A antagonists whereas the late IPSP was removed by GABAB antagonists. Aβ perfusion induced recorded cells to depolarize, increase their input resistance and decrease the late IPSP. Aβ action mechanism was localized at postsynaptic level and most likely linked to GABAB-related ion channels conductance decrease. In addition, it was found that the specific pharmacological modulation of the GABAB receptor effector, G-protein-coupled inward rectifier potassium (GirK) channels, mimicked all Aβ effects previously described. Thus, our findings suggest that Aβ altering GirK channels conductance in CA3 pyramidal neurons might have a key role in the septohippocampal activity dysfunction observed in AD.Last evidences suggest that, in Alzheimer's disease (AD) early stage, Amyloid-β (Aβ) peptide induces an imbalance between excitatory and inhibitory neurotransmission systems resulting in the functional impairment of neural networks. Such alterations are particularly important in the septohippocampal system where learning and memory processes take place depending on accurate oscillatory activity tuned at fimbria-CA3 synapse. Here, the acute effects of Aβ on CA3 pyramidal neurons and their synaptic activation from septal part of the fimbria were studied in rats. A triphasic postsynaptic response defined by an excitatory potential (EPSP) followed by both early and late inhibitory potentials (IPSP) was evoked. The EPSP was glutamatergic acting on ionotropic receptors. The early IPSP was blocked by GABA GABA mediates the inhibitory neurotransmission and accordingly, regulates excitatory activity preventing hyperexcitation, actions especially relevant to maintain neural network stability and oscillatory activity  raised in the Salamanca University Animal House . All animal procedures were reviewed and approved by the Ethical Committee for Use of Laboratory Animals of the University of Salamanca and University of Castilla-La Mancha, and followed the European Communities Council (86/609/EEC).in vitro preparation have been described elsewhere  artificial cerebrospinal fluid (ACSF), with sucrose (234 mM) replacing the NaCl (117 mM) to maintain osmolarity. In order to preserve the optimal connectivity from fimbria fibers on CA3 pyramidal neurons  and maintained at room temperature during the recordings.For recordings, a single septohippocampal slice was transferred to an interface recording chamber  and perfused continuously with ACSF comprising (in mM) 117 NaCl, 4.7 KCl, 2.5 CaClIntracellular sharp electrode recordings from CA3 pyramidal neurons were obtained with borosilicate glass microelectrodes  filled with a potassium acetate solution (3 M) and connected to the headstage of an intracellular recording amplifier . Only data from neurons with both, stable resting membrane potential (RMP) with values ≤ −60 mV in the absence of direct current (DC) holding currents, and presenting overshooting action potentials, were collected for analysis. Spike amplitude, afterdepolarization and afterhyperpolarizing potentials were measured relative to threshold.Excitatory and inhibitory postsynaptic potentials  were elicited orthodromically by stimulating the lateral fimbria where septal afferents to CA3 hippocampal neurons are mainly found , 6-cyano-nitroquinoxaline-2,3-dione , 2-amino-5-phosphonovalerate , Bicuculline Methiodide (specific blocker of GABAA receptors), (RS)-3-Amino-2-(4-chlorophenyl) propylphosphonic acid , (RS)-4-Amino-3-(4-chlorophenyl) butanoic acid , Tetrodotoxine , Tertiapin-Q (selective blocker of GirK channels) and 2-methyl-2,4-pentanediol .All chemicals used in this study were purchased from Sigma  and Tocris  and applied by superfusion in the ACSF. The chemicals used were amyloid-β peptides , accordingly with data distribution. When necessary, one-way ANOVA or equivalent non parametric test  and post-hoc analysis were performed. Statistical significance was determined at a level of p ≤ 0.05.Sharp electrode data were acquired online with the help of a CED 1401 interface , and stored on a personal computer (sample frequency 12.5 kHz). Analysis in both cases was performed using the MiniAnalysis Program, version 6.0.3 . Unless otherwise indicated, the electrophysiological data are always expressed as mean ± standard error of the mean (SEM), and n represents the number of averaged neurons. Synaptic potentials were averaged (≥5) before quantitative analysis. Statistical analysis of collected data was performed using either Student's n = 10) filled with biocytin is illustrated in Figure stratum pyramidale and the visible basal dendrites on stratum oriens  following fimbria stimulation. Finally a second hyperpolarization . The amplitudes of both, IPSPs and EPSP, varied with membrane potential , a specific blocker of GABAA receptors , a specific blocker of GABAB receptors , without any noticeable effect  for at least another 10 min at each concentration.In all cases, the recordings were stabilized for at least 10 min. During this time, characterization of firing pattern, membrane potential, Ri and synaptic responses were performed. The specificity of the Aβ Figures . Then, sF = 2.62, p = 0.062], threshold , ADP , fAHP , or sAHP  after perfusion with Aβ25–35 at increasing concentrations. However, as shown in Figures 25–35 was applied . The membrane potential of the recorded neurons was maintained at its RMP value by DC holding current injection to cancel out the depolarization induced by Aβ25–35  and 1.5 μM  concentrations and no difference was observed at 0.5 μM concentrations . Additionally, Aβ25–35 also produced a significant increase in the relative Ri   and 1.5 μM  whereas no differences were found at 0.5 μM  .To determine the synaptic location of these Aβg Figure  of intran = 20). In the experiment shown in Figure 25–35 produced a significant decrease of the late IPSP component  that was neither observed at 0.5 μM  nor on early IPSP . However, the late IPSP reduction was associated with an increase in the excitatory response observed at concentration of 1.5 μM  but not at lower concentrations , not only removed the GABAB component  as well as membrane depolarization , therefore mimicking all Aβ25–35 effects previously described.Depolarization caused by Aβ25–35 concentrations on Ri were evaluated  compared to control  at high concentrations , which was not observed at 0.5 μM . These results indicate a postsynaptic Aβ25–35 mechanism that quite likely involves a reduction in ion channels conductance.To determine the mechanism involved in the postsynaptic reduction of the late IPSP amplitude and Ri increase, pharmacological blockage of different receptors/channels was performed and effects of increasing Aβd Figure . The blo25–35-induced changes on Ri  or Ri , in contrast to Aβ25–35. Altogether, these results suggest that Aβ25–35 exerts its effects acting preferentially on postsynaptic GirK channels, instead of GABAB receptors.On the other hand, since saclofen perfusion prevented Aβ25–35 was found to be unable to generate additional increase on Ri after postsynaptic blockage of GirK channels by tertiapin-Q , suggesting that Aβ-induced Ri increase may be associated to a reduction in GirK channel conductance. However, because conductance depends, among others, on the number of channels, their open probability or membrane voltage, the methodology used in the present study has some limitations to determine the exact mechanism for Aβ-mediated Ri increase. In addition, in some experiments, it was necessary to inject DC to maintain a stable membrane potential and to prevent depolarization. Previous studies had reported that depolarization mechanisms induced by Aβ might involve activation of glutamatergic receptors . In contrast, perfusion with CNQX, APV and the GABAA blocker, bicuculline (10 μM), was not able to prevent the Ri increase induced by Aβ25–35  further suggesting an effect of Aβ25–35 on GirK channels.In accordance with this hypothesis, Aβ25–35 on GABAB response. To verify whether Aβ25–35 affects the postsynaptic response mediated by GABAB receptor activation, we used a drug cocktail including: TTX to block synaptic transmission, bicuculline to block GABAA receptors activation and baclofen to stimulate GABAB receptors , which confirms that Aβ25–35 reduces the postsynaptic GABAB response in a concentration and time dependent manner. But Aβ25–35 action on this GABAB response might also be explained by an effect on its final effector, GirK, which would also underlie the already described Aβ25–35 effects on Ri and membrane depolarization.Given that, Ri changes may depend on multiple factors and might be affected by Aβ acting on different ion channels and receptors, we designed a protocol to specifically evaluate the effects of Aβs Figure . Cocktai25–35 on GirK response, we used a GirK channel agonist, MPD . Then, perfusion of Aβ25–35 (1–1.5 μM) removed the hyperpolarization mediated by GABAB-GirK stimulation , while this effect was not evident at 0.5 μM . In fact, the hyperpolarization could be eliminated when the cocktail was washed , indicating that Aβ25–35 directly affects GirK channels conductance.In order to validate this hypothesis and evaluate the effect of Aβd Figure . FinallyDespite the importance that rhythms as an emergent property of neural network seem to have, few studies have investigated how Aβ induces injury and how it may contribute to impair septohippocampal oscillatory activity, which in turn may underlie the early symptoms typically observed in AD patients Colom, . The pre25–35 constitutes the biologically active fragment of Aβ  response in CA3 region after fimbria stimulation , and late (GABAB). This complex response has also been described in pyramidal neurons of basolateral amygdaloid nucleus (Washburn and Moises, A inhibition led to an increase in EPSP and late IPSP amplitudes whereas complete GABAergic component block induced an epileptic-like response mainly mediated by non-NMDA receptors, although NMDA antagonist reduced the response partially. Very similar results have been shown in neurons from CA3 hippocampal region (Scanziani et al., A receptors on the inhibitory cell. This machinery has been suggested as a self-protective mechanism for the control of recurrent activity when an imbalance of the system occurs (Scanziani et al., A biphasic (glutamatergic, non-NMDA, and GABAergic, GABAOn the other hand, although it has been reported that cholinergic septal neurons innervate pyramidal CA3 neurons and interneurons (Widmer et al., theta frequencies and Aβ increases this frequency (Leao et al., In the present study, pharmacological characterization of the complex septohippocampal synaptic response revealed the glutamatergic nature of the EPSP. Glutamatergic septohippocampal neurons (Colom et al., 25–35 to increase the EPSP. This result may be explained because excitatory response is strongly braked by the GABAB activity (Otmakhova and Lisman, 25–35 diminished late IPSP. Since late IPSP was generated by GABAB receptors stimulation, an Aβ25–35 effect on such receptors, on its intracellular signaling mechanism, or on its final effector, GirK channels, could be hypothesized. Assuming that the early GABAA component is not affected by Aβ25–35, the reduction of GABAB component would not be due to an inhibition of GABA release or other presynaptic mechanism. Together with Ri increase and membrane depolarization, our results lead to a reduction of the conductance of potassium channels coupled to GABAB receptor.We found AβB2 subunit in AD patients (Massone et al., B receptor signaling.Binding studies in postmortem AD patients have shown a reduction in GABA receptors density in the hippocampus (Chu et al., B receptors of CA3 pyramidal cells; Aβ25–35 behaves as a selective antagonist and reduces late IPSP. However, the GABAB system has a very low tonic activity in basal conditions, and only when the system is activated pharmacologically or by afferent stimulation, the antagonist effect becomes obvious (Bowery and Smart, B receptors induces little effects on membrane properties, i.e., membrane potential or Ri (Lambert et al., B receptor antagonism would not explain all the effects induced by Aβ25–35.When fimbria is stimulated, GABAergic interneurons activate GABAB receptors and GirK channels are coupled and co-expressed in the postsynaptic membrane of CA3 pyramidal neurons (Luscher et al., 25–35 action on the membrane should include the effector coupled to GABAB receptor. GirK channels exhibit a tonic basal activity, even without receptor signaling, due to their direct binding to the Gα subunit of G proteins (Lujan et al., 25–35 on the postsynaptic membrane, i.e., late IPSP reduction, Ri increase and membrane depolarization, even in the presence of GirK and GABAB agonists. Furthermore, Aβ25–35 was found to be unable to generate an additional significant increase in Ri after pharmacological blocking of GirK channels. These results not only suggest that GirK channels are functional in the basal state, but also that Aβ25–35 action seems to be more evident when these GirK channels are activated.Another aspect to consider is that both, GABAGirK channels activity alteration may have multiple implications for synaptic activity and neuronal network function. Numerous studies have emphasized its role in several pathological processes in the nervous system such as epilepsy, pain, addiction, Parkinson or Down syndrome (Luscher and Slesinger, The present study proposes a putative synaptic mechanism for neural network hyperactivity, which is considered as an early event in AD pathogenesis and is associated with early Aβ deposition in non-demented humans with or without mild cognitive impairment (Sperling et al., 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.


Neonatal exposure of rodents to an immune challenge alters a variety of behavioural and physiological parameters in adulthood. In particular, neonatal lipopolysaccharide  exposure produces robust increases in anxiety-like behaviour, accompanied by persistent changes in hypothalamic-pituitary-adrenal (HPA) axis functioning. Altered autonomic nervous system (ANS) activity is an important physiological contributor to the generation of anxiety. Here we examined the long term effects of neonatal LPS exposure on ANS function and the associated changes in neuroendocrine and behavioural indices. ANS function in Wistar rats, neonatally treated with LPS, was assessed via analysis of tyrosine hydroxylase (TH) in the adrenal glands on postnatal days (PNDs) 50 and 85, and via plethysmographic assessment of adult respiratory rate in response to mild stress (acoustic and light stimuli). Expression of genes implicated in regulation of autonomic and endocrine activity in the relevant brain areas was also examined. Neonatal LPS exposure produced an increase in TH phosphorylation and activity at both PNDs 50 and 85. In adulthood, LPS-treated rats responded with increased respiratory rates to the lower intensities of stimuli, indicative of increased autonomic arousal. These changes were associated with increases in anxiety-like behaviours and HPA axis activity, alongside altered expression of the GABA-A receptor α2 subunit, CRH receptor type 1, CRH binding protein, and glucocorticoid receptor mRNA levels in the prefrontal cortex, hippocampus and hypothalamus. The current findings suggest that in addition to the commonly reported alterations in HPA axis functioning, neonatal LPS challenge is associated with a persistent change in ANS activity, associated with, and potentially contributing to, the anxiety-like phenotype. The findings of this study reflect the importance of changes in the perinatal microbial environment on the ontogeny of physiological processes. Escherichia coli (E. coli)), and other agents, such as toxins  is associated with an increased likelihood of psychopathology in later life Activation of the immune system in early life is thought to play a role in predisposing to later life psychopathologies. Epidemiological evidence, for instance, indicates higher incidences of psychopathologies, including schizophrenia, negative emotionality and panic disorders in children exposed to viral or bacterial infections in-utero LPS exposure on postnatal days (PNDs) 3 and 5 is a well-documented rodent model used to examine the impact of “perinatal programming” on a variety of physiological and behavioural outcomes The system most commonly implicated in the development of anxiety-like behaviours, in the context of neonatal exposure to LPS, is the hypothalamic-pituitary-adrenal (HPA) axis. Alterations in circulating corticosterone have been reported in adult animals treated as neonates with LPS. This is particularly apparent when these animals are subjected to an additional acute stressor in adulthood The HPA axis is not, however, the only system affected by neonatal exposure to an inflammatory insult. The HPA axis and the autonomic nervous system (ANS) work in a coordinated fashion to modulate the stress response. Recent evidence from our laboratory indicates that neonatal exposure of rodent pups to LPS results in an immediate and sustained response of the ANS as indicated by an increase in phosphorylation and activity of tyrosine hydroxylase (TH) in the adrenal glands Exposure to LPS has been demonstrated to induce activation of the ANS during the acute-phase immune response. Specifically, parasympathetic nervous system signalling was evidenced via both the afferent and efferent vagal signals. Peripheral administration of LPS was reported to increase levels of circulating catecholamines and to cause a distinct activation of central catecholaminergic neurons that are likely to play a mediating role in the neuroendocrine responses to peripheral inflammation  facilitates the measurement of changes in respiration at thresholds far lower than that required to induce autonomically mediated changes in cardiovascular system In the current study neonatal programming of the ANS was investigated via the assessment of TH activation in the adolescent and adult adrenal glands. Autonomic function was further validated by the measurement of respiratory responses to mild stressful stimuli in adult rats. To confirm the presence of an anxiety-like phenotype, adult anxiety-like behaviours were evaluated, and HPA axis activity at both the peripheral and central levels was assessed. Brain factors examined included mRNA levels of CRH, CRH binding protein (CRHBP), CRH receptor type 1 (CRHR1), GRs and mineralocorticoid receptors (MRs) as well as GABA-A receptor subunit alpha-2 (GABA-Arα2), due to its role in modulation of the stress response and anxiety states Experimentally naive female Wistar rats were obtained from the University of Newcastle animal house and mated in the University of Newcastle Psychology vivarium, under conventional housing conditions. At birth (PND 1), pups were randomly allocated into either LPS or saline control conditions. Pups were briefly removed from their cages on PNDs 3 and 5, weighed and administered with LPS  or an equivolume of saline , intraperitoneally (i.p.). Neonatal drug administration protocols and housing conditions were previously reported At 4 h, 24 h and 48 h following the last neonatal drug administration on PND 5, animals were rapidly decapitated and trunk blood was collected into EDTA-coated tubes .On PND 50 animals were deeply anesthetized with an overdose of Lethabarb . Cardiac blood was obtained into EDTA-coated tubes. Adrenals glands were surgically removed, cleaned of any excess fat tissue, immediately frozen in liquid nitrogen and kept at −80°C until assayed.On PND 85, immediately following the respiratory testing blood was collected via the saphenous vein. All blood samples were centrifuged at 1000 × g for 15 min at 4°C, and plasma stored at −20°C until assayed. Blood and adrenal glands were collected from an additional subset of animals on PND 85, after a lethal injection of Lethabarb (2 ml/kg), following the previously described methods.Plasma corticosterone concentrations were assessed using a commercially available rat corticosterone 125I radioimmunoassay kit . The recovery of free corticosterone is 100%, with an inter- and intra-assay variability of 4.4% and 6.5%, respectively.Anxiety-like behaviours in adulthood were assessed on the elevated plus maze (EPM) and the holeboard apparatus, as per previously described protocols later solution , stored at 4°C overnight and then kept at −20°C until further analysis.Expression of CRH, CRHR1, CRHBP and GABA-Arα2 mRNA levels was determined by quantitative RT-PCR in the prefrontal cortex (PFC), hypothalamus and hippocampus of LPS and saline-treated animals. In addition, expression of GRs and MRs was determined in the hypothalamus and hippocampus. Two weeks following the respiratory testing, animals were deeply anesthetized with an overdose of Lethabarb , brains were rapidly removed and areas of interest isolated . Samples were then immersed in RNA−ΔΔC(t), where C(t) is the cycle at which fluorescence was first detected above background, and presented as a fold increase relative to the saline control.Total RNA extraction from brain tissue was carried out using RNeasy Lipid Tissue Mini Kit  according to the manufacturer’s instructions. RNA concentrations were determined by spectrophotometer, NanoDrop 2000c . First-strand cDNA was synthesized from 2 µg of total RNA using a SuperScript® VILO™ cDNA Synthesis Kit , according to manufacturers’ instructions. Real-time PCR was performed using SYBR Green PCR Master Mix  on a 7500 RT-PCR Fast instrument . Primer sequences are listed in Tyrosine hydroxylase phosphorylation, protein and activity levels were analysed as previously described with some modification Thirty µg of each sample was mixed with sample buffer  and subjected to SDS–polyacrylamide gel electrophoresis before being transferred to nitrocellulose. Membranes were incubated with blocking solution  for 2 h at 25°C. Membranes were washed in Tris-buffered saline with Tween (TBST)  and incubated with pSer19-, pSer31-, pSer40-, total-TH or β-actin-HRP antibodies for 1 h at 25°C. Membranes were washed in TBST and incubated with HRP-linked anti-IgG secondary specific antibodies for 1 h at 25°C. Membranes were visualized on Fugifilm Las-3000 imaging system  using Western HRP Substrate (Millipore). The densities of phospho-, total TH and β-actin bands were measured using a MultiGauge V3.0 . Total TH protein levels were expressed as the ratio of TH protein to β-actin as β-actin levels are used as an endogenous control. pSer19, pSer31 and pSer40 were expressed as the ratio to total TH protein to account for variability in total TH between samples.3H]-L-tyrosine, final volume 50 µL) and subjected to tritiated water release assay. The 50 µL reactions were initiated with the addition of 100 µM tetrahydrobiopterin in 5 mM HCl. Control representing background reactions were added with 5 mM HCl but did not contain tetrahydrobiopterin. Assays were performed for 20 min at 30°C and were stopped by addition of 700 µL charcoal slurry . Mixtures were vortexed for 1 min and were centrifuged at 16,000 rpm for 10 min at 30°C. 350 µL supernatants were added to 3 mL scintillation cocktail and were vortexed for 10 s. Mixtures were assayed by scintillation spectrometry for 10 min per sample. TH activity assays which were performed under these conditions were linear. The changes in TH activity were normalized to total TH protein levels and expressed as a fold increase relative to the saline control.Fifty µg of each sample was then mixed with reaction mixture  methods t test analyses on Δ respiratory rate were performed and complemented by Bayesian t tests, presented as posterior odds of preference for either null 0(H) versus alternative hypotheses (1H)  changes in respiratory rate were presented as a difference  attained by subtracting the baseline respiratory rate prior to the onset of stimulus from the peak respiratory response to the stimulus, as generated by the Change Detection model. Student t test analyses adjusted for multiple comparisons. The significance level was set at p≤0.05.Statistical analyses were conducted using the Statistical Package for the Social Sciences for Windows, Version 18 (SPSS Inc.). All data, except for the changes in respiration in response to acoustic stimuli (as described above), were analysed using analyses of variances (ANOVA) design. A nested ANOVA design, whereby “litter” is nested into “treatment” and analyses of covariance (ANCOVA), whereby “male-to-female ratio” and “litter size” are covaried in the analyses, were also employed where appropriate and reported only when significantly contributing to the data. Planned comparisons were performed when significant interactions were observed using F = 26.925, p<.001) following weaning (PND 22) to adolescence (PND 50). Significant weight gain in LPS treated animals, as compared to saline-treated controls, was also evident from adolescence (PND 50) to adulthood (PND 85)  = 17.623, p<.001). See Weight gain was recorded in all males born into the litters participating in this study. No differences in weight gain were observed between the LPS and saline-treated animals during the neonatal period. However, LPS-treated males gained significantly more weight, than saline-treated controls  = 4.09, p<.005), with LPS-treated males exhibiting significantly increased levels of circulating corticosterone  than saline-treated controls , and a significant interaction between the time point of blood collection and treatment  = 3.88, p<.005). Planned comparisons revealed LPS-treated neonates had significantly increased corticosterone levels at 4 h following LPS challenge on PND 5 (t(15)  = 2.89, p<.05), with no differences at later time point, as shown in Assessment of circulating corticosterone levels during the neonatal period revealed a significant effect of treatment  = 4.69, p<.05), and in adulthood, on PND 85  = 8.89, p<.05) as demonstrated in F  = 6.12, p = .05), see LPS-treated males exhibited increased baseline corticosterone levels on PND 50   = 3.39, p = .05), F = 5.03, p = .05). Trends, which approached significance (p = .06), indicated that LPS-treated males exhibited fewer exploratory head dips in the holeboard apparatus than their saline-treated counterparts, Neonatal LPS treatment resulted in significantly increased percentage of time spent in closed arms of the EPM, compared to saline-treated controls  = 12.02, p<.05; F = 6.09, p<.05, p = .16), p = .09), significantly decreased in the hypothalamus  = 19.24, p<.001), but significantly increased in the hippocampal region  = 6.04, p<.05), see F = 5.54, p<.05). Significantly increased GR mRNA levels were evident only in the hippocampus of LPS-treated animals  = 6.25, p<.05), see Significantly decreased CRHR1 mRNA levels were evident in LPS-treated males in the PFC and hypothalamus  = 13.72, p<.005; F = 9.86, p<.05; F = 22.52, p<.0001; F  = 5.36, p<.05, respectively, see F = 6.71, p<.05; F = 8.42, p<.05; F = 39.63, p<.001; F  = 22.73, p = .001, respectively, see A significant effect of treatment was observed in regards to TH phosphorylation at all 3 serine residues  and TH activity in adolescent t  = 1.42, p = .18), Bayes factor (B01 = .97) constitutes an approximately equal likelihood for the preference for either null or the alternative hypothesis. In response to the 60 dB stimulus, LPS-treated animals responded with a significantly increased respiratory rate ((15)t  = 2.88, p<.05; B01 = .15). This difference from saline-treated controls remained significantly increased when presented with the 70 dB stimulus ((15)t  = 2.57, p<.05; B01 = .24). No differences were observed at the highest intensity (80 dB) between the neonatally treated groups as reflected in both statistical approaches ((14)t  = .005, p = .99; B01 = 2.18). See Analysis of changes in respiration by using a Change Detection model revealed that LPS-treated males responded with a tendency to increase respiration when introduced to the stimulus of lowest intensity (50 dB). Despite that F = 3.61, p<.05), with no differences in baseline respiratory rate obtained prior to the presentation of the stimulus. See As demonstrated in The primary aim of this study was to investigate the long term consequences of neonatal immune challenge on ANS functioning, and its association with behavioural and neuroendocrine indices in an anxiety-like phenotype. Here, we replicate and confirm this model by demonstrating increased anxiety-like behaviours and alterations to the HPA axis following neonatal LPS challenge. A significant outcome of this study is the demonstration that an exposure to LPS during the neonatal period is also associated with persistent alterations to ANS functioning. This is demonstrated using a behavioural index of ANS activity  this response is mediated by an increase in TH mRNA levels and a subsequent protein synthesis in vivo studies through the demonstration that the sustained phase of TH activation in neonatal adrenal glands persists for up to 24 h after LPS administration LPS challenge in the neonate induces an acute increase in TH phosphorylation and activity in neonatal adrenals in vivo studies have reported a disassociation between alterations in TH phosphorylation and activity, and TH protein levels in response to an acute In the current study, increased phosphorylation at all three serine residues  and increased enzymatic activity were observed independent of changes in TH protein, in animals treated with LPS as neonates, at both the adolescent and adult time points. Previous Increased glucocorticoids, resulting from either exposure to stress or direct administration of synthetic glucocorticoids, increase TH activity in sites of catecholamine production, including the adrenal medulla Given that the TH analysis has indicated long term alterations in autonomic activity, we aimed to validate this observation using a behavioural paradigm that specifically targets autonomic function. Using a recently established model of respiratory testing, which assesses respiratory responses to mild sensory stimuli, the current study assessed respiration in rats as a measure of arousal. Previous studies have demonstrated that this behavioural measure is a valid, sensitive and reliable index of rapid changes in autonomic activity In the current study, alterations in respiratory responses exhibited by the LPS-treated animals were associated with increases in anxiety-like behaviours and HPA axis activity. This is consistent with previous reports from this laboratory Examination of CRHR1 and CRHBP mRNA levels revealed substantial downregulation of gene expression in the PFC and the hypothalamus, but upregulation in the hippocampus of LPS-treated animals. The CRH system has a regulatory role in HPA and ANS functioning CRHBP, together with CRH receptors, mediates the activity of CRH in the brain and the pituitary, presumably acting as a negative regulator of CRH. CRHBP expression can be modulated by glucocorticoids and is cell-dependent, with increased pituitary levels in response to stress, suggesting its role in a negative feedback on the HPA axis A model of HPA axis dysfunction in depression and anxiety, proposed by Reul and Holsboer (2002) Increased expression of GR mRNA content was found in the hippocampus of LPS-treated animals. Previously, neonatal LPS treatment has been reported to decrease GR binding and density in the hippocampus, hypothalamus and frontal cortex of adult animals, adrenalectomized prior to brain collection Our findings suggest that exposure to an immune challenge in the early postnatal period of life drives long term alterations in the neuroendocrine and autonomic systems, and is associated with alterations in behavioural functioning, overall indicative of an anxiety phenotype. While programming of the HPA axis activity and anxiety-like behaviours by neonatal LPS exposure has been previously demonstrated, this study provides novel insights into the effects of an early life immune challenge on ANS functioning. Since prolonged exposure to increased endocrine and autonomic activity is a known epidemiological risk factor for development of metabolic disorders, such as visceral obesity and insulin resistance, as well as cardiovascular diseases


Contradictory results have been reported regarding the association between leptin level and breast cancer. Therefore, a meta-analysis was performed to investigate this issue.CI) was calculated using fixed-effects or random-effects model analysis.Published literature from PubMed and the Chinese National Knowledge Infrastructure (CNKI) Database was retrieved. This study was performed based on different cases and control groups. The combined effect . B) For 4 studies comparing premenopausal breast cancer cases and healthy controls the Overall, the mean serum leptin level of case groups was significantly higher than that of control groups. A) For 9 studies comparing breast cancer cases and healthy controls the combined effect This meta-analysis suggests that leptin level plays a role in breast cancer and has potential for development as a diagnostic tool. Breast cancer is the most common cancer and the second-leading cause of cancer related death among women worldwide Leptin, a protein hormone produced mainly by adipocytes, placenta and mammary epithelium, plays a significant role in the control of metabolism, reproductive processes, immune processes, angiogenesis, haemopoiesis and oxidation of lipids Many studies have investigated the association between serum leptin level and breast cancer. However, the reported results have been contradictory. In this study, we performed a meta-analysis to assess the association between serum leptin level and breast cancer risk.To identify all studies that examined the relationship between leptin level and breast cancer, a systematic online databases search was conducted. The search was done on June 10, 2012. All published studies were found with PubMed/MEDLINE and China National Knowledge infrastructure (CNKI) using the following terms: (“leptin” or “ob gene product” or “ob protein” or “obese gene product” or “obese protein” ) and (“breast cancer” or “breast carcinoma” or “breast tumor” or “breast neoplasm” or “breast tumors” or “breast neoplasms” or “mammary carcinoma” or mammary neoplasm” or “mammary neoplasms” or “cancer of breast” or “cancer of the breast” or “human mammary carcinoma”). References from the retrieved articles were also screened to complete the data bank. No “language”, “publication year”, or other limits were used. If more than one article were published using the same case series, only the study with largest sample size was selected.Studies were eligible for inclusion if: (1) They could be defined as a case-control study or nested case-control study or a cohort study, (2) all patients were pathological diagnosed with breast cancer that had not undergone any previous treatment for tumors, (3) they included sufficient data for determining serum leptin level. We excluded studies that were not published as full reports, or studies without control subjects, or studies that included patients who received antitumor treatments.With the purpose of extracting the necessary characteristics, all relevant articles were collated independently by two reviewers (Jingping Niu and Liqin Wang). They checked for any encountered discrepancies and reached a consensus.SD) of leptin level in each group. If standard error of mean (SEM) was reported, SD was calculated according to the formula: The extracted data included: (1) publication details: first author’s last name, publication year, and origin of the studied population, (2) study design, (3) source of control subjects, (4) characteristics of the studied population: sample size, mean of age, mean of BMI, (5) methods of leptin measurement, and (6) means and standard deviation (MD)) was converted into standard mean difference (SMD). The combined effect  to assess the strength of the association between serum leptin level and breast cancer.All analyses were performed using Review Manager (version 5.1.2) and STATA (version 11.0). For comparing data from different sources, the absolute difference of leptin levels (mean difference (I2 statistic to investigate heterogeneity among studies P≤0.10), a random-effect model was selected to pool the data. Otherwise, a fixed-effect model was used.We used the To investigate the sources of heterogeneity, meta-regression analyses were conducted The flowchart summarizing the process of study search and selection is presented in I2 values were 92%, 72%, 95%, 97%, 65% and 62%, respectively; P values were <0.001, 0.001, <0.001, <0.001, 0.09 and 0.03, respectively). A random-effects model was applied for these groups. The combined effects CI were A) 0.66 , B) 0.41 , C) 1.09 , D) 0.96 , E) 0.33  and G) 0.59 . Meta-regression did not show a significant difference in published year, sample size, age, or BMI. For group A, heterogeneity could be attributed mainly to five studies I2 values were 33%, 0%, 0%, 0% and 14%, respectively; P values were 0.15, 0.51, 0.49, 0.88 and 0.32, respectively) in the remaining studies. The combined effect Heterogeneities were found within studies of groups A, B, C, D, E and G . The combined effect There was no heterogeneity within studies of groups F and H . However, it was unable to estimate publication bias for group E due to the limited quantity of the primary studies.According to the funnel plot and Egger's test, publication bias was not detected for groups A, B, C, D, F, G and H Click here for additional data file.Text S1PRISMA Flow Diagram.(DOC)Click here for additional data file.Text S2Calculation formalas for SMD and combined effect ((DOC)Click here for additional data file.


Loss of integrity of the basal forebrain cholinergic neurons is a consistent feature of Alzheimer’s disease, and measurement of basal forebrain degeneration by magnetic resonance imaging is emerging as a sensitive diagnostic marker for prodromal disease. It is also known that Alzheimer’s disease patients perform poorly on both real space and computerized cued  or uncued (idiothetic) recall navigation tasks. Although the hippocampus is required for allothetic navigation, lesions of this region only mildly affect idiothetic navigation. Here we tested the hypothesis that the cholinergic medial septo-hippocampal circuit is important for idiothetic navigation. Basal forebrain cholinergic neurons were selectively lesioned in mice using the toxin saporin conjugated to a basal forebrain cholinergic neuronal marker, the p75 neurotrophin receptor. Control animals were able to learn and remember spatial information when tested on a modified version of the passive place avoidance test where all extramaze cues were removed, and animals had to rely on idiothetic signals. However, the exploratory behaviour of mice with cholinergic basal forebrain lesions was highly disorganized during this test. By contrast, the lesioned animals performed no differently from controls in tasks involving contextual fear conditioning and spatial working memory (Y maze), and displayed no deficits in potentially confounding behaviours such as motor performance, anxiety, or disturbed sleep/wake cycles. These data suggest that the basal forebrain cholinergic system plays a specific role in idiothetic navigation, a modality that is impaired early in Alzheimer’s disease. Alzheimer’s disease results in cognitive impairment, including deficits in memory and spatial navigation Atrophy of the basal forebrain has been consistently correlated with generalized cognitive decline in Alzheimer’s disease NTR) was used to create a specific lesion of the cholinergic neurons of the basal forebrain in mice, and their uncued spatial navigational abilities were quantified. We found that mice with a basal forebrain lesion displayed very disorganized behaviour in a passive avoidance navigation task lacking extramaze cues, but performed no differently from control animals on a range of other tests including contextual conditioning and working spatial memory. These results demonstrate that impaired idiothetic navigation is directly attributable to the loss of cholinergic neurons of the basal forebrain.Loss of integrity of the basal forebrain cholinergic neurons is a consistent hallmark of Alzheimer’s disease at autopsy Experimentally naive male C57Bl6 mice (8–12 weeks of age) were housed in groups of four in plastic cages. Mice were maintained on a 12 hour light/dark cycle (lights on at 7∶00 a.m.), with food and water provided ad libitum. All procedures were approved by the University of Queensland Animal Ethics Committee and conducted in accordance with the Australian code of Practice for the Care and Use of Animals for Scientific purposes.NTR-saporin  or control rabbit-IgG-saporin  were performed using a 30G needle attached to a 5 µl Hamilton syringe. The needle was lowered into the lateral ventricle  8 week old male C57Bl/6J mice (22–24 g) were anaesthetized by intraperitoneal (i.p.) injection of ketamine (100 mg/kg) and the muscle relaxant xylazine (10 mg/kg). Each mouse was then placed in a stereotaxic frame (David Kopf Instruments), with the incisor bar maintained at −3.3 mm below horizontal to achieve a flat skull position. Bilateral infusions of murine-p75Fourteen to twenty-one days after surgery mice were subjected to a range of behavioural tests. Separate cohorts of mice were used to analyse performance in the place avoidance and contextual fear conditioning tasks. Prior to these behavioural tests mice were tested for levels of anxiety using the light/dark test, video tracking of sleep/wake cycles, or the Y-maze. Following contextual fear conditioning, tests of motor integrity were performed using a Rota-rod.The place avoidance task was a modification of test first described by Cimadevilla et al. On day one all mice underwent one exploration session lasting 5 minutes in which they could freely explore the arena without any shocks being applied. For each phase of the experiment the mice entered the arena directly opposite the shock zone, and were immediately returned to their home cage following trials. After 1 hour, mice were given a single 5 minute training session in which they learned to avoid a 60° stable shock zone. During training the mice received a 0.4 mV foot-shock for half a second when they entered the 60° shock zone, with an entrance delay of 500 ms, and an inter-shock interval of 1 second. To determine place memory for the shock zone, mice were allowed to freely explore the arena 24 hours after the training session without any shocks being applied. During testing, behaviour was measured by the number of entrances into the shock zone over a 5 minute period.The positional data from the testing session were used to measure avoidance of the shock sector. This analysis was based on the fraction of time each animal spent opposite the shock sector and the spread of the area explored. We first measured the angle between the shock area and the preferred position of the animal and the range of the area in which the animal spent most of its time. Subtracting these two angles provided a measure of the constraint of the animal’s position away from the shock area. This allowed us to differentiate the time spent away from the shock area from random navigation.The Y-maze was employed to test working spatial memory. The Y-maze was composed of two perpendicular arms connected by a start arm. Both of the arms and the starting arm were 45 cm long ×5 cm wide ×15 cm high. The maze was positioned 1 metre above the ground. The two perpendicular arms contained different optical cues . During training the mice were placed in the start arm with access to only one arm for a period of 3 minutes. The training arm alternated between mice. Following a 20 minute intermission in their home-cage, mice were placed back in the Y-maze for 3 minutes with access to both the training and the novel arm. Animals were tracked using Ethovision software (Noldus Information Technology) and the total time spent in the novel arm was analysed.The apparatus used for contextual fear conditioning consisted of a chamber (30 cm×20 m×20 cm) with a clear Perspex front wall and ceiling, three stainless steel walls, and a removable shock grid floor consisting of 32 stainless steel rods  with a stainless steel chamber underneath. Chambers were concealed within a sound-attenuating cubicle that contained a 12 V light, an exhaust fan and a video camera. Freezing and locomotor activity were measured using Video freeze software (Med Associates Inc) during both the training and testing sessions. The criterion for a freezing event was set at an observation interval of 2 seconds, where the mouse had to remain under the motion threshold  for a period of 500 milliseconds. During training, the mice were placed in the fear conditioning chamber for a period of 20 minutes, during which they received 5 random foot-shocks (0.8 mV). The conditioning context consisted of a 1% acetic acid odour with lights and fans on. Contextual memory was tested 24 hours after training. The mice were placed back in the conditioning chamber for 4 minutes with no shocks under the same contextual conditions as in the initial session.Twenty four hours after the conclusion of contextual fear conditioning, the motor integrity of the mice was tested using the accelerating Rota-rod . Mice were placed on the Rota-rod arm travelling at 2 rpm, accelerating at 0.4 rpm/sec up to a maximum of 20 rpm. Testing was conducted over a 3 minute period and falls, if they occurred before the 3 minute cut-off, were recorded automatically.As changes in the integrity of the sleep/wake cycle are a potential confound in the analysis of memory performance, a subset of mice were measured for sleep/wake patterns prior to behavioural testing. Changes in sleep/wake activity were determined by 48 hours of recording via an overhead video camera, and locomotor activity and velocity were measured using Ethovision software. Testing encompassed two full wake cycles and one sleep cycle, with the distance travelled by the mice in 1 hour time bins being used to assess the sleep/wake cycle.The light–dark test of anxiety was performed using a square open field (30 cm×30 cm×30 cm) made of opaque white acrylic, in which a cardboard insert with a roof was used to enclose half the box. A 5 cm×5 cm hole in the dividing wall allowed access to the enclosed side. Mice were placed in the enclosed side of the box and the number of entries to the open side and the total time spent in the open during a 5 minute test period were recorded.Immediately following behavioural testing mice were deeply anaesthetized with sodium pentobarbital (100 mg/kg i.p.) and transcardially perfused with 20 ml of 0.9% saline containing 1% sodium nitrite and heparin (5000 I.U./ml), followed by 100 ml of 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Brains were post-fixed overnight and, after repeated washing in phosphate buffered saline , placed in 20% sucrose solution for 24 hours. Brains were blocked using a matrix (Stoelting Co.), aligned to the atlas of Franklin and Paxinos One series of sections was used to reveal choline acetyl transferase (ChAT) using goat anti-ChAT antibody , biotinylated donkey anti-goat IgG  and ABC reagent . Black immunoreactive cytoplasm labelled for ChAT was revealed by a nickel-intensified diaminobenzidine reaction, with peroxide being generated by glucose oxidase. Sections were mounted onto chrome-alum/gelatin-treated slides, dehydrated, cleared in histolene, and coverslipped with DePeX (Sigma-Aldrich).A second series of sections from a subset of mice was used to reveal parvalbumin by immunofluorescence. Free-floating sections were immunostained using mouse anti-parvalbumin antibody , and donkey anti-mouse FITC . Sections were mounted onto chrome alum/gelatin-treated slides and coverslipped with buffered glycerol (pH 8.6).Bilateral counts of neurons immunoreactive for ChAT were conducted through the rostro-caudal extent of the basal forebrain. Analysed sections were 120 µm apart. Five sections were analysed for the medial septum, 4 sections for the vertical diagonal band of Broca (beginning 1.42 mm from Bregma), and 5 sections for the horizontal diagonal band of Broca (beginning 0.74 mm from Bregma) and the basal nucleus (Meynerts) (beginning −0.34 mm from Bregma). Four sections of the adjacent nucleus accumbens (beginning 1.42 mm from Bregma) were also analysed To determine the specificity of the lesion caused by mu-p75-SAP, counts of parvalbumin-positive neurons through the medial septum and the vertical diagonal band of Broca were performed as above. Eight-bit monochrome images were taken of the basal forebrain sections beginning 1.42 mm from Bregma Behavioural and anatomical data were analysed using one-way ANOVA with α set at 0.05 using SPSS software (IBM Corporation). When ANOVA revealed a significant difference between groups, a Tukey post-hoc analysis was performed.NTR antibody  = 40.427, p<0.001), vertical diagonal band of Broca  = 38.892, p<0.001), and horizontal diagonal band of Broca  = 19.148, p<0.001). A smaller but significant loss of ChAT-positive neurons was also detected in the basal nucleus (Meynerts)  = 29.88, p<0.001). In contrast, there was no loss of parvalbumin-positive GABAergic neurons in the medial septum or vertical diagonal band of Broca  = 0.065, p = 0.865; F = 0.089, p = 0.832, respectively;  = 0.004, p = 0.950), demonstrating that the toxin was targeted to p75NTR-expressing neurons in the basal forebrain.To selectively ablate cholinergic basal forebrain neurons we used a saporin-conjugated p75 = 0.802, p = 0.507) or speed  = 0.804, p = 0.506) between any of the groups during the 5 minute habituation period, indicating that the lesioned mice did not have any deficits associated with exploring a novel open field. During the training session, conducted 1 hour after habituation, all mice entered a 60° shock zone that was located directly opposite where the animals were introduced to the arena. Under these conditions, all mice that received foot-shocks (0.4 mV) successfully avoided the shock zone for the remainder of the 5 minute training session (data not shown), showing that lesioned animals detected and avoided the shock area during training.To assess idiothetic navigation, four groups of mice  were tested using the place avoidance task. There was no difference in either the distance travelled  = 8.079, p<0.01), no shock vs no surgery: p<0.05; not shock vs Rb-IgG-SAP: p<0.05]. There was no significant difference in the number of entries on test for lesioned mice as compared to no shock mice  = 6.389, p<0.01), no shock vs mu-p75-SAP, p<0.05], indicating that their memory for the context was intact. This reduction in exploratory behaviour displayed by the lesioned mice on test was similar to that of control mice that received a foot-shock  accurately avoided the shock zone during the 5 minute test as compared to the animals that did not receive a foot-shock during training . Both gr = 3.743, p<0.05; mu-p75-SAP vs no surgery+shock: p<0.05; mu-p75-SAP vs Rb-IgG-SAP: p<0.05). Moreover, some lesioned mice randomly entered and stayed within the shock zone as indicated by the large variability in the number of potential foot-shocks received by lesioned mice  = 0.363, p = 0.569), indicating that in this test of spatial working memory lesioned mice performed as well as control animals   = 4.407, p<0.05) and less locomotor activity  = 4.807, p<0.05) than control mice that received no shocks. No differences in the percentage of freezing or locomotor activity were detected in lesioned mice compared to the two control groups that received shocks during training  = 0.437, p = 0.671 and mu-p75-SAP vs no surgery+shock F = 0.951, p = 0.362; Locomotor: mu-p75-SAP vs Rb-IgG-SAP, F = 0.065, p = 0.949 and mu-p75-SAP vs no surgery+shock F = 0.859, p = 0.409). Similarly, all mice that received foot-shocks during training showed significantly higher levels of freezing and less locomotor activity when they were placed back into the training context  = 7.569, p<0.05 and F = 3.518. p<0.05, respectively). Furthermore, no differences in the percentage of freezing or locomotor activity during test were detected in lesioned mice compared to the two control groups that received foot-shocks on training  = 0.151, p = 0.883 and mu-p75-SAP vs no surgery+shock F = 0.529, p = 0.607; Locomotor: mu-p75-SAP vs Rb-IgG-SAP, F = 0.177, p = 0.863 and mu-p75-SAP vs no surgery+shock F = 0.544, p = 0.597 )  foot-shocks during the training session showed significantly more freezing (F 0.597 ) , indicat = 0.311 p = 0.587; F = 0.383, p = 0.547, respectively). Interestingly, lesioned mice made more entries into the light side of the apparatus than controls during the 5 minute test  = 6.133, p<0.05)  .As altered motor performance could be a potential confounding variable, mice that underwent contextual fear conditioning were also assessed for motor performance using the accelerating Rota-rod 24 hours after the conclusion of testing. All mice, regardless of lesion status, reached the 3 minute threshold without any falls from a Rota-rod that accelerated from 2 rpm to 20 rpm at a rate of 0.4 rpm/sec . = 0.005, p = 0.955; F = 0.008, p = 0.931, respectively)  was detected by ANOVA (2-way repeated measures with between group factors [mu-p75-SAP vs Rb-IgG-Sap] and repeated measure factor [time]) during the sleep/wake cycle (Fctively) .In this study we used intercerebroventricular application of mu-p75-SAP to induce a discrete and specific loss of basal forebrain cholinergic neurons in the mouse To test the animals’ ability to navigate they were subjected to an idiothetic version of the passive place avoidance test in which all extramaze cues were removed. The design of this task forces the mice to use kinaesthetic self-motion information to locate themselves in space, relative to their home area and the aversive shock zone. Ablation of the basal forebrain cholinergic neurons led to highly disorganized exploratory behaviour. Control mice that received foot-shocks during training successfully avoided the shock zone, and displayed organized exploratory behaviour that centred on the home area of the arena. In contrast, lesioned mice did not stay within the safe area but randomly entered the shock zone, even though they travelled a similar distance to control mice during their exploration.No deficits in contextual or working spatial memory were apparent following loss of the basal forebrain cholinergic neurons. Lesioned mice showed similar levels of freezing to control mice in contextual fear conditioning, and explored the novel arm of the Y-maze with the same intensity. Furthermore, although lesioned mice were unable to locate the shock zone or home region of the place avoidance arena on test, their behaviour was dramatically different from their performance during habituation, as well as in comparison with that of control mice that were never trained, indicating that their memory for the context was intact. Moreover, we found no deficits in motor performance, anxiety, or the sleep/wake cycle. These findings indicate that the basal forebrain cholinergic system is not critically involved in the cognitive abilities required for context recognition or working spatial memory.In general, animals use two different strategies to navigate, allothetic and idiothetic. In allothetic navigation the animal uses distal environmental cues to locate itself in space, and continually reorients its motion, whereas in idiothetic navigation the animal uses self-movement cues generated by internal sensory information such as vestibular and proprioceptive input coupled with memory In a previous study, Moreau et al. A major function of basal forebrain hippocampal innervation is the generation of the 4–10 Hz theta rhythm. Lesion studies targeting GABAergic or cholinergic inputs to the hippocampus have revealed that the GABAergic system is responsible for the pacing of theta rhythm synchronization, whereas the cholinergic system contributes to the amplitude or the power of the rhythm It is well established, based on single-cell recordings, that hippocampal neurons respond to movement, place, and head orientation Both basal forebrain atrophy and hippocampal volume loss, as measured by magnetic resonance imaging, are emerging as accurate markers for diagnosing at risk Alzheimer’s disease patients, with basal forebrain atrophy and hippocampal volume loss being highly correlated


The eyes and skin are obvious retinoid target organs. Vitamin A deficiency causes night blindness and retinoids are widely used to treat acne and psoriasis. However, more than 90% of total body retinol is stored in liver stellate cells. In addition, hepatocytes produce the largest amount of retinol binding protein and cellular retinoic acid binding protein to mobilize retinol from the hepatic storage pool and deliver retinol to its receptors, respectively. Furthermore, hepatocytes express the highest amount of retinoid x receptor alpha (RXRα) among all the cell types. Surprisingly, the function of endogenous retinoids in the liver has received very little attention.Based on the data generated from chromatin immunoprecipitation followed by sequencing, the global DNA binding of transcription factors including retinoid x receptor α (RXRα) along with its partners i.e. retinoic acid receptor α (RARα), pregnane x receptor (PXR), liver x receptor (LXR), farnesoid x receptor (FXR), and peroxisome proliferator-activated receptor α (PPARα) has been established. Based on the binding, functional annotation illustrated the role of those receptors in regulating hepatic lipid homeostasis. To correlate the DNA binding data with gene expression data, the expression patterns of 576 genes that regulate lipid homeostasis were studied in wild type and liver RXRα-null mice treated with and without RA. The data showed that RA treatment and RXRα-deficiency had opposite effects in regulating lipid homeostasis. A subset of genes (114), which could clearly differentiate the effect of ligand treatment and receptor deficiency, were selected for further functional analysis. The expression data suggested that RA treatment could produce unsaturated fatty acids and induce triglyceride breakdown, bile acid secretion, lipolysis, and retinoids elimination. In contrast, RXRα deficiency might induce the synthesis of saturated fatty acids, triglyceride, cholesterol, bile acids, and retinoids. In addition, DNA binding data indicated extensive cross-talk among RARα, PXR, LXR, FXR, and PPARα in regulating those RA/RXRα-dependent gene expression levels. Moreover, RA reduced serum cholesterol, triglyceride, and bile acid levels in mice.We have characterized the role of hepatic RA for the first time. Hepatic RA mediated through RXRα and its partners regulates lipid homeostasis. The eyes and skin are obvious retinoid target organs. Vitamin A deficiency causes night blindness and retinoids are widely used to treat acne and psoriasis. However, more than 90% of total body retinol  is stored in liver stellate cells . In addiThe broad and complicated roles of retinoids can be explained by the presence of multiple receptors for retinoic acid (RA), the biological active form of retinol. The receptors for RA are retinoic acid receptor (RAR) as well as retinoid x receptor (RXR) . In addiRXRα is highly expressed in the liver . Liver sTo understand the global roles of RXRα and RARα at the genomic level, chromatin immunoprecipitation using anti-RXRα and -RARα antibodies followed by sequencing (ChIP-seq) was performed. Since RXRα is an essential partner for other nuclear receptors, we compared ChIP-seq data to RXRα binding locations with locations from previous studies for PXR , LXR 1919, FXR , and PPATo understand the global roles of RXRα and RARα at the hepatic genome level, ChIP-seq was performed using anti-RXRα and -RARα antibodies. Single read sequencing yielded 18 and 32 million uniquely mapped reads for RXRα and RARα, respectively. After filtering by including peak scores that were greater than 20 and distance within 10 kb from the transcriptional start site, 17,973 peaks were detected for RXRα and 18,697 peaks for RARα. Since RXRα is an essential partner for other nuclear receptors, we compared ChIP-seq data of RXRα with those of PXR , FXR 20. Our datCluster and principal component analysis (PCA) were performed using the binding data. Overlapping bindings between RXRα and RARα, PPARα, FXR, LXR, and PXR were analyzed. According to the length of the vertical branches Figure A, LXR anTo understand the potential biological role of genes likely to be targeted by RXRα-heterodimers, DAVID functional annotation was performed. The top ten processes for each of the heterodimers formed by RXRα and other nuclear receptors were investigated in this study Table . AlthougHepatic RXRα KO mice have elevated serum cholesterol and triglyceride levels , and theTaken together, ligand (RA) treatment and hepatic RXRα deficiency resulted in opposite effects. Figure Additional analysis was done to understand which other nuclear receptors may be involved in regulating the expression of these 114 RA/RXRα target genes, which have a role in lipid homeostasis. The binding data generated in the current study (RXRα and RARα) were compared with the binding data of PXR, LXR, FXR, and PPARα. Figure ChIP-Seq and RNA expression profiling indicate the role of RA in controlling lipid homeostasis in the liver. Serum cholesterol, triglyceride, and bile acid levels were quantified to test the genetic findings. The data showed that RA reduced serum cholesterol, triglyceride as well as bile acid levels in wild type mice Figure . HoweverThis study establishes the role of nuclear receptors and RA in regulating lipid homeostasis in the liver. In addition, the mechanisms by which nuclear receptors and RA regulate lipid homeostasis were illustrated at the gene, pathway, and systemic levels. Furthermore, relationships between RXRα and PXR, LXR, FXR, as well as PPARα in regulating lipid homeostasis were analyzed. These nuclear receptors depend on RXRα to execute their functions and more than 85% of their bound genes overlap with those bound by RXRα. The nuclear receptor binding data were strengthened by profiling the expression patterns of 576 lipid genes, which showed that RA treatment and RXRα-deficiency had an opposite effect in regulating lipid homeostasis. Nuclear receptor binding data also illustrated extensive cross talk among the studied nuclear receptors. Thus, our novel in vivo data provided extensive evidence showing the role of RA in dictating lipid homeostasis in the liver.It is intriguing that more than 85% of the PXR, LXR, FXR, and PPARα binding sites overlapped with those of RXRα. In contrast, only 43% of RARα binding sites overlapped with those of RXRα. It has been shown that RARα can form homodimers . It is aClustering and PCA showed that the genome-wide binding pattern of RARα is not similar to that of PPARα, LXR, PXR, and FXR. Biological function annotation also showed that RARα has some unique features including protein processing, protein localization, and RNA processing. However, the five studied nuclear receptors also demonstrated functional redundancy. For example, there are four pathways, including oxidation reduction, carboxylic acid catabolic process, organic acid catabolic process, and cofactor metabolic process, that can be regulated by more than four nuclear receptors. This finding suggests the importance of these four pathways in the liver, and the role of RARα in them.Although PPARα, LXR, FXR, and PXR have extensive roles in regulating lipids, they also have specific roles in regulating different types of lipids. RXRα/PPARα prefers to bind to genes that participate in neutral lipids, glycerol ether, and organic ether as well as fatty acid metabolism processes. All of which are either fatty acid-derived products or precursors for the biosynthesis of fatty acids. Another pathway bound by RXRα/PPARα is the acylglycerol metabolic process, which is involved in triglyceride homeostasis. RXRα/LXR tends to regulate genes involved in sterol metabolism, which is consistent with its known role . RXRα/FXAll of the 114 genes that showed differential effects of RA treatment and RXRα deficiency are bounded by RXRα and RARα, PXR, LXR, FXR, and PPARα heterodimers. These findings indicate that those studied nuclear receptors retain RA response in vivo and the effect of RA is dependent upon those nuclear receptors. RA has a broad spectrum of effects including biosynthesis of retinoids, phospholipids, and unsaturated fatty acids. It also has a role in eliminating retinoids, oxidizing saturated fatty acids, and breaking down triglycerides. It seems that RA has extensive beneficial effects in maintaining the health of the liver. Specifically, RA induced the expression of Cyp2c37/38/50/54/70 and Cyp2j5. These genes encode enzymes involved in the generation of epoxyeicosatrienoic acids , which hTaken together, the differentiation and morphogenetic effect of RA is well known. However, the current study provides a comprehensive analysis of the role of RA in lipid homeostasis. All-trans RA is the most abundant retinoid that can be easily detected in the liver. Thus, RA is likely to act as a regulator to control hepatic lipid metabolism. Since the effect of RA is broad, it is important to develop specific retinoids in order to target specific pathways. Such efforts may allow us to identify compounds that can be used to treat or prevent metabolic syndromes and other lipid-related health issues.Male wild type mice (12 weeks old) and hepatocyte RXRα-deficient mice (KO) ,11, whicAll ChIP-grade antibodies except anti-RNA Pol II  were purchased from Santa Cruz Biotechnology, Inc. . DNA purification kit was purchased from Qiagen Co. . All other ChIP-related reagents were obtained from Invitrogen Co. .Retinoic acid  was given to wild type and hepatic RXRα-deficient mice at a dosage of 150 mg/kg diet for 7 days. As controls, mice were fed with normal diet.ChIP was performed according to our previously published study . After fBy using the End-It DNA End Repair Kit , DNA fragments prepared from ChIP were ligated with specified adaptors and amplified, then size-selected (175-225 bp) on an agarose gel followed by sequencing .http://hgdownload.cse.ucsc.edu/goldenPath/mm10/bigZips/) by Bowtie 0.12.7 [The target sequences were aligned to the mouse genome  was used to determine the genome-wide mRNA expression levels. Microarray data were annotated using Affymetrix Expression Console (MAS5). The probe signal with http://david.abcc.ncifcrf.gov/) [All data were treated with the same cut off criteria. The generated RXRα binding data were compared with the data for RARα, PXR, LXR, FXR, and PPARα. The principle component analysis (PCA) and cluster analysis package in SPSS program was used to analyze the global binding data. For both PCA and cluster analysis, called peaks were assigned the value of 1. Not called peaks were assigned the value of 0. Genes with overlapping binding sites of RXRα and each of RARα, PXR, LXR, FXR, and PPARα at the same location were functionally analyzed by the DAVID (rf.gov/) .http://www.genome.jp/kegg/). The expression of those 579 genes were determined in wild type and liver RXRα KO mice treated with and without RA (n = 3) for 7 days. After multiple comparisons, only 30 and 36 out of all 579 lipid homeostasis genes showed significant change at the corrected p-value of 0.05 after RA treatment and RXRα knockout, respectively. Therefore, IBM SPSS PCA package was used to differentiate groups based on the global expression pattern of all 579 lipid homeostasis genes.Genes (579) involved in regulating lipid homeostasis were extracted from the KEGG database  that was modified to a 96-well format. Spectrophotometric analysis was conducted with a Bio-Tek microtiter plate reader .http://www.ncbi.nlm.nih.gov/geo/).The microarray and ChIP-Seq data supporting the results of this article are available with accession numbers of GSE50028 and GSE46762, respectively, in the GEO repository (RA: Retinoic acid; ChIP: Chromatin immunoprecipitation; seq: Sequencing; RXRα: Retinoid x receptor alpha; RARα: Retinoic acid receptor alpha; PXR: Pregnane x receptor; LXR: Liver x receptor; FXR: Farnesoid x receptor; PPARα: Peroxisome proliferator-activated receptor alpha; DAVID: Database for annotation, visualization and integrated discovery; PCA: Principal component analysis; WT: Wild type; KO: Knockout; NR: Nuclear receptors; KEGG: Kyoto encyclopedia of Genes and genomes; PE: Phosphatidylethanolamine; PC: Phosphatidylcholine.The authors declare that they have no competing interests.YH: Performed experiments, analyzed data, generated figures and tables as well as prepared manuscript. LG: Microarray experiments and analysis. YF: Sequence alignment and call peaks. QZ: Functional annotation. H-XL: Animal experiments. YL: Statistics analysis. GLG: FXR ChIP Seq data generation. LL-M: Microarray data generation. JF: Sequence alignment and call peaks. Y-JYW: Generated idea and supervised the all overall performance of the project. All authors read and approved the final manuscript.


P ≥ 0.40). The association between walking speed and mortality was partially explained by baseline inflammatory markers (percentage reduction of the association 22.8%), height and body mass index (16.6%), chronic diseases (14.0%), and health behaviors (13.4%). Together these and other baseline factors  explained 48.5% of the association . In conclusion, walking speed measured in late midlife seems to be an important marker of mortality risk; multiple factors, in particular inflammatory markers, partially explain this association.Slow walking speed is associated with increased mortality in the elderly, but it is unknown whether a similar association is present in late midlife. Our aim was to examine walking speed in late midlife as a predictor of mortality, as well as factors that may explain this association. Data are drawn from the Whitehall II longitudinal cohort study of British civil servants. The analyses are based on 6,266 participants  for whom “walking speed at usual pace” was measured over 8 ft (2.44 m) at baseline. Participants were followed for all-cause and cause-specific mortalities during a mean of 6.4 (SD = 0.8) years. During this period, 227 participants died. Participants in the bottom sex-specific third of walking speed  had an increased risk of death compared to those in the middle and top thirds  = 1.45–2.46), with no evidence of effect modification by age or sex  contains supplementary material, which is available to authorized users. Lower walking speed in the elderly has been associated with increased all-cause  over a clearly marked 8-ft (2.44 m) walking course using a standardized protocol  were traced for mortality through the national mortality register  using the National Health Service identification number assigned to British citizens. Our analyses are based on deaths that occurred between phase 7 (2002–4) and January 31st, 2010. We examined all-cause, cardiovascular, and cancer mortalities; international classification of disease (ICD) codes identified cancer  and cardiovascular  deaths.Sociodemographic variables included age, sex, and socioeconomic status (SES) . Smoking history was assessed using questions on smoking status and current amount of tobacco smoked categorized as “current smoker at phase 7,” “recent ex-smoker” (stopped smoking between phases 1–7), “long-term ex-smoker” (stopped smoking before phase 1), and “never smoker.” Alcohol consumption was assessed via questions on the number of alcoholic drinks  consumed in the last 7 days at each phase. This was converted to number of units (1 unit = 8 g) of alcohol. The frequency of fruitand vegetableconsumption was assessed on an eight-point scale (ranging from “seldom or never” to “two or more times a day”). The number ofhours ofmoderate andvigorous physicalactivity perweek was calculated from a question on the number of hours of physical activity at different levels (phases 1 and 3) and from a 20-item questionnaire on frequency and duration of participation in walking, cycling, sports, gardening, housework, and home maintenance (phases 5 and 7)  was calculated as weight divided by height squared (kg/m²) measured at phase 7 clinical examination  , stroke, arthritis, respiratory illness, diabetes, and depression. CHD  was based on clinically verified events  were measured at phases 3 and 7 ; higher scores correspond to better function  and SAS 9.1 . Descriptive statistics were used to examine participants’ characteristics as a function of vital status and tertiles of walking speed. As men walked faster than women, walking speed was categorized using sex-specific tertiles, and these groups were compared using linear or logistic regression with adjustment for age and sex.For the survival analyses, participants were followed-up until death or until the date of censoring for those who were alive , whichever occurred first. The cumulative risk of all-cause death was plotted by tertiles of walking speed which are defined using sex-specific cutoffs , and the logrank test was used for statistical testing. Cox proportional hazards models with age as the time axis estimated hazard ratios (HR) and 95% CI. The proportional hazards assumption was verified using Schoenfeld residuals. Interactions were tested by including multiplicative terms in the models.i was calculated using the formula 100 × (βModel 1 − βiModel ) / (βModel 1), where β is the coefficient obtained from the Cox proportional hazards model.Model 1 included walking speed and sex as predictors and was controlled for age by considering it as the timescale. We examined the extent to which the association between walking speed and mortality was attenuated by adding the following covariates (models 2–8): SES; height and BMI, health behaviors, cardiovascular risk factors, chronic conditions, cognitive function, and inflammatory markers. The final model (model 9) included all covariates. The percentage reduction (PR) of the association between walking speed and mortality attributed to covariates included in model P < 0.001). Two hundred seventeen participants examined at phase 7 were not included in the analysis due to missing walking speed data: these persons were older , more likely to be women , and were from the lower SES group  than those who were included. Excluded participants had higher mortality (6.0% vs 3.6%), but this difference was not statistically significant after adjustment for age, sex, and SES (P = 0.15).The analyses were based on 6,266 participants, with complete data on walking speed and covariates, contributing to a total of 39,892 person-years. Compared to the baseline of the Whitehall II study , those included in the analysis were younger, more likely to be men, and were from the higher SES group  years, 227 of the 6,266 participants included in the analyses died : 111 from cancer  and 62 from cardiovascular disease . Table P < 0.001). There was a significant linear association between all other covariates and walking speed, except for alcohol consumption and history of respiratory disease. Subjects who walked slower had higher levels of inflammatory markers (P < 0.001). As shown in Fig. P < 0.001). The curves began to separate during the first year of follow-up and continued to diverge over the follow-up. Differences in cumulative risk of death were also evident for cardiovascular (P < 0.001) and cancer (P < 0.001) deaths (results not shown).Table P = 0.18) for participants in the middle tertile  and 2.19  for participants in the bottom . Because there was no statistically significant difference between the top and middle tertiles of walking speed for all-cause , further analyses combined these two groups. In sensitivity analyses, we also modelled walking speed as a continuous variable  showed the HR of death to be 1.30 . The association between walking speed and mortality was stronger in men (HR = 2.00 [1.46–2.75]) than women (HR = 1.68 [1.04–2.71]), but the difference was not statistically significant (P = 0.40); therefore, all analyses combine men and women and are adjusted for sex and age. SES (model 2), cardiovascular risk factors (model 5), and cognitive function (model 7) explained a small part of the walking speed–mortality association (PR = 3.3–6.4%). Adjustment for height and BMI (model 3), health behaviors (model 4), and chronic conditions (model 6) led to PRs of a similar magnitude (13.4–16.6%). The highest PR (22.8%) was on adjustment for inflammatory markers (model 8). All the covariates together explained nearly half (PR = 48.5%) the association; in the fully adjusted model, higher IL-6 (P = 0.04) and CRP (P = 0.04) remained associated with mortality. Analyses based on continuous walking speed yielded similar findings  or sex . For both causes, adjustment for inflammatory markers led to an important reduction in HRs , and all the covariates explained nearly half of the association . Analyses based on continuous walking speed yielded similar findings  are shown as supplementary results Table . The HR gs Table .N = 44); the association between walking speed and mortality remained unchanged for all-cause (HR = 1.80 [1.34–2.42]). Cardiovascular (HR = 1.99 [1.13–3.51]) and cancer mortalities (HR = 1.51 ) and cardiovascular mortalities (1.85 [0.90–3.79]) decreased, but adjustment for inflammatory markers still led to an important attenuation of the association . Using CES-D score rather than antidepressant use as a surrogate for depression, adjustment for forced expiratory volume/height² instead of history of respiratory disease, exclusion of NSAIDs users (N = 320), or participants with CRP level greater than 10 g/L (N = 103) on the day of examination led to conclusions similar to those in main analyses.In sensitivity analyses, we excluded deaths occurring within the first 2 years of follow-up  and was present in all age groups. Among a large number of covariates, inflammatory markers, and, to a lesser extent, chronic diseases, health behaviors, and height and BMI contributed to this association.Several studies have shown that slower walking speed is associated with increased mortality  Below is the link to the electronic supplementary material.


Arabidopsis thaliana co-expression data, we adopted a novel reverse-complementary distance-sensitive n-gram profile algorithm. This algorithm converts each upstream sub-sequence into a high-dimensional vector data point and transforms the prediction task into a classification problem using support vector machine-based classifier. Our approach showed significant improvement compared to other computational methods based on the area under curve value of the receiver operating characteristic curve using 10-fold cross validation. In addition, in the light of the highly skewed structure of the dataset, we also evaluated other metrics and their associated curves, such as precision-recall curves and cost curves, which provided highly satisfactory results.Biological prediction of transcription factor binding sites and their corresponding transcription factor target genes (TFTGs) makes great contribution to understanding the gene regulatory networks. However, these approaches are based on laborious and time-consuming biological experiments. Numerous computational approaches have shown great potential to circumvent laborious biological methods. However, the majority of these algorithms provide limited performances and fail to consider the structural property of the datasets. We proposed a refined systematic computational approach for predicting TFTGs. Based on previous work done on identifying auxin response factor target genes from  Transcription factors can also function as activators, which promote the expression of TFTGs. In addition to post-transcriptional gene regulations, there are also post-translational gene modification regulations, including biochemical alteration and RNA interference Unraveling the gene regulatory networks is regarded as one of the fundamental problems challenging biologists et al.n-gram profile algorithm based on the fact that a specific TF may bind on either a DNA strand or its reverse complement and produced satisfactory results. Following their work, Dai et al.et al.De novo methodology-based predictions did not require any model training based on the prior knowledge of TFTGs thus showing its advantages in terms of computational cost and classification accuracy et al.In order to circumvent the laborious biological experiments for screening TFBSs and their corresponding TFTGs, a number of computational algorithms have been proposed in the last decade on the basis of pre-established biological results n-gram profiling (RCDSNGP) algorithm designed to better capture the patterns of potential conserved motifs and their corresponding positions relative to recognized TFBSs. For feature selection, we adopted Monte Carlo simulation based on information gain (IG) measurements to select features that have a p-value smaller than 0.01. Finally, each upstream sequence of either TFTGs or non-TFTGs was represented by a single data point in a multi-dimensional feature space and was later fed to SVM to build prediction models.Using the well-documented information domain of the corresponding TFs, TFBSs, and TFTGs, we constructed a binary classifier based on support vector machines (SVM). A standard feature extraction, feature selection, model construction, and dataset testing paradigm was followed. The feature extraction region was limited to within 1000-bp upstream from the transcription start point. This frame was verified to contain the most amounts of TFBSs from previous biological studies Feature extraction, selection, model training, and testing were performed on the basis of a 10-fold cross validation (10-fold CV). That is, the entire dataset is randomly and evenly split into 10 disjoint subsets. Each subset contains a proportion of TFTG sequences similar to that of the pre-split dataset, e.g. 19 TFTGs +260 non-TFTGs  = 279. The final result is calculated from a composite of 10 trials. Within each trial, a different subset of samples is selected for testing and the other nine subsets of samples are used for training. Feature extraction and selection are performed within the nine training subsets during each trial, thus, selected features are different in each trial.et al.et al.A. thaliana treated with auxin and brassinosteroid and reported that only 186 out of 2787 genes containing the conserved motif ('TGTCTC' or 'GAGACA') in their 1000-bp upstream region were TFTGs.The procedures for generating sequence datasets were described previously by Dai et al.ftp://ftp.arabidopsis.org/home/tair/Genes/TAIR6_genome_release), the 1000-bp gene upstream sequences of A. thaliana with the conserved motif (186 TFTGs +2601 non-TFTGs  = 2787) were extracted from the genome sequences (ftp://ftp.arabidopsis.org/home/tair/home/tair/Sequences/whole_chromosomes). The entire dataset can be downloaded from the Samuel Roberts Noble Foundation online supplementary data source (available via http://bioinfo.noble.org/manuscript-support/TF_Supp/dataset/).By referring to the accession IDs verified by Goda n-gram profiling algorithm was used previously to represent a sequence stream by a set of n continuous characters and their corresponding frequencies k-mer approach used in other gene sequence studies Each upstream sequence has to be converted into a series of numerical values corresponding to its coordinates in a high-order feature space for SVM training and prediction purposes. An et al.Because of the double helix structure and base-pairing property of DNA, TFs may bind on either strand of a DNA molecule. Thus, a conserved motif and its reverse complement should be treated equally for each TF. In the light of this, He m-length sequence s = s1, s2…ms, the RCNP of s is a set of K 2-tuples, denoted as RCNP(s) = {,…}, kf being a distinct n-gram, kr being the reverse complement of kf, and kc being the sum of frequency counts of kf and kr in s. Additionally, {kf, kr}  includes all possible combinations of an n-gram and its reverse complement in s.Definition 1 (RCNP): Given an n-gram or its reverse complement will be counted equally as one increment of that feature . In addition, by limiting the feature extraction region within a finite window evenly neighboring the center motif (such as 100-bp window size with 50 bp on each flank), He et al.n-gram is to the primary TFBSs, the stronger its influence on regulating TF binding processes. An optimal area under curve (AUC) value of 0.8949 was obtained on a similar dataset using this RCNP algorithm The essence of RCNP was that an occurrence of either an et al.'s work, Dai et al.n-gram profile (RCPSNP) algorithm by incorporating a positional information parameter and a position-sensitive parameter into the RCNP. The position sensitive parameter was introduced to mainly account for the possibility that two identical n-grams extracted from a certain window flanking the center motif on the same DNA strand may have similar impacts on regulating TF binding processes regardless of their positional differences. This feature generation scheme yielded an AUC value of 0.73 for the receiver operating characteristic (ROC) curve Immediately after He n-gram with a signed integer representing its direction and distance relative to the primary TFBS as proposed by Dai et al.n-gram profile (RCDSNGP), formalized as follows.In this study, we propose an improved feature generation algorithm. Studies have shown the existence of a composite structure containing constitutive elements adjacent to the 'TGTCTC' binding site for ARFs m-length sequence s = s1, s2… is…i+js…sm, the RCDSNGP of s with respect to a j-length reference subsequence x = is…i+j−1s is a set of K 2-tuples, denoted as RCDSNGP(s) = {,…}, kf being a distinct n-gram, kr being the reverse complement of kf, kd being the relative distance parameter, and kc being the sum of frequency counts of kf and kr with the same kd relative to x in s. Additionally, {kf, kr, kd}  include all possible combinations of an n-gram, its reverse complement, and its distance to x in s.Definition 2 (RCDSNGP): Given an kf or kr as an n-gram, g = t, st+1s…t+n−1s (n−1<t+n−1<i or m−n+2>t>i+j−1), then its relative distance to x is calculated as follows.If we denote either kf, kr, kd}) within a 2-tuple of an RCDSNGP is a reverse-complementary distance-sensitive n-gram (RCDSNG), synonymously a feature in our study.Each set .By adopting RCDSNGP, an occurrence of either an n-grams of n = 4–9 for profiling each upstream sequence using RCDSNGP algorithm because n = 4–9 were verified (data not shown) to give optimal performances and can be handled efficiently in a moderate computing environment. Given the maximum distance maxd, a total number of maxd×4n features at most can be generated for each n, which is less than half of the number produced by Dai et al. We used maxd, 10 trials were conducted to generate the final result (10-fold CV). Within each trial, a separate IG-based feature ranking was used on the nine training subsets S, which contained e.g. N = 2509 (168 TFTGs +2341 non-TFTGs  = 2509) upstream sequences and two distinct class labels y = 2), can be calculated asS belonging to TFTGs and non-TFTGs, respectively.Given a f, we can partition the original set S into two distinct subsets: f; f. Thus, S = {y = 2. The entropy of S with respect to f is evaluated asf and no occurrence of f are denoted by f and no occurrence of f are denoted by S according to f is calculated using:After observing a specific feature p-value for a specific feature was calculated according toN is the total number of shuffling (N = 10 000). The smaller the p-value is, the stronger the contribution the feature would provide to differentiate a sample between two classes. In our study, features were considered important at p<0.01 level.Instead of ranking features based on IGs and subjectively choosing the cutoff value, we further evaluated each feature using Monte Carlo simulations F features whose p-values were smaller than 0.01 from the total of 493 781 all possible features . Each 1000-bp upstream sequence was represented by an RCDSNGP consisting of these F features and their corresponding occurrences. The whole set of N sequences (e.g. N = 168 TFTGs +2341 non-TFTGs  = 2509) were then represented by an N×(F+1) matrix (1 extra column for class labels).Within each trial, we selected Support vector machine-based approaches have been widely used in various problem domains, such as bioinformatics N = 2509); n is the dimension of For model training, given a set of vector-label pairs et al.C of SVM and a kernel width This is equivalent to finding the maximum-margin hyperplane that separates TFTG samples (labeled as 1) and non-TFTG samples (labeled as -1) with minimized measures of errors. Predictions are made based on the geometric location of an unknown sample when fed into the model. A label is assigned to a sample according to which side of the hyperplane it resides. In order to obtain better classification accuracies, data are often projected into a high-dimension feature space with a kernel function. We evaluated other possible kernels in addition to the linear kernel suggested by Dai F1First, we measured the traditional accuracy defined asFurthermore, we adopted the ROC curve technique, which demonstrated satisfactory performances across different classifiers and datasets from previous studies et al.maxd = half of the window size) on each flank of the central TFBSs, including maxd = 25, 50, 75, 100, 125, 150, 175, and 200 bps.Dai maxd settings within each trial on the basis of 10-fold CV. Within each maxd setting during each trial, an IG-based p-value selection procedure was adopted to select the important (p<0.01) features based on the training dataset (nine folds out of 10). Immediately following that, we located the optimal combination of C for SVM and σ for the RBF kernel using the grid  search. Thus, the optimal combination of features, C, and σ depended on the training dataset within each trial. The final results, including accuracy, precision, recall, and F1 as well as the corresponding AUC values were generated from the combined classification results containing all 2787 sequences according to different maxd settings.To summarize, we evaluated eight different maxd = 150 and n = 4–9 for building RCDSNGP, 2 455 252 unique features were generated from 1000-bp upstream of 2787 sequences (186 TFTGs +2601 non-TFTGs). We eliminated singleton features that contain only 1 occurrence across all sequences to reduce the feature size down to 735 624. The same technique was applied to all other settings of maxd as well.Using a maximum distance p-value for that feature. We selected those features that had p-values smaller than 0.01. For different maximum distances, maxd = 25, 50, 75, 100, 125, 150, 175, and 200, we selected 893, 1870, 2732, 3502, 4255, 4949, 5622, and 6136 unique features, respectively on the basis of 10-fold CV , with n-grams of n = 4–9. The RBF kernel provided the best performances regardless of measurement metrics used. In our study, the best accuracy (0.9602), precision (0.8319), and F1 (0.6288) values were obtained with maxd = 150. The best recall (0.5215), ROC-AUC (0.7739), and PR-AUC (0.5879) values were obtained with maxd = 100, 75, and 200, respectively . Combining different evaluation metrics tends to provide comprehensive assessment of classification on imbalanced datasets. When maxd = 25, our model suffered from low recall rate because of its poor accuracy when classifying the positive samples (TFTGs). Starting from maxd = 50, a boost in performances was observed in recall and F1 values as a result of increased accuracy in predicting positive samples. At maxd = 150, the model reaches the highest accuracy value of 0.9602. The best precision value is 0.8319 when maxd = 150 and declines a little as maxd increases. Starting from maxd = 100, recall values are above 0.5 and peak at maxd = 100 with a value of 0.5215. Likewise, the F1 scores are above 0.6 when maxd is bigger or equal to 100 and reach the greatest value of 0.6288 when maxd = 150. maxd curve based on both the ROC curve and the PR curve. The ROC-AUC value arrives at 0.7739 when maxd = 75. A slight decrease is detected when maxd>100. Furthermore, by varying maxd the PR-AUC value responds more obviously than the ROC curve. Beginning at maxd = 100, our model produces above-0.56 PR-AUC values, which gradually decrease from maxd = 100 to maxd = 125 and peaks at 0.5879 when maxd = 200. Overall, a 150-maxd setting is likely to give a superior performance with limited complexity of computation compared to other maximum distance settings. Although it fails to produce the optimal AUC values for either ROC or PR curve, it provides the best accuracy, precision, and F1 values. Considering the fact that most maxd value settings perform well in detecting the correct negative samples (non-TFTGs), the model that is most capable of identifying the correct positive samples  yields the best results. A maximum distance maxd = 200 provides great PR-AUC values. However, it adds great computational cost for feature generation and selection processes . It represents the proportion of positive samples when the classifier is deployed. Thus, at x = 0 (no positive samples), the only possible misclassification errors are FPs. Likewise, at x = 1 (no negative samples), the only possible misclassification errors are FNs. The straight line connecting these two points represents the trend of misclassification cost as percentage of positive samples varies. The lower envelope generated by a non-discrete classifier such as SVM or Artificial Neural Network is the counterpart for the upper convex hull of the ROC curve. At each probability cost value, the closer the curve is to the x-axis, the better the classifier performs (a lower expected cost). As presented in p<0.05) from RCPSNP-based model within the 4.8 to 65 percent range of positive samples using the method proposed by Drummond and Holte Finally, we evaluated the cost curve of each model. The cost curve, which emphasizes the expected misclassification cost for performance measure, is proposed to better visualize the misclassification cost and compare performances on the basis of statistical significance A. thaliana based on published datasets maxd = 150 using the RBF kernel based on an average of 2395 features. We adopted the ROC measure for efficacy evaluation and obtained an ROC-AUC value of 0.7626 (SE = 0.0021), accuracy value of 0.9602 (SE = 0.0023), precision value of 0.8319 (SE = 0.0331), recall value of 0.5054 (SE = 0.0231), and F1 score of 0.6288  based on the same dataset but with a different 10-fold CV split.To summarize, using new feature generation and selection strategies to predict TFTGs of ARFs in et al.et al.maxd = 150 setting and the sequence conservation was evaluated using a four (four nucleotides) by 300 (2×maxd) position frequency matrix (150-bp flanking each side of the primary conserved motif). At each residue (1 out of 300), we considered 20-bp window size  to construct the frequency count for each nucleotide. The standard SVM-based training and testing was performed based on generated PSSMs. Likewise, the algorithm only provided an ROC-AUC value of 0.5569 and a PR-AUC value of 0.0801 using the same 10-fold CV split as our RCDSNGP algorithm  and applied it to the same 10-fold CV split as our RCDSNGP algorithm, which yielded an ROC-AUC value of 0.5055, PR-AUC value of 0.0773, accuracy value of 0.9300, precision value of 0.2000, recall value of 0.0161, and F1 score of 0.0299 , a top F representative features based on IG, and a position sensitive factor P . The best result was obtained when n = 4–9, C = 100, P = 100, and F = 1000. Their detailed results containing prediction scores can be found in their supplementary web data C equals P. In other words, RCPSNP behaves almost the same as RCNP C and P hold the same value. The significant performance increase based on our RCDSNGP algorithm indicated that ARFs function by recognizing multiple consensus motifs that might be co-occurring TFBSs or subsequences functioning coordinately. More importantly, the relative distance from each motif to the binding site should always play an important role in the gene regulation process. The structural complexities of protein and DNA may result in a type of mutual recognition that relies more on the distance from the conserved motif to the TFBS, regardless of where the motif lies . The PSSM-based approaches may be useful for TFTG identification when more associated TFBSs are known.We deployed a novel feature extraction method (RCDSNGP) that incorporated a relative distance parameter into each feature to count for the positional information of each motif relative to the central TFBS. For feature selection, we adopted a Monte Carlo simulation-based statistical approach rather than arbitrarily choosing thresholds. We compared our results with the RCPSNP-based approach n-grams is a computationally expensive work. The complexity becomes even greater when a distance parameter is included. Therefore, better feature selection methods become necessary. We employed a statistical systematic approach. Based on a given feature, two class samples are different from each other if, and only if, the probability of obtaining a bigger IG value than original is below a certain level . This probability value is obtained using Monte Carlo simulations p-values (between 0.005 and 0.01). However, when the p-value becomes bigger, feature number increases drastically, which greatly increases computational cost. Moreover, we also evaluated a number of important features that have a p-value smaller than the 0.01 threshold versus different maxd values (data not shown). The slope of the curve reached the maximum value between maxd = 25 and maxd = 50 and began to dwindle when maxd>50, suggesting that flank regions closer to the core motif contain more important features for predicting TFTGs.Identifying patterns of other potential TFBSs and the binding property of ARFs by enumerating all possible Precision-recall curve is used in information retrieval as an alternative to ROC curve when analyzing imbalanced datasets A. thaliana. It might also provide new insights into the understanding of gene regulatory networks.Taken altogether, the RCDSNGP algorithm combined with statistical feature selection methods provides an efficient and highly accurate way to predict TFTGs on the basis of well-studied TFBSs. We believe that this improved methodology can be employed when analyzing other species besides