share a highly specialized sound production mechanism independent of air movement.

Results

Here, we performed behavioral observations in the poorly known African pipid genus Pseudhymenochirus and document that the sound production in this aquatic frog is almost certainly air-driven. However, morphological comparisons revealed an indisputable pipid nature of Pseudhymenochirus larynx. To place this paradoxical pattern into an evolutionary framework, we reconstructed robust molecular phylogenies of pipids based on complete mitochondrial genomes and nine nuclear protein-coding genes that coincided in placing Pseudhymenochirus nested among other pipids.

Conclusions

We conclude that although Pseudhymenochirus probably has evolved a reversal to the ancestral non-pipid condition of air-driven sound production, the mechanism through which it occurs is an evolutionary innovation based on the derived larynx of pipids. This strengthens the idea that evolutionary solutions to functional problems often emerge based on previous structures, and for this reason, innovations largely depend on possibilities and constraints predefined by the particular history of each lineage.
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9.

Introduction

Due to dangers associated with potential accidents from nuclear energy and terrorist threats, there is a need for high-throughput biodosimetry to rapidly assess individual doses of radiation exposure. Lipidomics and metabolomics are becoming common tools for determining global signatures after disease or other physical insult and provide a “snapshot” of potential cellular damage.

Objectives

The current study assesses changes in the nonhuman primate (NHP) serum lipidome and metabolome 7 days following exposure to ionizing radiation (IR).

Methods

Serum sample lipids and metabolites were extracted using a biphasic liquid–liquid extraction and analyzed by ultra performance liquid chromatography quadrupole time-of-flight mass spectrometry. Global radiation signatures were acquired in data-independent mode.

Results

Radiation exposure caused significant perturbations in lipid metabolism, affecting all major lipid species, including free fatty acids, glycerolipids, glycerophospholipids and esterified sterols. In particular, we observed a significant increase in the levels of polyunsaturated fatty acids (PUFA)-containing lipids in the serum of NHPs exposed to 10 Gy radiation, suggesting a primary role played by PUFAs in the physiological response to IR. Metabolomics profiling indicated an increase in the levels of amino acids, carnitine, and purine metabolites in the serum of NHPs exposed to 10 Gy radiation, suggesting perturbations to protein digestion/absorption, biological oxidations, and fatty acid β-oxidation.

Conclusions

This is the first report to determine changes in the global NHP serum lipidome and metabolome following radiation exposure and provides information for developing metabolomic biomarker panels in human-based biodosimetry.
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10.

Introduction

Histologically lung cancer is classified into four major types: adenocarcinoma (Ad), squamous cell carcinoma (SqCC), large cell carcinoma (LCC), and small cell lung cancer (SCLC). Presently, our understanding of cellular metabolism among them is still not clear.

Objectives

The goal of this study was to assess the cellular metabolic profiles across these four types of lung cancer using an untargeted metabolomics approach.

Methods

Six lung cancer cell lines, viz., Ad (A549 and HCC827), SqCC (NCl-H226 and NCl-H520), LCC (NCl-H460), and SCLC (NCl-H526), were analyzed using liquid chromatography quadrupole time-of-flight mass spectrometry, with normal human small airway epithelial cells (SAEC) as the control group. The principal component analysis (PCA) was performed to identify the metabolic signatures that had characteristic alterations in each histological type. Further, a metabolite set enrichment analysis was performed for pathway analysis.

Results

Compared to the SAEC, 31, 27, 34, 34, 32, and 39 differential metabolites mainly in relation to nucleotides, amino acid, and fatty acid metabolism were identified in A549, HCC827, NCl-H226, NCl-H520, NCl-H460, and NCl-H526 cells, respectively. The metabolic signatures allowed the six cancerous cell lines to be clearly separated in a PCA score plot.

Conclusion

The metabolic signatures are unique to each histological type, and appeared to be related to their cell-of-origin and mutation status. The changes are useful for assessing the metabolic characteristics of lung cancer, and offer potential for the establishment of novel diagnostic tools for different origin and oncogenic mutation of lung cancer.
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11.

Background

Cells are open complex thermodynamic systems. They can be also regarded as complex engines that execute a series of chemical reactions. Energy transformations, thermo-electro-chemical processes and transports phenomena can occur across the cells membranes. Moreover, cells can also actively modify their behaviours in relation to changes in their environment.

Methods

Different thermo-electro-biochemical behaviours occur between health and disease states. But, all the living systems waste heat, which is no more than the result of their internal irreversibility. This heat is dissipated into the environment. But, this wasted heat represent also a sort of information, which outflows from the cell toward its environment, completely accessible to any observer.

Results

The analysis of irreversibility related to this wasted heat can represent a new approach to study the behaviour of the cells themselves and to control their behaviours. So, this approach allows us to consider the living systems as black boxes and analyze only the inflows and outflows and their changes in relation to the modification of the environment. Therefore, information on the systems can be obtained by analyzing the changes in the cell heat wasted in relation to external perturbations.

Conclusions

The bioengineering thermodynamics bases are summarized and used to analyse possible controls of the calls behaviours based on the control of the ions fluxes across the cells membranes.
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12.

Background

Human cancers are complex ecosystems composed of cells with distinct molecular signatures. Such intratumoral heterogeneity poses a major challenge to cancer diagnosis and treatment. Recent advancements of single-cell techniques such as scRNA-seq have brought unprecedented insights into cellular heterogeneity. Subsequently, a challenging computational problem is to cluster high dimensional noisy datasets with substantially fewer cells than the number of genes.

Methods

In this paper, we introduced a consensus clustering framework conCluster, for cancer subtype identification from single-cell RNA-seq data. Using an ensemble strategy, conCluster fuses multiple basic partitions to consensus clusters.

Results

Applied to real cancer scRNA-seq datasets, conCluster can more accurately detect cancer subtypes than the widely used scRNA-seq clustering methods. Further, we conducted co-expression network analysis for the identified melanoma subtypes.

Conclusions

Our analysis demonstrates that these subtypes exhibit distinct gene co-expression networks and significant gene sets with different functional enrichment.
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13.

Introduction

Immediate responses towards emotional utterances in humans are determined by the acoustic structure and perceived relevance, i.e. salience, of the stimuli, and are controlled via a central feedback taking into account acoustic pre-experience. The present study explores whether the evaluation of stimulus salience in the acoustic communication of emotions is specifically human or has precursors in mammals. We created different pre-experiences by habituating bats (Megaderma lyra) to stimuli based on aggression, and response, calls from high or low intensity level agonistic interactions, respectively. Then we presented a test stimulus of opposite affect intensity of the same call type. We compared the modulation of response behaviour by affect intensity between the reciprocal experiments.

Results

For aggression call stimuli, the bats responded to the dishabituation stimuli independent of affect intensity, emphasising the attention-grabbing function of this call type. For response call stimuli, the bats responded to a high affect intensity test stimulus after experiencing stimuli of low affect intensity, but transferred habituation to a low affect intensity test stimulus after experiencing stimuli of high affect intensity. This transfer of habituation was not due to over-habituation as the bats responded to a frequency-shifted control stimulus. A direct comparison confirmed the asymmetric response behaviour in the reciprocal experiments.

Conclusions

Thus, the present study provides not only evidence for a discrimination of affect intensity, but also for an evaluation of stimulus salience, suggesting that basic assessment mechanisms involved in the perception of emotion are an ancestral trait in mammals.
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14.

Background

The development of ethologically meaningful test paradigms in young animals is an essential step in the study of the ontogeny of animal personality. Here we explore the possibility to integrate offspring separation (distress) calls into the study of consistent individual differences in behaviour in two species of mammals, the domestic cat (Felis silvestris catus) and the mound-building mouse (Mus spicilegus). Such vocal responses in young mammals are a potentially useful test option as they represent an important element of mother-offspring communication with strong implications for offspring survival. In addition, the neural control of vocalisation is closely associated with emotional state.

Results

We found marked similarities in the pattern of individual responses of the young of both species to separation from their mother and littermates. In the domestic cat as well as in the mound-building mouse, individual differences in the frequency of calls and to a lesser extent in locomotor activity were repeatable across age, indicating the existence of personality types. Such consistencies across age were also apparent when only considering relative individual differences among litter siblings. In both species, however, individual patterns of vocalisation and locomotor activity were unrelated. This suggests that these two forms of behavioural responses to isolation represent different domains of personality, presumably based on different underlying neurophysiological mechanisms.

Conclusions

Brief separation experiments in young mammals, and particularly the measurement of separation calls, provide a promising approach to study the ontogeny of personality traits. Future long-term studies are needed to investigate the association of these traits with biologically meaningful and potentially repeatable elements of behaviour during later life.
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15.

Introduction

Stable isotopic labeling experiments are powerful tools to study metabolic pathways, to follow tracers and fluxes in biotic and abiotic transformations and to elucidate molecules involved in metal complexing.

Objective

To introduce a software tool for the identification of isotopologues from mass spectrometry data.

Methods

DeltaMS relies on XCMS peak detection and X13CMS isotopologue grouping and then analyses data for specific isotope ratios and the relative error of these ratios. It provides pipelines for recognition of isotope patterns in three experiment types commonly used in isotopic labeling studies: (1) search for isotope signatures with a specific mass shift and intensity ratio in one sample set, (2) analyze two sample sets for a specific mass shift and, optionally, the isotope ratio, whereby one sample set is isotope-labeled, and one is not, (3) analyze isotope-guided perturbation experiments with a setup described in X13CMS.

Results

To illustrate the versatility of DeltaMS, we analyze data sets from case-studies that commonly pose challenges in evaluation of natural isotopes or isotopic signatures in labeling experiment. In these examples, the untargeted detection of sulfur, bromine and artificial metal isotopic patterns is enabled by the automated search for specific isotopes or isotope signatures.

Conclusion

DeltaMS provides a platform for the identification of (pre-defined) isotopologues in MS data from single samples or comparative metabolomics data sets.

Graphical Abstract

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16.
17.

Background and aims

Biocrust morphology is often used to infer ecological function, but morphologies vary widely in pigmentation and thickness. Little is known about the links between biocrust morphology and the composition of constituent microbial community. This study aimed to examine these links using dryland crusts varying in stage and morphology.

Methods

We compared the microbial composition of three biocrust developmental stages (Early, Mid, Late) with bare soil (Bare) using high Miseq Illumina sequencing. We used standard diversity measures and network analysis to explore how microbe-microbe associations changed with biocrust stage.

Results

Biocrust richness and diversity increased with increasing stage, and there were marked differences in the microbial signatures among stages. Bare and Late stages were dominated by Alphaproteobacteria, but Cyanobacteria was the dominant phylum in Early and Mid stages. The greatest differences in microbial taxa were between Bare and Late stages. Network analysis indicated highly-connected hubs indicative of small networks.

Conclusions

Our results indicate that readily discernible biocrust features may be good indicators of microbial composition and structure. These findings are important for land managers seeking to use biocrusts as indicators of ecosystem health and function. Treating biocrusts as a single unit without considering crust stage is likely to provide misleading information on their functional roles.
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18.

Background

Understanding the role of avian vocal communication in social organisation requires knowledge of the vocal repertoire used to convey information. Parrots use acoustic signals in a variety of social contexts, but no studies have evaluated cross-functional use of acoustic signals by parrots, or whether these conform to signal design rules for different behavioural contexts. We statistically characterised the vocal repertoire of 61 free-living Lilac-crowned Amazons (Amazona finschi) in nine behavioural contexts (nesting, threat, alarm, foraging, perched, take-off, flight, landing, and food soliciting). We aimed to determine whether parrots demonstrated contextual flexibility in their vocal repertoire, and whether these acoustic signals follow design rules that could maximise communication.

Results

The Lilac-crowned Amazon had a diverse vocal repertoire of 101 note-types emitted at least twice, 58 of which were emitted ≥5 times. Threat and nesting contexts had the greatest variety and proportion of exclusive note-types, although the most common note-types were emitted in all behavioural contexts but with differing proportional contribution. Behavioural context significantly explained variation in acoustic features, where threat and nesting contexts had the highest mean frequencies and broad bandwidths, and alarm signals had a high emission rate of 3.6 notes/s. Three Principal Components explained 72.03 % of the variation in temporal and spectral characteristics of notes. Permutated Discriminant Function Analysis using these Principal Components demonstrated that 28 note-types (emitted by >1 individual) could be correctly classified and significantly discriminated from a random model.

Conclusions

Acoustic features of Lilac-crowned Amazon vocalisations in specific behavioural contexts conformed to signal design rules. Lilac-crowned Amazons modified the emission rate and proportional contribution of note-types used in each context, suggesting the use of graded and combinatorial variation to encode information. We propose that evaluation of vocal repertoires based on note-types would reflect the true extent of a species’ vocal flexibility, and the potential for combinatorial structures in parrot acoustic signals.
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19.

Introduction

Previous metabolomics studies have revealed perturbed metabolic signatures in esophageal squamous cell carcinoma (ESCC) patients, however, most of these studies included mainly late-staged ESCC patients due to the difficulties of collecting the early-staged samples from asymptotic ESCC subjects.

Objectives

This study aims to explore the early-staged ESCC metabolic signatures and potential of serum metabolomics to diagnose ESCC at early stages.

Methods

Serum samples of 97 ESCC patients (stage 0, 39 cases; stage I, 17 cases; stage II, 11 cases, stage III, 30 cases) and 105 healthy controls (HC) were enrolled and randomly separated into training data (77 ESCCs, 84 HCs) and validation data (20 ESCCs, 21 HCs). Untargeted metabolomics was performed to identify ESCC-related metabolic signatures.

Results

The global metabolomics profiles could clearly distinguish ESCC from HC in training data. 16 ascertained metabolites were found to be disturbed in the metabolic pathways characterized by dysregulated fatty acid biosynthesis, glycerophospholipid metabolism, choline metabolism in cancer and linoleic acid metabolism. The AUC value in validation data was 0.895, with sensitivity 85.0 % and specificity 90.5 %. Good diagnostic performances were also achieved for early stage ESCC, with the values of area under the curve (AUC) 0.881 for the ESCC patients in both stage 0 and I–II. In addition, six metabolites were found to discriminate ESCC stages. Among them, three biomarkers, dodecanoic acid, LysoPA(18:1), and LysoPC(14:0), exhibited clear trend for ESCC progression.

Conclusion

These findings suggest serum metabolomics, performed in a minimally noninvasive and convenient manner, may possess great potential for early diagnosis of ESCC patients.
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20.

Background

This study is mainly motivated by the need of understanding how the diffusion behavior of a biomolecule (or even of a larger object) is affected by other moving macromolecules, organelles, and so on, inside a living cell, whence the possibility of understanding whether or not a randomly walking biomolecule is also subject to a long-range force field driving it to its target.

Method

By means of the Continuous Time Random Walk (CTRW) technique the topic of random walk in random environment is here considered in the case of a passively diffusing particle among randomly moving and interacting obstacles.

Results

The relevant physical quantity which is worked out is the diffusion coefficient of the passive tracer which is computed as a function of the average inter-obstacles distance.

Conclusions

The results reported here suggest that if a biomolecule, let us call it a test molecule, moves towards its target in the presence of other independently interacting molecules, its motion can be considerably slowed down.
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