In addition to dopamine neuron firing, cholinergic interneurons (ChIs) regulate dopamine release in the striatum via presynaptic nicotinic receptors (nAChRs) on dopamine axon terminals. Synchronous activity of ChIs is necessary to evoke dopamine release through this pathway. The frequency-dependence of disynaptic nicotinic modulation has led to the hypothesis that nAChRs act as a high-pass filter in the dopaminergic microcircuit. Here, we used optogenetics to selectively stimulate either ChIs or dopamine terminals directly in the striatum. To measure the functional consequence of dopamine release, D2-receptor synaptic activity was assessed via virally overexpressed potassium channels (GIRK2) in medium spiny neurons (MSNs). We found that nicotinic-mediated dopamine release was blunted at higher frequencies because nAChRs exhibit prolonged desensitization after a single pulse of synchronous ChI activity. However, when dopamine neurons alone were stimulated, nAChRs had no effect at any frequency. We further assessed how opioid receptors modulate these two mechanisms of release. Bath application of the κ opioid receptor agonist U69593 decreased D2-receptor activation through both pathways, whereas the μ opioid receptor agonist DAMGO decreased D2-receptor activity only as a result of cholinergic-mediated dopamine release. Thus the release of dopamine can be independently modulated when driven by either dopamine neurons or cholinergic interneurons. Striatal dopamine is a critical component of basal ganglia dependent movement and motivational behaviors1256785Studies using local electrical stimulation of the striatum have shown that inhibiting nicotinic receptors modulates the release of dopamine to a greater extent at low frequencies of stimulation61212415To test the role of nicotinic receptors in frequency-dependent dopamine release onto D2-receptors in MSNs, we used optogenetics to separate the contribution of cholinergic and dopaminergic dopamine release while recording D2-receptor mediated inhibitory postsynaptic currents (D2-IPSCs). Overexpressed G-coupled inwardly rectifying potassium (GIRK) channels provided an electrophysiological sensor of D2-receptor activation in striatal MSNsAll animal procedures and protocols were approved by the Institutional Animal Care and Use Committee at Case Western Reserve University . All experiments were performed in accordance with the appropriate guidelines of the Institutional Animal Care and Use Committee at Case Western Reserve University.Wild-type C57BL6, ChAT-internal ribosome entry site-Cre heterozygote (ChAT-Cre), or DAT-Cre transgenic heterozygote mice were injected at post-natal day 21. All animals were obtained from the Jackson Laboratory . Sterotaxic surgeries were performed using a model 1900 stereotax (Kopf). Sterotaxic surgeries were performed under isoflurane anesthesia. Briefly, a small craniotomy was made using a 33-gauge drill bit above the desired coordinate. A small pulled glass pipette containing AAV was attached to a Nanoject II (Drummond) and was then inserted to the appropriate depth. Injections were performed at a rate of 90 nl/minute. The coordinates used for striatal injections were (relative to bregma): AP +1.15 mm, ML −1.85 mm, DV −3.325 mm. A volume of 300 nL of AAV9.hSyn.tdTomato.T2A.mGIRK2-1-A22A.WPRE.bGH was injected into one hemisphere of all mice. To optogenetically activate ChIs, 300 nL of AAV5.EF1a.DIO.hChR2(H134R)-EYFP.WPRE.hGH was combined with the 300 nL of AVV.GIRK2 and injected at the same time in ChAT-Cre mice. To optogenetically activate dopamine terminals, 500 nL of AAV5.EF1a.DIO.hChR2(H134R)-EYFP.WPRE.hGH was separately injected into one side of the midbrain of DAT-Cre mice. Midbrain coordinates for the SNc/VTA (relative to bregma) were: AP −2.3 mm, ML −0.55 mm, DV −4.7 mm. All AAVs were obtained from the University of Pennsylvania Viral Core. Animals were allowed to recover for ~2–4 weeks to allow for viral expression.2, 0.1 CaCl2, 1.2 NaH2PO4, 25 NaHCO3, 2.5 D-glucose, and 50 sucrose; bubbled with 95% O2 and 5% CO2. Slices were incubated at 35 °C for at least 45 minutes in ACSF containing (in mM): 126 NaCl, 2.5 KCl, 1.2 MgCl2, 2.4 CaCl2, 1.2 NaH2PO4, 21.4 NaHCO3, and 11.1 D-glucose; and 10 μM MK-801 bubbled with 95% O2 and 5% CO2. Slices were transferred to a recording chamber and perfused with warm ACSF (34 ± 2 °C) at 2 mL/minute. Perfusion solution contained picrotoxin (100 μM), DNQX (10 μM), CGP 55845 (300 nM), SCH 23390 hydrochloride (1 μM), and scopolamine (500 nM) to block GABAA, AMPA, GABAB, dopamine D1 and muscarinic receptors respectively. MSNs were visualized using a BXWI51 scope (Olympus) with custom-built gradient contrast optics using a near-IR LED (Thor Labs).Coronal slices (240 μM) containing the striatum were made in ice-cold cutting solution containing (in mM): 75 NaCl, 2.5 KCl, 6 MgCl2, 10 HEPES(K), 10 BAPTA-tetrapotassium. Patch pipettes for ChIs contained (in mM): 135 D-gluconate(K), 10 HEPES(K), 0.1 CaCl2, 2 MgCl2, 0.1 EGTA. All internal patch solution contained: 1 mg/mL ATP, 0.1 mg/mL GTP, and 1.5 mg/mL phosphocreatine . Membrane potential was held at −60 mV during voltage clamp recordings. Cells were discarded if their series resistance exceeded 15 MΩ, as series resistance was not compensated. ChIs were identified by the presence of an h-current when stepped to −90 mV. Data was acquired with Axograph X (Axograph Scientific) at 5 kHz. Voltage clamp recordings were filtered to 2 kHz. Electrical stimulation (0.7 ms) was applied with a monopolar extracellular stimulating electrode filled with ACSF (World Precision Instruments). Optogenetic stimulation was evoked with 2 ms widefield illumination (470 nm) using a custom made LED . All drugs were applied via bath application unless otherwise specified. All recordings were made in the dorsal striatum.Whole-cell recordings were performed using either Axopatch 200 A or Axopatch 200B amplifiers (Molecular Devices). Membrane potentials were not corrected for liquid junction potentials. Patch pipettes (1.5–2 MΩ) were made from borosciliate glass capillary tubes (World Precision Instruments). Patch pipettes for MSNs contained (in mM): 115 K-methylsulfate, 20 NaCl, 1.5 MgClDNQX, picrotoxin, MK-801, and naloxone were obtained from Ascent Scientific. SCH 23390, CGP 55845, scopolamine hydrobromide, ambenonium, dihydro-b-erythroidine hydrobromide (DHβE), mecamylamine, sulpiride, and DAMGO were obtained from Tocris Bioscience. K-methylsulfate was from Acros Organic. BAPTA was from Invitrogen. All other chemicals were from Sigma-Aldrich.Data are shown as mean ± SEM. Statistical significance was assessed using either Wilcoxon matched-pairs signed rank test, Mann-Whitney test, or one-way ANOVA where appropriate .+ (GIRK2) channels in striatal MSNs1717A, GABAB AMPA, NMDA, dopamine D1, and muscarinic receptors. Td-Tomato+ MSNs were voltage-clamped at a holding potential of −60 mV. As about half of striatal MSNs belong to the D2-receptor expressing indirect pathway+ MSNsTo examine D2-receptor activation in response to nicotinic receptor activity, we overexpressed G-protein coupled inwardly rectifying K+ MSNs that was attenuated by the α4β2 nicotinic receptor subtype antagonist DHβE   in indirect pathway GIRK2ilcoxon) . Saturatilcoxon) . As α7 s+ MSNs  into either the substantia nigra (SNc) of DAT-Cre mice or into the striatum of ChAT-IRES-Cre mice. Photostimulation of either dopamine terminals (DAT-Cre:ChR2) or ChIs (ChAT-Cre:ChR2) with a single flash of blue light  was sufficient to evoke D2-IPSCs in indirect pathway GIRK2+ MSNs . In both+ MSNs  and tetr+ MSNs  but did + MSNs . Thus, w+-expressing MSNs (voltage clamp). ChIs were hyperpolarized to just below threshold during these recordings to prevent tonic firing. To increase the resolvable amplitude of paired IPSCs, dopamine transporters were inhibited with the dopamine transport blocker cocaine (10 μM). While electrical stimulation evoked robust D2-IPSCs, single action potentials in ChIs failed to elicit D2-IPSCs  and GIRK2D2-IPSCs . Single Past work has demonstrated that nicotinic receptors provide stronger modulation of dopamine release during low frequency electrical stimulationIn vivo, ChIs and dopamine neurons fire in opposing patterns during reward-learning tasks15222324To determine the kinetics of nAChR desensitization, we next examined the time course of recovery of D2-IPSCs. Paired pulse experiments revealed that ChAT-Cre:ChR2 evoked IPSCs took significantly longer to recover than DAT-Cre:ChR2 evoked IPSCs . As D2-IMu, kappa and delta opioid receptors are expressed in the striatum where they regulate transmission through multiple circuits27283129333436As dopamine release in the striatum is critical for motivated behaviors and action selection, modulation of this release is an important regulator of striatal circuitry. Independent of dopamine neuron impulse activity, nicotinic receptors on striatal dopamine terminals also facilitate dopamine release5in vivo. We found that, similar to the electrochemical measurements of Zhang and Sulzer (2004), nicotinic receptors more strongly modulated dopamine release at low frequency electrical stimulation. However, by segregating these two pathways using optogenetics, we found that nicotinic receptors only act as high pass filters when both pathways are activated simultaneously. Selective stimulation of only dopamine terminals by activating ChR2 expressed in SNc axons of DAT-Cre mice revealed that D2-IPSCs exhibited no change in nicotinic receptor-mediated facilitation at any frequency examined. Thus, under conditions when dopamine neurons and ChIs are not synchronously active, nicotinic receptors failed to modulate the release of dopamine that underlies the activation of D2-receptors.Dopamine neurons typically fire at tonic low frequencies, and their firing switches to a bursting pattern in response to rewards and their cues12111222High-affinity α4β2 nicotinic receptors on midbrain dopamine neurons rapidly desensitize in response to low concentrations of ACh or nicotine3940416395As DAT-Cre:ChR2 evoked D2-IPSCs facilitated during high frequency optogenetic stimulation and recovered from paired-pulse depression at a faster rate than ChAT-Cre:ChR2 evoked IPSCs, our results reveal a ChI-mediated mechanism of depression rather than dopamine vesicle depletion or D2-receptor desensitization. Similar to the observed recovery of ChAT-Cre:ChR2 evoked IPSCs, human α4β2 nAChRs expressed in heterologous systems can take ~20 seconds to recover their activity to normal levels in response to ACh and nicotinein vivo and in the absence of synaptic inputs442+ in axon terminals compared with AP triggered release that has been shown to alter probability of release at other synapsesChIs are tonically active both ACh tone may not affect dopamine release because acetylcholinesterase (AChE), the degradative enzyme of ACh, is enriched in the striatum and highly efficient, effectively terminating ACh signaling before it can activate striatal nicotinic receptorsWithin the striatum, mu, kappa and delta opioid receptors are widespread inhibitory modulators of striatal circuitry . Enkepha419in vivo microdialysis studies that have shown that nicotinic receptor antagonism specifically in the striatum causes a small decrease in dopamine concentration over timeTogether, this work suggests that the role of the burst-firing pattern of ChIs common following reward stimuli is to synchronize firing, allowing for transient, phasic dopamine release, rather than to alter gain of dopamine release in the striatum. This parallels How to cite this article: Mamaligas, A. A. et al. Nicotinic and opioid receptor regulation of striatal dopamine D2-receptor mediated transmission. Sci. Rep.6, 37834; doi: 10.1038/srep37834 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Levodopa is the most effective medication for motor symptoms in Parkinson's disease. However, various motor and non-motor complications are associated with levodopa treatment, resulting from altered levodopa-dopamine metabolism with disease progression and long-term use of the drug. The present review emphasizes the role of monoamine transporters other than the dopamine transporter in uptake of extracellular dopamine in the dopamine-denervated striatum. When dopaminergic neurons are lost and dopamine transporters decreased, serotonin and norepinephrine transporters compensate by increasing uptake of excessive extracellular dopamine in the striatum. Organic cation transporter-3 and plasma membrane monoamine transporter, low affinity, and high capacity transporters, also potentially uptake dopamine when high-affinity transporters do not work normally. Selective serotonin reuptake inhibitors and serotonin norepinephrine reuptake inhibitors are often administered to patients with Parkinson's disease presenting with depression, pain or other non-motor symptoms. Thus, it is important to address the potential of these drugs to modify dopamine metabolism and uptake through blockade of the compensatory function of these transporters, which could lead to changes in motor symptoms of Parkinson's disease. Parkinson's disease (PD) is a neurodegenerative disease associated with progressive loss of nigrostriatal dopaminergic neurons. A patient with PD presents motor symptoms such as tremor, akinesia, hypokinesia, rigidity, and postural disturbance, and non-motor symptoms such as pain, sleep disturbance, apathy, depression, and constipation -A, MAO-B, aldehyde dehydrogenase (ALD.DH), and COMT into homovanillic acid , and plasma membrane monoamine transporter (PMAT) in the striatum, which can also reuptake extracellular dopamine and are gaining increasing attention.SERT has a high affinity for serotonin uptake and the majority of these transporters exist on the axonal plasma membranes of serotonin fibers  6A family. They are the primary targets for drugs used to treat depression and other psychiatric disorders and may transport some neurotoxins in patients with drug addiction and animal models of the diseases  family of transporters. Decynium-22 (D-22) can block OCT. OCT-1 and OCT-2 are mainly expressed in the liver and kidney, respectively  treatment. The same mouse model also shows elevated extracellular dopamine concentration after high-dose methamphetamine treatment, but not after low-dose treatment (Cui et al., These studies suggest OCT-3 plays a role in the regulation of dopamine concentration in the parkinsonian striatum and could be a target for PD treatment.PMAT is a member of the SLC 29, equilibrative nucleoside transporter family. PMAT is also one of the D-22 sensitive transporters, which can be blocked by D-22, similar to OCT-3. In mice, PMAT is expressed exclusively in neurons, not in astrocytes (Dahlin et al., As mentioned in the preceding subsection, Sader-Mazbar et al.  demonstrWe reviewed levodopa-dopamine metabolism and pathways in which extracellular dopamine is reuptaken in the dopamine-denervated striatum. Most of the studies addressed were performed using rodent models of PD induced with neurotoxin. Further studies are needed to expand the discussion in this review to higher species.To date, various drugs that modify dopamine metabolism and uptake, especially monoamine transporter inhibitors, have been investigated for treatment of PD motor and non-motor symptoms. Since the recent comprehensive review by Huot et al. , new stuHN: reviewing the concept, compiling sources, interpreting the data, and writing the manuscript. MT: reviewing the concept, compiling sources, reviewing and critiquing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Both cultivars were annotated using the same method to allow comparison of gene content. We identified genes unique to each cultivar and differentiate these from artefacts due to variation in the assembly and annotation. We demonstrate that using a common annotation pipeline can result in different gene predictions, even for closely related cultivars, and repeat regions which collapse during assembly impact whole genome comparison. After accounting for differences in assembly and annotation, we demonstrate that the genome of Darmor‐bzh contains a greater number of genes than the genome of Tapidor. Our results are the first step towards comparison of the true differences between B. napus genomes and highlight the potential sources of error in future production of a B. napus pangenome.As an increasing number of plant genome sequences become available, it is clear that gene content varies between individuals, and the challenge arises to predict the gene content of a species. However, genome comparison is often confounded by variation in assembly and annotation. Differentiating between true gene absence and variation in assembly or annotation is essential for the accurate identification of conserved and variable genes in a species. Here, we present the  Brassica is a genus which contains economically important crop species, including condiments such as mustards; vegetable crops including broccoli, Brussels sprouts, cabbage, cauliflower; and commercially important oilseeds which are used as both a food and biofuel. The genomes of the Brassica family are related as described in the triangle of U , B. nigra , and B. oleracea , and these genomes evolved from a common ancestor which underwent at least one whole genome triplication , B. carinata  and B. napus . Several Brassica genome assemblies have now been published, including the A genome of the B. rapa cultivar Chiifu . Variable genes can be split into two groups: copy number variations (CNVs), in which the number of copies of a gene differs between individuals, and the presence/absence variations (PAVs), an extreme form of CNV in which a gene is present in some individuals but absent in others  revealed around 1224 unique genes in each of the three genomes , of which nine were shared between the genomes.Gene presence/absence variation is important for crop improvement as some variable genes have been shown to be associated with agronomic traits. In rice, comparison of three divergent lines led to the identification of several variable genes including the submergence tolerance gene et al., et al., et al.  (Table The SkimGBS pipeline (Bayer %) Table .B. napus cultivar Tapidor was assembled de novo using paired end and mate paired Illumina sequencing data with a range of insert sizes. The sequence data were first cleaned prior to assembly. From 417 527 199 Tapidor read pairs, 54 030 058 (12.9%) were identified as clonal and removed. Quality control using sickle  unplaced contigs. Using this Tapidor assembly, the SkimGBS pipeline predicted 945 406 SNPs and called 35 829 337 genotypes for the Tapidor × Ningyou‐7 population. ContigPlacer then placed an additional 452 Tapidor contigs into pseudomolecules, bringing the total size of the Tapidor pseudomolecules to 625.9 Mbp, with 2741 contigs (8.2 Mbp) remaining unplaced, none of which contained SNPs  repeats in Darmor‐bzh and 223 636 559 bp (35.15%) repeats in Tapidor. LTR/Gypsy was the most common repetitive element in both assemblies, with 97 597 copies in Darmor‐bzh and 92 747 copies in Tapidor , or carrying transposase‐related PFAM domains were removed.‐bzh , and 70 162 predicted genes for Tapidor   located on pseudomolecules had sequence identity with Tapidor genes. Of these, 65 280 were located in collinear blocks of at least five genes in the respective Tapidor pseudomolecule. Of the 11 688 genes with no partners in collinear blocks 2458 matched sequence on the expected chromosome, of which 701 matched the expected chromosomal region. In Tapidor, 69 372 of 69 531 genes (99.7%) showed sequence identity with Darmor‐bzh genes, with 59 099 genes in collinear blocks of at least five genes. Of the 10 903 genes with no partners in collinear blocks, 2103 matched with the expected chromosome, of which 524 matched within the expected range.We compared the gene content between assemblies across each pair of homologous chromosomes. In Darmor‐bzh, while 73 Darmor‐bzh genes were predicted to be absent in Tapidor  showed no expression in Darmor‐bzh or Tapidor, 19 (26%) were expressed in Darmor‐bzh but neither Tapidor nor Ningyou‐7, 27 (37%) were expressed in Darmor‐bzh and Ningyou‐7 but not in Tapidor, and expression segregated in the Tapidor × Ningyou‐7 DH population, 2 (2.7%) were expressed in Tapidor and Ningyou‐7 but not in Darmor‐bzh, while 4 (5.5%) were expressed in all tissues in cases  were located in collapsed regions and 5703 (7.1%) were located in repetitive regions, while in Tapidor, 2651 genes (3.8%) were located in collapsed regions and 246 (0.4%) were located in repetitive regions . In Darmor‐bzh, the chromosomes A3 and C3 carried the largest number of repetitive genes  while chromosomes A1 and C1 carried the most repetitive genes in Tapidor .Genes were identified in both the collapsed and repetitive regions of the Darmorns Table . In Tapi‐bzh and Tapidor in order to identify any common patterns that may lead to problems in genome assemblies. The largest number of shared Pfam domains and shared Swiss‐Prot hits was between genes located in repetitive regions in Darmor‐bzh and genes located in collapsed regions in Tapidor. In repetitive genes in Darmor‐bzh, 10% of Pfam domains overlapped with collapsed genes in Tapidor and Pfam domains collapsed in Darmor‐bzh, while only 0.5% of genes located in collapsed and repetitive regions in Tapidor shared Pfam domains   Table . A simils Figure . As withe’ Table .Brassica napus canola is an important global crop and a major source of vegetable oil for human consumption. While genetic markers have been used for crop improvement for several years  and Tapidor (1335 Mbp). In contrast, the predicted assembly size was similar to, although smaller than the actual assembly size with an estimated 808 Mbp for Darmor‐bzh and 697 Mbp for Tapidor. The predicted genome size estimate accounts for coverage, sequencing depth and unique k‐mers, while the genome assembly estimates account only for unique k‐mers. The Tapidor sequence data contains fewer unique k‐mers than the Darmor‐bzh sequencing data, which suggests that the Tapidor genome is actually smaller than the Darmor‐bzh genome. Both estimated genome sizes here are larger than the flow‐cytometry based estimations for different B. napus (AACC) cultivars of 1129–1235 Mbp  compared with an assembly size of 850 Mbp for Darmorzh Table . Using k‐bzh assembly having more gene copies than Tapidor. This suggests that some gene copies have collapsed in the Tapidor assembly which have not collapsed in the Darmor‐bzh assembly. The increased collapsing of regions in the Tapidor assembly compared to the Darmor‐bzh assembly is probably due to the use of long read 454 and Sanger sequence data in the production of the Darmor‐bzh assembly.While there appears to be a real difference in the genome size of these two homozygous individuals, we hypothesize that the different assembly methods cause the majority of observed differences between the assemblies. The CEGMA results suggest that both assemblies contain the majority of the gene content and that both assemblies are of high quality. Both assemblies contain all core eukaryotic genes (CEGs), although two CEGs were only partially present in Tapidor, which suggests that this assembly may be more fragmented. Similarly, the BUSCO results show that both assemblies carry the majority of gene content, with the Darmor‐bzh is lower in our study than the 101 040 gene models originally predicted by Chalhoub et al.  and in Tapidor 10 903 genes (15.5%) were not in collinear blocks and were not identified in their expected regions, suggesting that they are either misplaced in the assembly or are nonsyntenic in the genome. Wicker et al.  were expressed in Darmor‐bzh and Ningyou‐7 but not in Tapidor, with clear segregation in the Tapidor × Ningyou‐7 DH population, suggesting that these genes have been truly lost in Tapidor but are present in Ningyou‐7  predicted to be absent in Tapidor and present in Darmor‐bzh appear to be expressed in Tapidor but not in Darmor‐bzh, and four genes (5.5%) appear to be expressed in all cultivars. These results may be due to the more relaxed mapping parameters for the single RNASeq reads compared to the paired genomic sequence reads, leading to erroneous read mapping and false calling of gene presence in the RNASeq analysis. Genes which showed no expression in any of the cultivars assessed may be pseudogenes, misannotated or only expressed in certain conditions or tissues which were not sampled during RNA‐Seq library preparation , and chromosome C2 contains several breeding related QTLs such as seed yield, seed weight and heterosis  are frequently observed in polyploid species, and the number of genes located in repetitive and collapsed regions can be used for the detection of HE between the A and the C genome . MSTMap  together with Repbase v were used to mask repeats. Gene models were produced using Tophat v2.1.0 and cufflinks v2.2.0  reads were aligned using bowtie2   identified collapsed and repetitive regions with a minimum block size of 50 bp. R v3.2 . The Tapidor × Ningyou‐7 DH data are available at http://www.ncbi.nlm.nih.gov/bioproject/PRJNA274890All raw sequence reads for the cultivar Tapidor used for assembly have been submitted to NCBI BioProject  and the Tapidor assembly (x‐axis) pseudomolecules.Click here for additional data file.Figure S2 Comparison of ranks between 19 linkage maps from MSTMap and physical placement using the Darmor‐bzh genome assembly as reference.Click here for additional data file.Figure S3 Number of shared Swiss‐Prot hits between genes located in collapsed and repetitive regions between Darmor‐bzh and Tapidor.Click here for additional data file.Table S1 Overlap between collections of previously unplaced contigs and contigs placed in the improved Darmor assembly.Table S2 Repeats in Darmor and Tapidor.Table S3 Gene expression values for 73 genes present in Darmor and predicted to be absent in Tapidor using RNA‐Seq data from Darmor, Tapidor, Ningyou‐7 and the Tapidor x Ningyou‐7 population.Table S4 Gene expression values for 3 genes present in Tapidor and predicted to be absent in Darmor using RNA‐Seq data from Darmor, Tapidor, Ningyou‐7 and the Tapidor x Ningyou‐7 population.Table S5 TopGO enriched terms for genes absent in Tapidor but present in Darmor.Table S6 Number and total sizes of collapsed regions in Darmor and Tapidor.Table S7 Number and total sizes of repetitive regions in Darmor and Tapidor.Table S8 Number of genes in predicted repetitive and collapsed regions in Darmor and Tapidor.Table S9 Shared domains between Darmor genes located in repetitive regions and Tapidor genes located in collapsed regions (only ten most common domains).Table S10 Ten most common Swiss‐Prot hits for repetitive genes in Darmor and collapsed genes in Tapidor with an e‐value cutoff of 1e‐5.Click here for additional data file.


Coleoid cephalopod molluscs comprise squid, cuttlefish and octopuses, and represent nearly the entire diversity of modern cephalopods. Sophisticated adaptations such as the use of colour for camouflage and communication, jet propulsion and the ink sac highlight the unique nature of the group. Despite these striking adaptations, there are clear parallels in ecology between coleoids and bony fishes. The coleoid fossil record is limited, however, hindering confident analysis of the tempo and pattern of their evolution. Here we use a molecular dataset  of 26 cephalopod species to explore the phylogeny and timing of cephalopod evolution. We show that crown cephalopods diverged in the Silurian–Devonian, while crown coleoids had origins in the latest Palaeozoic. While the deep-sea vampire squid and dumbo octopuses have ancient origins extending to the Early Mesozoic Era, 242 ± 38 Ma, incirrate octopuses and the decabrachian coleoids (10-armed squid) diversified in the Jurassic Period. These divergence estimates highlight the modern diversity of coleoid cephalopods emerging in the Mesozoic Marine Revolution, a period that also witnessed the radiation of most ray-finned fish groups in addition to several other marine vertebrates. This suggests that that the origin of modern cephalopod biodiversity was contingent on ecological competition with marine vertebrates. ChariAnatomical evolution is in part shaped by the ecological relationships between predator– and prey species. Cephalopods (and in particular oceanic squid) fill a niche that largely overlaps with fishes as active mesopredators . ConsideBy contrast, the fossil record leaves limited insight on the providence of modern coleoid groups , despiteShimanskya [Pohlsepia [The first molecular divergence times of cephalopod evolution recovered very ancient divergences for the coleoids , suggestimanskya  and Pohlohlsepia , for whiohlsepia . Subsequohlsepia ,21. Thesohlsepia ,23 and sohlsepia  in the Eohlsepia . These oohlsepia  coincideohlsepia . The jawohlsepia . The coiTo explore the tempo and mode of coleoid evolution, we assembled a dataset of 180 nuclear genes of consistent rate of molecular evolution, representing crown diversity across Coleoidea. Phylogenetic and molecular divergence time analyses were carried out in a Bayesian framework, applying a molecular evolution model accommodating rate and compositional heterogeneity.2.hyloBayes MPI v. 1.5a [Γ. The maximum-likelihood software RAxML MPI v. 8.1.15 [Γ.For full details of experimental procedures, see the electronic supplementary material. We compiled a supermatrix with data from 56 species  for 180 genes. Phylogeny was inferred from this superalignment using the software package P v. 1.5a  under CA. 8.1.15  was applhyloBayes 3.3f was used to infer molecular divergence times under the CIR [hyloBayes MPI v. 1.5a. A prior was applied to the root of 565 ± 10 Ma, representing the root of lophotrochozoa. Eleven fossil calibration points were applied to the analysis, as shown in table .P the CIR  clock mo3.Nautilus as sister group to coleoids [Spirula), in agreement with morphology and previous molecular studies [Vampyroteuthis and the cirroctopod Grimpoteuthis represent cirrate octopuses, branching deep as successive sister groups to the incirrate octopuses  of West Greenland [The oldest unequivocal crown group coleoids appear in the latest Triassic, with belemnites representing stem group decabrachians, and phragmoteuthidids (Early Triassic or latest Permian) proposed to represent stem group Octobrachia . Our divreenland . Moleculreenland ) and we 4.Our molecular divergence estimates show that the coleoid fossil record ,39 belieThe iconic shell has had a shifting functional role through cephalopod evolution, and is informative as to lifestyle and ecology. Subsequent to ancestral internalization of the phragmocone through the Carboniferous and Devonian, the decabrachian and octobrachian lineages independently evolved towards shell reduction ,16, alloDecabrachian coleoids are nektonic predators with streamlined morphology, high metabolic rates and shoaling behaviour; adaptations in common with teleost fishes . The majAmmonoids are stem group coleoids, which were common throughout the Late Palaeozoic until the end of the Mesozoic. Evidence from their radula morphology ,46 sugge5.Taken together, molecular divergence times and the cephalopod fossil record are consistent with a scenario in which predator–prey arms races shaped the coleoid body plan, biodiversity and ecology. The coincidence with the evolution of jawed vertebrates and teleost fishes during the Devonian Nekton Revolution and the Mesozoic Marine Revolution, suggests that nektonic marine vertebrates have been key antagonists towards cephalopods throughout most of their evolution.


The present study assessed the therapeutic effect of exenatide and metformin as the initial therapy on overweight/obese patients with newly diagnosed type 2 diabetes (T2D).  The prospective, nonrandomized, interventional study enrolled a total of 230 overweight or obese patients with newly diagnosed T2D who were administrated exenatide or metformin hydrochloride for 12 weeks. P < 0.05). The reduction in HbA1c and the proportion of patients with HbA1c < 7.0% (53 mmol/mol) were higher in the exenatide group than in the metformin group . The exenatide treatment caused a greater decline in the body weight and BMI as compared to the metformin treatment . The exenatide treatment  and baseline HbA1c level  were independent influencing factors for the decrease in HbA1c level.  224/230 patients, including 106 in the exenatide group and 118 in the metformin group, completed the 12-week treatment. Both exenatide and metformin significantly decreased the HbA1c levels in overweight/obese patients with newly diagnosed T2D (all NCT03297879. For an initial therapy in overweight/obese patients with newly diagnosed T2D, exenatide causes a better glycemic control than metformin. This trial is registered with  Type 2 diabetes (T2D) is a common metabolic disease with high morbidity and mortality due to T2D-related complications . ObesityHowever, it is yet unclear whether as initial treatment, GLP-1 receptor agonist contributes to an improved therapeutic efficacy than metformin in overweight/obese patients with newly diagnosed T2D. Exenatide is a classical drug of a GLP-1 receptor agonist. In the present study, we assessed the therapeutic effect of exenatide and metformin as the initial therapy on overweight/obese patients with newly diagnosed T2D.2  (P < 0.05)  ; however < 0.01] . The pro < 0.05)  3]..P < 0.05β in the two groups after exenatide or metformin treatment   versus 14.03 (6.34–21.72), P < 0.01]. However, the decrease in the HOMA-IR value was similar after 12 weeks of exenatide or metformin treatment (P > 0.05)  . The inc > 0.05) .P > 0.05). However, the greater increase in the HOMA-β value was still observed in the exenatide group than in the metformin group postadjustment for the decrease in FBG (P < 0.05).Interestingly, exenatide did not cause a greater decrease in the HbA1c level than metformin after adjustment for the reduced body weight following the 12-week treatment (P < 0.05) (P < 0.05) (P < 0.01) (Both exenatide and metformin significantly reduced the TC levels after 12 weeks of treatment ( < 0.05) . Moreove < 0.05) . In the  < 0.01) .β = 0.41, P < 0.01) and baseline HbA1c level  were independent influencing factors for the decrease in HbA1c level.Multivariate regression analysis was used to further assess the relationships between the decrease in the HbA1c level and related variables. The influence of six variables such as age, gender, body mass index, baseline HbA1c, baseline FBG, and drug treatment was assessed. The analysis indicated that the exenatide treatment  were higher in the exenatide group than in the metformin group. The exenatide treatment caused a greater decline in the body weight and BMI as compared to the metformin treatment. Multivariate regression analysis indicated that the exenatide treatment and baseline HbA1c level were independent influencing factors for the decrease in HbA1c level.In our study, metformin reduced the FBG levels by 2.17 mmol/L and the HbA1c levels by 1.44% from baseline, which was consistent with previous studies , 10. HowObesity is associated with T2D by inducing endoplasmic reticulum stress, chronic inflammatory state, and insulin resistance , 18. MetInsulin resistance plays a major role in the pathogenesis of T2D and its complications . The preβ-cell dysfunction [β values, and the increase in the exenatide group was higher than that in the metformin group. The alleviation of glucose toxicity, rather than a direct drug effect, might underlie the HOMA-β improvement in the two groups [β value was still observed in the exenatide group than in the metformin group. Previous studies showed that metformin indirectly increased β-cell function through decreasing glucotoxicity, lowering insulin resistance, inhibiting the formation of advanced glycation end products, and reducing oxidative stress [β-cells [β-cells as compared to the insulin glargine in a similar glycemic control, and this effect was sustained after a 4-week off-drug period [β-cells and inhibits their apoptosis [Compared to the Western population, Asians with T2D showed a distinct function . In the o groups . Howevere stress , 26. In [β-cells –30. A prg period . Animal poptosis , 30. Celβ-cell function and insulin sensitivity. Furthermore, the treatment duration was relatively short. Long-term observation of these treatments is needed to evaluate the persistence of beneficial effects over time. Moreover, the cost/benefit ratio is another aspect that should be considered. In terms of medical expenses, metformin might be more preferable for a lifelong treatment. Despite these limitations, one implication of the present study is that for a patient with newly diagnosed T2D, exenatide might contribute to a better effect on glycemic control, weight management, and β-cell function, which at least partly represents an avenue for investigation in the treatment of T2D.Notably, our study has several limitations. The major limitation was that the present study was not a randomized controlled trial. In addition, our study also lacked accurate methods, such as hyperglycemic and hyperinsulinemic clamp techniques, to assess β-cell function.In conclusion, for an initial therapy in overweight/obese patients with newly diagnosed T2D, exenatide causes a better glycemic control than metformin, whose mechanism might potentially be related to the better weight loss effect and amelioration of


Kaposi’s sarcoma (KS) is generally considered a neoplastic disorder of vascular origin and occurs in patients with acquired immunodeficiency syndrome (AIDS) or who have received immunosuppressive treatments after an organ transplant :1276–80, 1995; Viejo-Borbolla and Schulz, AIDS Rev 5(4):222–9, 2003; Schulz, J Antimicrob Chemother 45(Suppl T3):15–27, 2000; Aversa et al. Crit Rev Oncol Hematol 53(3):253–65, 2005; Mbulaiteye and Engels, Int J Cancer 119(11):2685–91, 2006; Tessari et al., Eur J Dermatol 16(5):553–7, 2006). Several Kaposi’s sarcoma case reports involving eyelids and conjunctiva have been published :221–2, 2012; Baumann et al., Ger J Ophthalmol 4(4):239–45, 1995).we report a 13 years old asian male patient rare case of ocular KS that was initiated from the sclera and progressed into the cornea and conjunctiva without an human Immunodeificiency Virus (HIV) or HHV-8 infection after a peripheral blood stem cells transplantation. In this case, anti- vascular endothelial growth factor (VEGF) therapy was attempted to stop the advance of ocular lesions and failed. Eventually, the KS was cured by a limbo-corneal lamellar graft, an amniotic membrane and scleral allograft transplantation plus intraoperative mitomycin C(MMC) after the complete excision of the tumors.A compete surgical excision combined with the intraoperative application of MMC, as well as grafts to repair the scleral, conjunctival, and corneal surfaces, could prevent a recurrence of KS. Kaposi sarcoma (KS) is probably a neoplastic disorder of vascular origin and occurs in four forms , which almost have the same histological appearance associated with evidence of human herpes virus type 8 (HHV-8) infection –3. KaposWe report here a HIV-negative case of extensively involved ocular  KS which occurred at 8 months after peripheral blood stem cell (PBSC) transplantation. No recurrence of KS was noted three years after excision of neoplasm combined with lamellar keratoplasty, allogeneic scleral transplantation, intraoperative mitomycin-C application and amniotic membrane transplantation.A 13-year-old boy presented with a red progressive neoplasm that had been on his right eye for 9 months. The boy had slightly decreased visual acuity without bleeding or obvious irritation before the initial onset Fig. . Before Treponema pallidum infection or viral infection, such as HIV or hepatitis viruses B and C, was also negative.A telangiectatic, reddish, and slightly elevated lesion of 4 × 5 mm was first noted on the nasal sclera near the corneal limbus of the right eye 8 months after the PBSC transplantation. He was then treated for episcleritis in another hospital with 0.3% tobramycin /0.1% dexamethasone eye drops  four times a day, TobraDex eye ointment every night, and pranoprofen eye drops  four times daily. When the patient initially came to our clinic, the visual acuity of his right eye became worse. The neoplasm presented as are dish elevated lesion that was 10 × 10 mm in size and had invaded into the cornea and extended close to the margo pupillaris Fig. . Three mA biopsy of the neoplasm was done with the patient’s and parents’ permissions. Tissues from the involved conjunctiva, sclera, and limbus were excised for histopathological examinations. The hematoxylin-eosin (HE)–stained slides indicated the possibility of angiogenic tumors and a capillary or lymphatic hemangioma. There were a few proliferating spindle cells and other irregular cells both inside and outside the neovessels, which were characterized by deep-dyed large and distinct nucleoli, nuclear membrane coarsening, pleomorphism, and a few mitotic figures in the nuclei Fig. . ImmunohBased on the pathological and clinical manifestations, a diagnosis of KS was established. Surgery was then recommended. During the operation, all of the involved nasal conjunctiva and about 1/2 the thickness of the involved sclera was removed. When the corneal lesions were dissected, only a small amount of posterior corneal stroma remained transparent under the surgical microscope. Before repair of the ocular surface defects, a pre-immersed cotton swab with 0.2 mg/ml MMC was placed onto the exposed residual sclera and medial rectus muscle for 5 min. Then, a full thickness limbo-corneal lamellar corneal graft without Descemet’s membrane and endothelium was transplanted onto the corneal surface. An alcohol-preserved full-thickness heterogeneous sclera was tailored and sutured to fit the scleral defect. Finally, the conjunctival defect was repaired with an amniotic membrane graft Fig. . One monThis was a rare case of ocular KS in which the conjunctiva, cornea, and sclera were extensively involved after a PBSC transplantation without an HIV infection.KS was first described in 1872. It has four clinical forms: classic, endemic, epidemic, and a post-transplant form . The claThere are several reports of KS after solid organ transplantations, especially after renal transplantations –18. HoweViral infections are considered to be a contributory factor that may activate neoplasm formation . RecentlReports of eye wall involvement in KS are rare. Conjunctival or eyelid KS usually grows slowly and can often be cured with a simple excision. Radiotherapy, chemotherapy, amniotic membrane transplantation, and intraoperative application of MMC have also been introduced mostly as adjunctive interventions combined with a local excision to treat KS –28. In tIn summary, we have reported a rare case of ocular KS that was initiated from the sclera and progressed into the cornea and conjunctiva without an HIV or HHV-8 infection after a PBSC transplantation. A compete surgical excision combined with the intraoperative application of MMC, as well as grafts to repair the scleral, conjunctival, and corneal surfaces, could prevent a recurrence of KS. There were no risks of distant metastasis during the years of follow-up examinations.


Tumor location served as an important prognostic factor in glioma patients was considered to postulate molecular features according to cell origin theory. However, anatomic distribution of unique molecular subtypes was not widely investigated. The relationship between molecular phenotype and histological subgroup were also vague based on tumor location. Our group focuses on the study of glioma anatomic location of distinctive molecular subgroups and histology subtypes, and explores the possibility of their consistency based on clinical background.We retrospectively reviewed 143 cases with both molecular information (IDH1/TERT/1p19q) and MRI images diagnosed as cerebral diffuse gliomas. The anatomic distribution was analyzed between distinctive molecular subgroups and its relationship with histological subtypes. The influence of tumor location, molecular stratification and histology diagnosis on survival outcome was investigated as well.Anatomic locations of cerebral diffuse glioma indicate varied clinical outcome. Based on that, it can be stratified into five principal molecular subgroups according to IDH1/TERT/1p19q status. Triple-positive (IDH1 and TERT mutation with 1p19q codeletion) glioma tended to be oligodendroglioma present with much better clinical outcome compared to TERT mutation only group who is glioblastoma inclined . Five molecular subgroups were demonstrated with distinctive locational distribution. This kind of anatomic feature is consistent with its corresponding histological subtypes.Each molecular subgroup in glioma has unique anatomic location which indicates distinctive clinical outcome. Molecular diagnosis can be served as perfect complementary tool for the precise diagnosis. Integration of histomolecular diagnosis will be much more helpful in routine clinical practice in the future. Glioma is the most common malignant brain tumor with heterogeneous growth pattern which can be found in different cerebral lobes , 2. ThisWe searched for Molecular Database and Image Bank in Department of Neurosurgery, Huashan Hospital and retrospectively selected 143 glioma samples for further study. H&E slides of all cases were reviewed by 2 individual neuropathologists to confirm the diagnosis of glioma according to WHO 2016 brain tumor guideline. Every patient has molecular diagnostic information. 140 out of 143 patients got complete follow-up. This research work was approved by Ethic Committee of Huashan Hospital and informed consents signed. Patients characteristic are listed in Table Paraffin blocks of each case were prepared. Four 4um slides and six 10um slides were sectioned. DNA extraction was performed by commercial DNA extraction kit  using 10um slides. IDH1 and TERT mutational analysis were done by Sanger Sequencing with method reported previously . The stahttp://www.braintumorsegmentation.org/).Image segmentation is an important pre-processing step for location analysis. Convolutional neural network (CNN) was proved to be an effective method for medical image segmentation . In our In order to study the location features in same coordinate system, the segmentation results were registered to MNI152 ) brain atlas . A resear value closed to 1. Median overall survival time (OS) was defined as the duration from the diagnosis and death or the last follow-up. Kaplan-Meier method was used to draw survival curve and analyzed by Log-rank test.Correlation coefficient was calculated to figure out the location relation between specific histology stratification and molecular phenotype. IBM SPSS statistic 20.0 software  was chosen as the analysis tool. A strong association would be found out with The information of histology diagnosis and WHO grade among 143 glioma patients can been found in Table P < 0.01). Median overall survival are 33 months for astrocytoma and 20.5 months for glioblastoma   Fig. .Fig. 2MeP < 0.01). Tumors located in hemisphere demonstrate better clinical outcome than those in central region even though no significance exists (Fig. Previous to studying anatomic preference to special molecular subgroups, we analyzed survival outcome between different tumor location. Herein, we found tumor located in frontal lobe indicated longer overall survival time of 66.1 months compared to tumors located in other cerebral regions (sts Fig. .Fig. 3SuFor triple-positive gliomas, tumor location tends to aggregate in bilateral frontal lobes. On the contrary, triple-negative tumors were more likely to locate in bilateral basal ganglia regions. In spite of that, IDH1/TERT mutation subgroup inclined to grow in left frontal lobe close to midline region. IDH1 mutation only subgroup was commonly seen in left frontal lobe and bilateral insular lobes. TERT mutation glioma apparently present with non-midline distribution while sitting in right frontal-insular lobe and left basal ganglia region. Meanwhile, TERT mutation only glioma has deep-seated location than triple-positive cases Fig. .Fig. 4DiThus glioma histology stratification strongly associated with molecular phenotype, we investigate whether anatomic distribution remains consistent within these two classification systems. We compared triple-positive samples with oligodendroglioma, IDH1 mutation only subgroup with astrocytoma and TERT mutation only tumors with glioblastoma since these genetic events highly represent histological diagnosis. It’s interested to find out that the tumor location and growth pattern is quite similar to each other based on MR images. The location correlation coefficients are 0.97, 0.94 and 0.85 accordingly Fig. .Fig. 5TuIn 2016 revised WHO diffuse glioma classification, the diagnosis of oligoastrocytoma was gone due to 1p19q can help clearly subdivide tumor into oligo-lineage or astroglial family. We have 14 oligoastrocytoma cases, 10 of them showed 1p19q codeletion with tumor likely to locate in bilateral frontal lobe which is similar to oligodendroglioma. On the other hand, tumor with 1p19q intact tends to grow in left insular lobe where is commonly seated by astrocytoma (Fig. Cerebral diffuse glioma is a biological heterogeneous tumor . PatientIn order to verify our hypothesis, we tried to study the interconnection between anatomic location and genetic biomarkers in our patient cohort. Beforehand, many papers published have put forward the cell origin theory underlying possible relationship between these two factors , 21. ManOn the other aspect, methods by using MR images to predict molecular biomarkers are popular recently, which was so-called Radiomics study. Ellingson et al. compared tumor volume ratio of T2 hyperintensity to contrast enhancement and central necrosis to differentiate mesechymal and non-mesenchymal molecular subtype in glioblastoma . His resAlthough molecular biomarkers are getting involved in routine pathological diagnosis of cerebral diffuse gliomas, more evidence should be provided to validate perfect match between molecular subtypes and classic histological diagnosis. Our study showed distinct anatomic distribution among different molecular phenotypes which is consistent with corresponding histological subtypes. Integration of molecular biomarkers with histology diagnosis will not only contribute to precise diagnosis but also predict patient clinical outcome. This kind of pathological diagnosis system was highly recommended in future clinical practice. Moreover, we developed a simple and cost-effective method to predict biomarkers which was supposed to be widely used in Radiomics study in glioma.


We show how faceted search using a combination of traditional classification systems and mixed-membership topic models can go beyond keyword search to inform resource discovery, hypothesis formulation, and argument extraction for interdisciplinary research. Our test domain is the history and philosophy of scientific work on animal mind and cognition. The methods can be generalized to other research areas and ultimately support a system for semi-automatic identification of argument structures. We provide a case study for the application of the methods to the problem of identifying and extracting arguments about anthropomorphism during a critical period in the development of comparative psychology. We show how a combination of classification systems and mixed-membership models trained over large digital libraries can inform resource discovery in this domain. Through a novel approach of “drill-down” topic modeling—simultaneously reducing both the size of the corpus and the unit of analysis—we are able to reduce a large collection of fulltext volumes to a much smaller set of pages within six focal volumes containing arguments of interest to historians and philosophers of comparative psychology. The volumes identified in this way did not appear among the first ten results of the keyword search in the HathiTrust digital library and the pages bear the kind of “close reading” needed to generate original interpretations that is the heart of scholarly work in the humanities. Zooming back out, we provide a way to place the books onto a map of science originally constructed from very different data and for different purposes. The multilevel approach advances understanding of the intellectual and societal contexts in which writings are interpreted. Just as Britain and America have been described as two nations separated by a common language, different academic disciplines often use the same words with divergent meanings . InterdiWidespread digitization of monographs and journals by HathiTrust , 17 and Our goal is to develop computational methods for scholarly analysis of large-scale digital collections that are robust across both the technological inconsistency of the digitized materials and the variations of meaning and practice among fields and across time. A further goal of our approach is that these methods should inform interdisciplinary research by suggesting novel interpretations and hypotheses. The methods should support scholars who wish to drill down from high level overviews of the available materials to specific pages and sentences that are relevant for understanding the various responses of scholars and scientists to contentious issues within their fields.In this paper, we provide a case study that focuses on meeting these challenges within the interdisciplinary field of History and Philosophy of Science (HPS). HPS must not only bridge the humanities and the sciences, but also the temporal divide between historically-significant materials and the present –28. We sUsing a novel approach of drill-down topic modeling—simultaneously reducing both the size of the corpus and the unit of analysis—we demonstrate how a set of 1,315 fulltext volumes obtained by a keyword search from the HathiTrust digital library is progressively reduced to six focal volumes that did not appear in the top ten results in the initial HathiTrust search. Topic modeling of these volumes at various levels, from whole book down to individual sentences, provides the contexts for word-sense disambiguation, is relatively robust in the face of OCR errors, and ultimately supports a system for semi-automatic identification of argument structure. We show how visualizations designed for macroanalysis of disciplinary scientific journals can be extended to highlight interdisciplinarity in arguments from book data . This guWhile the extension of computational methods such as these to various questions in the humanities may eventually provide ways to test specific hypotheses, the main focus of such research is likely to remain exploratory and interpretative, in keeping with the humanities themselves , 30. Thiprobabilistic topic models on these volumes. (2) We exploited the mixed-membership property of the topic models to identify the multiple contexts of the selected volumes and reduce the original search space even further. (3) Because topic models define the notion of a document flexibly, we drilled down further by constructing page-level topic models of the reduced set of volumes selected at the previous stage. (4) We used the page-level results to rank books and select pages from them for closer analysis, demonstrating an approach to semi-automatic argument extraction which showcases the interpretive results of our search process. (5) We exploited the close reading of arguments for exploratory investigation of drilling down even further, to sentence-level topic modeling within a single volume. (6) We used scientific mapping to locate relevant volumes Here, then, we see the author cautioning the reader not to jump to a high-level interpretation of the spider behavior. The spiders may perceive objects at various distances but they don’t judge it. The term ‘judge’ here is philosophically interesting, as it suggests an influence of Immanuel Kant on framing the debate. While Kant’s name does not appear in Washburn’s book, the term ‘judgment’ is important to Kant’s theory of cognition, and fundamental to the cognitive divide he posits between humans and animals. We emphasize that this is just a speculative suggestion about Washburn’s influences, but it does show how the topic modeling process can bring certain interpretive possibilities to the fore, moving the digital humanities another step closer to the goal of generating new insight into human intellectual activity.HT86 and HT6 corpora managed to target books in appropriate areas of interest.Finally, we found Library of Congress classification records for 776 out of 1,315 books and we used the LCCO classification crosswalk that we constructed to locate these books on the UCSD Map of Science, as shown in Ultimately, the map overlay provides a grand overview and a potential guide to specific books that were topic modeled, although without further guidance from the topic models, the map does not fully meet the desired objective of linking a high-level overview to more detailed textual analysis.The notion of “distant reading”  has captIn this paper we have motivated, introduced, and exemplified a multi-level computational process for connecting macro-analyses of massive amounts of documents to micro-level close reading and careful interpretation of specific passages within those documents. Thus we have demonstrated how existing computational methods can be combined in novel ways to go from a high-level representation of many documents to the discovery and analysis of specific arguments contained within documents.We have also shown how to zoom out to a macro-level overview of the search results. We presented a novel classification crosswalk between the Library of Congress Classification Outline (LCCO) and the UCSD Map of Science, which was constructed using only journal data, to extend the data to books. Because of the mismatch between the book data and the journal metadata, the crosswalk is not perfect, and the method of averaging locations places many books in uninterpretable regions of the map. Nevertheless, the visualization provides some useful information about the effectiveness of a simple keyword search in locating items of interest within a collection of hundreds of thousands of books.That our method succeeded in discovering texts relevant to a highly specific interdisciplinary inquiry shows its robustness to inconsistent and incomplete data. The HathiTrust Digital Library had OCR errors in 2.4% of volumes as of May 2010 . While tMulti-level topic modeling combined with appropriate measures of distance can efficiently locate materials that are germane to a specific research project, going from more than a thousand books, to fewer than a hundred using book-level topic models, and further narrowing this set down to a small number of pages within a handful of books using page-level topic models. The similarity measures used to span the word-topic and topic-document distributions are mediated by the topics in the model, and because every topic assigns a probability to every word in the corpus, this approach is highly adept at finding implicit relationships among the documents. Typical applications of topic modeling used elsewhere, such as graphing the rise and fall of topics through time, may show large-scale trends, but do not directly facilitate the interplay between distant reading and close reading that leads to deeper understanding. By connecting abstract, machine-discovered topics to specific arguments within the text, we have shown how topic modeling can bridge this gap.HT1315 and HT86 corpora. For users conditioned by online search engines, the word-centric search paradigm of LSA is more familiar than the topic-centric retrieval methods introduced here. Nonetheless, once users become aware of the potential for topic models, they provide more information about retrieved documents. For example, Washburn’s The Animal Mind is significant for its mixture of topics. (The topic models of the HT1315 collection can be explored using Washburn’s book as an entry point to the InPhO Topic Explorer page here: http://bit.ly/2qQZCJm.) Indeed the most prominent topic in the book  points to the importance of perceptual systems in her discussion of anthropomorphic attributions, and it is noteworthy that her dismissal of amoeba consciousness is grounded in what she argues is the lack of crossmodal associations  that are part of the content of human perceptual experiences—viz. the sentence previously discussed concerning why our human experience in the dark must be “wholly different in quality” from what an amoeba might experience.Could the similar outcomes have been accomplished with alternative methods such as Latent Semantic Analysis  or otheHT1315 corpus, to the roughly 32,000 pages in the HT86 collection, to the over 17,000 sentences of the HT6 collection, to smaller set of the 108 pages selected for close reading and argument markup. This reduction allowed us to filter out discussions of anthropomorphic deities and zero in on key elements of late 19th and early 20th Century arguments about anthropomorphizing nonhuman organisms.Using a six-step process we progressively reduced 300,000 public domain volumes from the HathiTrust Digital Library to the 1,315 books in the HT6 corpus revealed the surprising taxonomic range of these arguments, to include consideration even of consciousness in amoebae. This was surprising even to the domain experts on the team. This discovery raises new questions about the context for contemporary discussions of slime mold intelligence [The application to the history of comparative psychology described in our case study was successful in at least three ways. First, team members unfamiliar with the domain of comparative psychology were effectively guided towards highly relevant material in an unreadably large set of books. Second, team members were introduced to the work of Margaret Washburn, a pioneer of scientific comparative psychology, who wrote an important textbook of the early 20th Century —a book tlligence , 35 and HT1315, as documented in his Reading Notebooks. At the unlikely rate of one book a day, it would take nearly four years to read this set of books in its entirety. Even allowing for the fact that one fifth of the volumes retrieved in our were course catalogs, eliminating those would nonetheless leave a daunting, if not quite Olympian, reading task. As the majority of the volumes selected by keyword search were not directly relevant to the research project, the payoff made possible by more sophisticated computational analysis of the full texts was critical for the present task of finding arguments about anthropomorphizing animals.It took Charles Darwin 23 years to read a number of books comparable in size to Although we do not claim that our methods provide the only possible approach, we are unaware of any other approach providing the kind of multilevel macro-to-micro approach needed to assist digital humanities researchers who wish to leverage computational methods against large data collections to support both distant reading and more traditional close reading analyses in the humanities. Nevertheless, these methods suffer from the problem already noted by Blei  and othehttp://sci.cns.iu.edu/ucsdmap/The UCSD Map of Science is available at https://github.com/inpho/loc-ucsd.The LoC-UCSD Crosswalk is available via GitHub at https://hdl.handle.net/2022/21636 (doi: 10.5967/K8251GBZ). The models comprise of:A tar.gz file containing the HTRC-1315, HTRC-86 corpora and trained models supporting the conclusions of this article is available via the Indiana University ScholarWorks at HTRC-1315, volume-level, 60 topicsHTRC-86, page-level, 60 topicsAll materials in each corpus are in the public domain. All models are released under a Creative Commons Attribution 4.0 International (CC BY 4.0) License.vsm semantic modeling library are available on GitHub at https://github.com/inpho/topic-explorer and https://github.com/inpho/vsm, respectively. Both are published under the MIT License, a maximally-permissive open-source license.The InPhO Topic Explorer and https://hdl.handle.net/2022/21636. A read-only version of the notebooks, without the underlying models, may be viewed at https://github.com/inpho/digging. All notebooks are released under a Creative Commons Attribution 4.0 International (CC BY 4.0) License.The notebooks used for the HT1315 and HT86 analyses are included in the Indiana University Scholar Works at


Aspergillus niger, AnFaeA, by comparing its activity toward methyl and arabinose hydroxycinnamate esters. kcat/Km ratios revealed that AnFaeA hydrolysed arabinose ferulate 1600-fold, and arabinose caffeate 6.5 times more efficiently than their methyl ester counterparts. Furthermore, small docking studies showed that while all substrates adopted a catalytic orientation with requisite proximity to the catalytic serine, methyl caffeate and methyl p-coumarate preferentially formed alternative non-catalytic conformations that were energetically favoured. Arabinose ferulate was unable to adopt the alternative conformation while arabinose caffeate preferred the catalytic orientation. This study demonstrates that use of short alkyl chain hydroxycinnnamate esters can result in activity misclassification. The findings of this study provide a basis for developing a robust classification system for FAE and form the basis of sequence-function relationships for this class.Ferulic acid esterases  cleave the arabinose hydroxycinnamate ester in plant hemicellulose and other related substrates. FAE are commonly categorised as type A-D based on catalytic activities towards model, short alkyl chain esters of hydroxycinnamates. However, this system correlates poorly with sequence and structural features of the enzymes. In this study, we investigated the basis of the type A categorisation of an FAE from  O-[5-O-trans-feruloyl)-β-L-arabinofuranosyl]-D-xylopyranose) and it is the primary covalent interaction between lignin and hemicellulose1 which, of themselves, are two of the three main polymers made by plants. FAE have received much recent attention for their potential use in industrial transformations; the substrate promiscuity of some FAE has been employed for the deconstruction of plant biomass for biofuels and animal feed applications3 and for transesterification reactions utilising hydroxycinnamic esters4.Ferulic acid esterases  cleave the ester linkage between hydroxycinnamate and the C(O)5 position of an arabinose residue in hemicellulose, a plant-derived sugar polymer. This linkage is referred to as FAX , methyl p-coumarate (MpCA), methyl caffeate (MCA) and methyl sinapate (MSA). Initially, FAE candidates were divided into either type A or B depending on their activity towards MSA or MCA respectively6, and this was based on the activities of two classical FAE, AnFaeA and AnFaeB, derived from Aspergillus niger. The type A/B classification was later extended to include type C and D FAEs7. In general, type A FAEs show increased activity against model phenolic substrates containing a methoxy substitution at carbon-3 or -5, such as MSA and MFA. Type B FAE prefer small polar or no substitutions at carbon-3 or -5 and thus have strong activity towards MCA and MpCA. Type C and D are active against all of these model short chain hydroxycinnamic esters but differ in their ability to hydrolyse esters of diferulic acid. The FAE type A-D classification system is summarised in Fig. As the degree of reactivity of different FAE towards hydroxycinnamic ester linkages in biomass has become important in itself and valuable for their commercial applications, classification systems based on substrate specificity have been developed for the enzyme family. The most common hydroxycinnamic acids found in plant biomass are ferulic, ing Fig. . One of 9. For example, Benoit et al. categorised fungal FAE into 7 sub families which were later extended to 13 groupings based on phylogenetic analysis11. Subfamily 1 contained both B and C FAE, including the well characterised FAE, AnFaeB, as well as FAE from Aspergillus oryzae, AoFaeB and AoFaeC. The difficulty in aligning activity and sequence may be due, in part, to the diversity of the FAE enzyme class, with some putative members having close sequence similarity to lipase, acetyl xylan esterase, xylanase and chlorogenate esterase enzymes. Regardless, our ability to predict the activity of a novel FAE based on sequence similarity to a characterised FAE is limited.Genome sequencing programs have generated a large diversity of putative FAE sequences from bacteria and fungi and it would be very valuable to be able to build amino acid sequence-activity relationships for this expanding class of enzymes. However, attempts to link the type A-D activity classification system, which is based on hydrolysis of short chain esters of hydroxycinnamic acids, and the amino acid sequences of biochemically characterised FAE have hitherto been unsuccessful, resulting in groupings that contain mixed FAE activity types12 and CAZy13. While classification can take many forms including structural or sequence similarity, substrate specificity and activity are important factors and thus the selection of substrates as a basis for classification should be sound. As described above, the most commonly used FAE classification system is based on enzyme activity toward model substrates, which, while similar to the enzyme’s natural substrates of sugar-hydroxycinnamate linkages present in plant biomass, differ mostly in the alcohol component of the ester. In the natural substrate, the alcohol is a pentose monosaccharide such as arabinose, while the model esters used for activity-based classification have short chain alkyl groups, especially methyl. Structures of two caffeate ester substrates are compared in Fig. Enzyme classification is a useful tool in reducing the complexity of whole enzyme families and their applications, but may also be a basis for sequence, structure and function relationships for that enzyme group. Classification systems can be a valuable basis for rational design of enzymes as catalysts in biotechnological applications and extensive databases have been created to support these purposes including BRENDAet al.14 determined the crystal structure of one of the most studied FAE obtained from Aspergillus oryzae, AoFaeB, and used AutoDockTools (ADT) to simulate docking of the 4 short chain ester model substrates into the active site. The researchers were able to compare the active centre and docking to that of the related A. niger enzyme, AnFaeA, concluding that the residue TYR356 was most likely responsible for the severely reduced activity of AoFaeB toward MFA and MSA through steric hindrance to the larger methoxy group14. Furthermore, Goldstone et al. used GOLD to predict the binding mode of ferulic acid and a feruloyl arabinose ester to a previously unknown FAE, Est1E, identified from a ruminant bacterium15. Correct binding orientations for hydrolytic activity were modelled for feruloyl arabinose ester and extrapolated for short chain model esters; enzyme assays verified the enzyme’s activity toward ethyl ferulate and ferulic-containing hemicellulose fractions.Alternatives to the FAE type A-D classification system may be more revealing in terms of understanding substrate binding and more robust in terms of classification based on enzyme activity using more diverse substrates. Molecular modelling tools such as small molecule docking (SMD) have been used to investigate the activities of FAE toward short chain ester substrates. In particular, research by Suzuki et al. 2007 and Kelle et al.17 compared with model substrates. Thus with both molecular modelling tools and more authentic enzyme substrates available, alternative approaches to FAE classification can be explored.In terms of the synthesis of more diverse substrates to assist in the classification of FAE by activity, FAE themselves have been used to synthesise a variety of sugar esters via transesterification, or reverse hydrolysis, in non-conventional media such as organic solvents. Some of the synthesised esters are closer in structure to the linkages that FAE hydrolyse in plant biomass such as phenolic acid sugar esters prepared by Vafiadi A. niger, AnFaeA, toward the model hydroxycinnamic acid substrates, methyl ferulate and -caffeate, and their arabinose ester counterparts. The arabinose ester substrates were synthesised by transesterification and were fully characterised. AnFaeA was specifically chosen as this was the main enzyme for which the A-D classification was based on. Furthermore, the type A category is the most robustly defined. The second most relevant enzyme to the A-D classification, AnFaeB from A. niger, which was initially categorized as type B has since been re-categorised as belonging to in a family of type C FAE due to its sequence similarity11. This goes to further highlight the complexity of the current classification systems based on enzymatic activity. In addition to its prominence in the classification of FAE, AnFaeA is a suitable candidate for the focus of this manuscript as it has a large body of literature defining its substrate specificity, has a detailed solved crystal structure and is highly similar to many of the other type A FAE. A major contributing factor to the selection of AnFaeA is the small number of significantly distinct FAE with solved crystal structures. The Protein Database (PDB) contains only 10 records under EC of 3.1.1.73 (feruloyl esterase) with only approximately three other listed outside this set. Of these AnFaeA has the most significant volume of literature relating to activity with a clear distinction of no activity on methyl caffeate. As such, we propose that the primary assessment of enzymatic characterization should be done on AnFaeA.Here we compare the catalytic activities of the type A FAE from While an enzyme from type A such as AnFaeA would be expected to demonstrate good activity toward ferulate but negligible activity toward caffeate esters, we found this was only true for methyl esters; both arabinose hydroxycinnamic acid esters were good substrates for the enzyme. The basis for the stark contrast in enzyme activities measured for the methyl and arabinose-based esters was explored further by SMD and this provided new insights into substrate binding to FAE. The findings of this study will aid in developing a robust classification system for FAE and form the basis of a structure-function relationship for this enzyme class, whereby features of FAE sequence, structure and activity are aligned.Clostridium thermocellum and Neocallimastix sp. had increased activity toward FAXX and PAXX (2-O-[5-O-trans-feruloyl)-β-L-arabinofuranosyl]--O-β-D-xylopyranosyl--D-xylopyranose and 2-O-[5-O-trans-p-coumaroyl)-β-L-arabinofuranosyl]--O-β-D-xylopyranosyl--D-xylopyranose respectively) over MFA19.AnFaeA exhibited activity toward all methyl and arabinose esters of ferulic and caffeic acid as shown in Table 7, that is, if restricting the discussion to methyl esters, the activity toward caffeate was almost undetectable (72-fold lower) which is the main characteristic of type A FAE specificity. By contrast, the activity of AnFaeA against ACA was only 6-fold lower than MFA activity showing the enzyme can also hydrolyse caffeate esters at reasonable rates, which is usually characteristic of type B, C and D FAEs.In terms of hydroxycinnamate linkage, ferulate esters were better substrates than caffeate for AnFaeA which is in agreement with the current classification systemm) were determined from the fit of the Michalis-Menten reaction to the data  revealing an important difference in AnFaeA affinity towards sugar-linked hydroxycinnamate esters to that of model substrates. The kcat/Km ratio revealed that AnFaeA hydrolyses AFA 1600 times more efficiently than MFA, and ACA 6.5 times more than MCA. The extremely high kcat/Km ratio for AFA is attributed to the very fast reaction (high kcat) and the high substrate affinity (low Km) for this substrate.The enzyme velocity versus substrate concentration curves for AnFaeA towards the methyl and arabinose esters of hydroxycinnamic acids are shown in Fig. ta Table ; AnFaeA 9. Nevertheless, in the present study a low level of activity was detected with MCA which is attributed to the high enzyme load used for the MCA assays, approximately 100 times higher than the one used for MFA. The comparative protein concentrations for ester hydrolysis are shown in Fig. The characterisation of AnFaeA shows that the enzyme has greater activity on substrates containing a more bulky sugar linkage, such as AFA and ACA, than the respective short alkyl chain ester. Generally, type A FAEs show highest activity against phenolic substrates containing a methoxy substitution at C-3 and C-5 such as MFA and MSA while no activity has been detected for MCA according to previous reportset al. and Vafiadi et al.21 who used various alkyl ferulates to characterise FAE. In those reports, FAE activity was detected against ferulic acid esters ranging from methyl to butyl. Interestingly, the kcat/Km of MtFae1a towards ethyl ferulate was less than half of the same measure for either methyl ferulate or n-propyl ferulate. Other studies have shown FAE with activity towards esters of p-nitrophenyl or alpha-naphthyl of up to C8 in length23. However, while some attention has been drawn towards the effect of the alcohol-side constituent of the ester group on FAE activity, its influence when combined with a range of hydroxycinnamic acids has been overlooked. Thus an explanation for the large difference in activity demonstrated by AnFaeA towards MCA and ACA is not currently forthcoming.From the characterisation of AnFaeA on both MFA and AFA it is clear that the moiety attached via the ester linkage can have a significant effect on the catalytic activity of the FAE. This observation is supported by studies conducted by Topakas et al.9 revealed that the methoxy residue of ferulic acid occupies a hydrophobic hole in the binding cavity of the enzyme (PDB: 1UZA) which is made from the residues PRO161, PRO200, ILE199 and TYR80 as shown in Fig. p-coumarate and caffeate.Investigation of the AnFaeA protein structure by Faulds 9. Simulated docking of hydroxycinnamic esters was undertaken with the enzyme to investigate the basis for lack of activity of the closely related structures. The conventional reasoning has been that a polar hydroxyl side group of the phenolic ring of the hydroxycinnamic acid cannot occupy the hydrophobic hole9. However, with the knowledge that one methoxy group of sinapinate points outwards from the enzyme , for MCA and MpCA there was a more favourable alternative docking conformation. This alternative conformation occupied nearly the same space as the catalytic binding orientation but the placement of the aromatic ring was on the opposite side of the binding cavity. These reversed solutions for MCA and MpCA are unlikely to be catalytic as the estimated distance between the catalytic serine and the carbonyl carbon of the ester bond is 6.49 Å compared with 2.40 Å between the serine and carbonyl carbon in the catalytic orientation. Furthermore, the binding energy of MCA in the reversed orientation is 0.85 kcal/mol lower than for the catalytic orientation, and almost twice as represented in robustness  as well as the number of solutions within each clustered solution, the latter being a test of robustness of the derived docking, were considered in the simulations Table . While tss Table . Similarss Table . In factUmg−1 protein25 for MpCA, compared with 67.2 for MFA), marginally higher than the now observed activity on MCA. A more comprehensive study of the factors that contribute to docking orientation would require the use of molecular dynamics simulation which was outside the scope of this manuscript.When visualised as docked substrates in the active site of AnFaeA, MpCA and MCA show an interesting phenomenon; the hydrophobic pocket that accommodates the methoxy side group of ferulate and sinapate is occupied by the methyl alcohol of MpCA or MCA. The residues that stabilise the 4′-OH of MCA in the catalytic orientation instead stabilise the carbonyl oxygen and residue TYR100 stabilises the ester bond Fig. . That isTo further investigate the effect of the residues in the ester linkage on substrate docking and compare these results to the catalytic activity profile measured with AnFaeA, the alkyl esters were replaced with arabinose in the docking simulations. Arabinose is particularly relevant as enzyme characterisation revealed that AnFaeA was significantly more active on the arabinose esters of ferulate and caffeate than methyl esters. Both AFA and ACA dock favourably in the forward, catalytic orientation Fig.  with binIn this study we replaced a small alkyl ester with a larger, more polar sugar residue and this had a considerable effect on the simulated docking of the esters into the enzyme active site and catalytic activities. These findings support our proposition that the previously determined low activity of AnFaeA toward MCA, and by extension to MpCA, may be due to the more favoured non-catalytic binding orientation in the active site. The docking simulation for MpCA, given in Fig. The docking simulations provide a possible mechanism for the difference in AnFaeA activities toward MCA and ACA: by replacing the small alkyl with the arabinose ester, the reversed orientation is much less favourable, with poorer MBE and clustering Table . The lar27) that arabinose hydroxycinnamates, as natural substrates, offer better binding and therefore higher activity. Given this observation, AnFaeA, previously classified as type A, does indeed show activity towards caffeate-type esters that have 3′-hydroxy substitution on the phenolic ring. In revision, the enzyme could well be classed as type C or D FAE with broad activity. This offers an insight into the binding of methyl and arabinose esters to AnFaeA and subsequent catalytic activity, which can in turn be extended to other FAE types. In essence, arabinose based substrates are a more appropriate ligand for determining substrate specificity and thus can aid in the classification of FAE. While by no means are the authors proposing a complete novel classification for FAE, as this is an objective of future work, such as classification system could combine the idea of endo and exolytic FAE activity28 with the observation that all FAE could potentially demonstrate broad substrate activity with a preference for moiety but not exclusion. Thus the structural elements of the β-clamp and the hydrophobic hole are better rectified with activity profiles. However, the establishment of such a classification system is subject to further investigation of other arabinose esters to further characterised FAE with known crystal structures. Ad Interim, this manuscript provides evidence that arabinose esters are superior and therefore a novel classification system can be created in the near future based on these specific substrates.In this manuscript, we prove through systematic experiments and validation of circumstantial reports  was expressed in Pichia pastoris as described previously28. For the synthesis of arabinose esters, a type B FAE from Myceliopththora thermophila ATCC 42464 (MtFae1a) was expressed in P. pastoris, as described previously20. The enzymatic preparations were concentrated and exchanged for 100 mM MOPS-NaOH pH 6.0. Protein concentration was determined by the PierceTM BCA Protein Assay . The FAE purity (%) of the enzyme preparations was determined by SDS-PAGE using a Novex Sharp pre-stained protein standard , followed by quantification using the JustTLC software .Recombinant FAE type A from −1 were introduced in the form of concentrated stock solutions in buffer. Reactions were carried out at 35 °C for 24 h without agitation.MFA and MCA (99%) were purchased from Alfa Aesar (Germany). Arabinose ferulate and arabinose caffeate  were synthesized through enzymatic transesterification at preparative scale (10 mL) in sealed flasks containing 70:30 v/v 100 mM MOPS-NaOH pH 6.0: dimethyl sulfoxide (DMSO). 50 mM of the donor (104 mg MFA or 97 mg MCA) was diluted in DMSO while 700 mM acceptor  and 0.08 mg MtFae1a mLμ column (250 mm × 21.2 mm) (Phenomenex) according to Vafiadi et al.16. The reaction mixture after evaporation under vacuum and mild heating (50 °C) was diluted in methanol (1 mL). Fractions containing AFA  or ACA  were pooled and evaporated. Proton NMR spectroscopy was done in DMSO-d6 on a Bruker Ascend Aeon WB 400 spectrometer.Isolation of the arabinose ester from each flask was performed by preparative HPLC on a C18 Luna 5 1H NMR  5-O-(trans-feruloyl)-L-arabinofuranose: δ 9.60 , 7.57 , 7.34–7.32 , 7.12 , 6.79 , 6.49 , 6.26–6.24 , 5.32–5.28 , 4.96 , 4.30–4.25 , 4.12–4.08 , 4.00 , 3.82 , 3.74–3.72 , 3.68–3.64 .d6, 400 MHz) 5-O-(trans-caffeoyl)-L-arabinofuranose: δ 7.48 , 7.04 , 7.01–6.98 , 6.74 , 6.24 , 4.95 , 4.32–4.29 , 4.11–4.06 , 4.00 , 3.74–3.72 , 3.67–3.64 .1H NMR  in MOPS-NaOH 100 mM pH 6.0. Samples were incubated for 30 min at 45 °C without agitation. The kinetic constants  were determined by fitting the Michaelis-Menten equation to the data using nonlinear regression . All assays were carried out in duplicate and concomitant with appropriate blanks. There was no hydrolysis observed in the absence of enzyme.For the characterization of AnFaeA, stock solutions of substrate  (5–10 mM) were prepared in DMSO. Each reaction was initialized by introducing enzyme in the reaction mixture in the form of concentrated stock solution in buffer. The AnFaeA activity was assayed on 0.5 mM substrate in MOPS-NaOH 100 mM pH 6.0 using different enzyme loads. Samples were incubated for 10 min at 45 °C without agitation. All reactions were terminated by incubation at 100 °C for 10 min. One unit (1 U) is defined as the amount of enzyme releasing 1 Quantitative analysis of samples was performed by HPLC on a C18 Nucleosil 100–5 column (250 mm × 4.6 mm) . Elution was conducted with a linear gradient method using water (solvent A) and acetonitrile (solvent B) at 0.6 mL/min and ambient temperature. Total running time was 20 min during which the following proportions of solvent B were used: 0–15 min 28–72% and 28% 15–20 min. Detection was achieved by a PerkinElmer Flexar UV/VIS detector at 300 nm based on calibration curves prepared using standard solutions of the detected compounds in 1:1 water:acetonitrile.et al.14 and consistent for all substrates. Here the rigid macromolecule was Chain A of the crystal structure of AnFaeA (PDB: 1UZA). Substrates used were the methyl esters , the ethyl ester EFA, and the arabinose 5-O esters . All were generated using Avogadro29 and structure optimised using UFF.Docking simulations were conducted as per the earlier report of Suzuki 30, keeping backbone atoms stationary. The PDB file was also cleaned for non-standard residues and water molecules were removed. AutoDockTools (ADT)31 was used to add polar hydrogen and to assign Gasteiger charges to all atoms within Chain A. A grid box of 80 × 80 × 80, centred on the catalytic serine was used as the search space with a grid spacing of 0.375 Å.Briefly, PDB entry 1UZA was first prepared by relaxing side group atoms using the amber suite31 with random starting conformations of the ligand with the maximum number of torsions available. Results were visualised using ADT or Chimera32.AutoDock (AD) was used for docking via a Lamarckian genetic algorithmA. niger (AnFaeA) and from Myceliopththora thermophila ATCC 42464 (MtFae1a) are available from NCBI (Accession: O42807.1 & G2QND5.1 respectively). The structure of AnFaeA is available from PDB (1UZA). The hydroxycinnamic ester substrates used in the SMD as well as the output AutoDock log files containing the docked solutions are available on reasonable request, from the corresponding author.The protein sequences for FAE from