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https://f1000research.com/articles/3-108/v1
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13 May 14
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{
"type": "Research Article",
"title": "Dynamin1 concentration in the prefrontal cortex is associated with cognitive impairment in Lewy body dementia",
"authors": [
"Julie Vallortigara",
"Sindhoo Rangarajan",
"David Whitfield",
"Amani Alghamdi",
"David Howlett",
"Tibor Hortobágyi",
"Mary Johnson",
"Johannes Attems",
"Clive Ballard",
"Alan Thomas",
"John O’Brien",
"Dag Aarsland",
"Paul Francis",
"Sindhoo Rangarajan",
"David Whitfield",
"Amani Alghamdi",
"David Howlett",
"Tibor Hortobágyi",
"Mary Johnson",
"Johannes Attems",
"Clive Ballard",
"Alan Thomas",
"John O’Brien",
"Dag Aarsland",
"Paul Francis"
],
"abstract": "Dementia with Lewy Bodies (DLB) and Parkinson’s Disease Dementia (PDD) together, represent the second most common cause of dementia, after Alzheimer’s disease (AD). The synaptic dysfunctions underlying the cognitive decline and psychiatric symptoms observed throughout the development of PDD and DLB are still under investigation. In this study we examined the expression level of Dynamin1 and phospho-CaMKII, key proteins of endocytosis and synaptic plasticity respectively, as potential markers of molecular processes specifically deregulated with DLB and/or PDD. In order to measure the levels of these proteins, we isolated grey matter from post-mortem prefrontal cortex area (BA9), anterior cingulated gyrus (BA24) and parietal cortex (BA40) from DLB and PDD patients in comparison to age-matched controls and a group of AD cases. Clinical and pathological data available included the MMSE score, neuropsychiatric history, and semi-quantitative scores for AD pathology (plaques - tangles) and for α-synuclein (Lewy bodies).Changes in the expression of the synaptic markers, and correlates with neuropathological features and cognitive decline were predominantly found in the prefrontal cortex. On one hand, levels of Dynamin1 were significantly reduced, and correlated with a higher rate of cognitive decline observed in cases from three dementia groups. On the other hand, the fraction of phospho-CaMKII was decreased, and correlated with a high score of plaques and tangles in BA9. Interestingly, the correlation between the rate of cognitive decline and the level of Dynamin1 remained when the analysis was restricted to the PDD and DLB cases, highlighting an association of Dynamin1 with cognitive decline in people with Lewy Body dementia.",
"keywords": [
"Alzheimer’s disease",
"Dementia with Lewy bodies",
"Parkinson’s disease with dementia",
"synaptic dysfunction",
"vesicle recycling",
"synaptic plasticity",
"beta amyloid",
"tau",
"cognitive impairment"
],
"content": "Introduction\n\nDementia with Lewy Bodies (DLB) and Parkinson’s disease dementia (PDD) are together the second most common cause of dementia after Alzheimer’s disease (AD) and account for 15–25% of dementias1. They are both characterised by progressive cognitive decline, visual hallucinations and Parkinsonism2,3. Lewy body dementias (including DLB and PDD) are neuropathologically defined by insoluble α-synuclein aggregates in neuronal somata, forming Lewy bodies and Lewy neurites in neuronal processes2. However, it has been widely reported that Alzheimer-type pathology (most often amyloid plaques, but also to lesser extent neurofibrillary tangles) often coexists with Lewy body pathology4–6. When compared to the large-scale cortical atrophy of AD and the dopaminergic neuronal cell loss of PD, cortical cell loss in DLB and PDD is less extensive7, suggesting that alternative mechanisms may be responsible for the symptoms associated with DLB and PDD. In addition to Lewy bodies and Lewy neurites, small aggregates of α-synuclein have been identified pre-synaptically in DLB and PDD, raising the possibility of a deleterious effect on synaptic function8,9. Alongside this, interactions between α-synuclein and synaptic vesicle (SV) recycling proteins have previously been described10, suggesting that SV proteins are affected by the alterations in concentration of pre-synaptic α-synuclein11. It has also been shown that dysfunctional vesicle regulation can lead to dementia-like cognitive deficits12.\n\nIn AD there is substantial synaptic loss, which was revealed to provide a better indicator for cognitive impairment than the classical AD morphological changes13. Although there is emerging evidence of synaptic pathology in PD14, much less is known regarding the molecular basis and clinical consequences of the synaptic pathology associated with PD and DLB compared to AD, which may provide an opportunity for therapeutic intervention, through restoration of neurotransmitter tone.\n\nDynamin1 is a ~ 100kDa protein with GTPase activity, that is involved in many intracellular trafficking processes including SV recycling, neurotransmitter reuptake and receptor internalization15,16. A considerable body of evidence supports a key role in synaptic transmission. For example, Dynamin1 interacts with other endocytotic proteins including amphiphysin, endophilin and syndapin through its C-terminal proline rich domain (PRD), and as a consequence, pharmacological inhibition leads to disturbance of normal SV- and endosome formation17. Furthermore, Dynamin1 knockout mice demonstrate defects in SV endocytosis during strong, but not mild, neuronal activity18. In addition, Dynamin1 may play a key role in establishing and maintaining mature neuronal structure19,20. For example, dynamin1 is upregulated during new neurite formation21 and is down-regulated during neurite retraction, and furthermore, silencing the initiation codon for dynamin significantly hampers the formation of axon-like structures.\n\nCa2+/calmodulin-dependent protein kinase II-α (CaMKII) is a protein kinase highly concentrated in the brain and implicated in synaptic plasticity mechanisms22–24. Synaptic activity-triggered Ca2+ influx through NMDA receptor channels can activate CaMKII and promote its autophosphorylation at Thr286, which results in a persistently active form of the kinase25,26. The resulting CaMKII activation is likely to occur at both pre-and postsynaptic sites. At the presynaptic site, CaMKII-mediated phosphorylation of synapsin1 promotes its dissociation from synaptic vesicles, causing increased neurotransmitter release27. Activation of a calcineurin-dependent phosphatase pathway, however, can dephosphorylate CaMKII and reduce its activity28. Interestingly, Calcineurin, which is also activated by calcium, can dephosphorylate Dynamin1 and therefore regulates the SV retrieval processes16.\n\nThe synaptic dysfunctions underlying the cognitive decline and psychiatric symptoms in DLB and PDD are still poorly understood. The relative lack of frank neurodegeneration in DLB and PDD combined with the potential importance of synaptic pathology, together with the key role Dynamin1 and CaMKII play in synaptic neurotransmission led us to propose the hypothesis that dysfunctional synaptic plasticity and disrupted vesicle recycling may contribute to cognitive decline. We therefore investigated the concentrations of these two key proteins in prefrontal cortex, anterior cingulate, and parietal cortex regions of DLB and PDD in comparison to controls and AD in relation with cognitive decline as assessed by serial measurements of the Mini-Mental State Examination (MMSE) together with semi-quantitative assessments of plaques, tangles and Lewy bodies within those regions.\n\n\nMaterials and methods\n\nPost-mortem brain tissue was obtained from several sources; University Hospital Stavanger (Norway), the MRC London Neurodegenerative Diseases Brain Bank, the Thomas Willis Oxford Brain Collection and the Newcastle Brain Tissue Resource. The UK brain banks are part of the Brains for Dementia Research Network. All participants gave informed consent for their tissue to be used in research and the study had ethics approval from the National Research Ethics Service (08/H1010/4). Neuropathological assessment was performed according to standardised neuropathological scoring/grading systems, including Braak staging, Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) scores, Newcastle/McKeith Criteria for Lewy body disease, National Institute on Aging - Alzheimer’s Association (NIA-AA) guidelines and phases of amyloid-β (Aβ) deposition (Aβ-phases)2,29–32. Controls were cognitively normal, with only mild age-associated neuropathological changes (e.g., neurofibrillary tangle Braak stage <II) and no history of neurological or psychiatric disease.\n\nPatients were followed prospectively with annual assessments including standardized instruments of cognitive, motor and neuropsychiatric symptoms. Cognitive impairment data consisted of the last Mini-Mental State Examination (MMSE) scores a maximum of two years prior to death33. Final diagnoses for patients are clinico-pathological consensus diagnoses incorporating the one-year rule to differentiate DLB and PDD2. Table 1 shows the demographic details of the patients and controls. Biochemical and histopathological analysis was undertaken on prefrontal cortex (Brodmann area, BA9), anterior cingulate gyrus (BA24) and parietal cortex (BA40). BA9 was selected due to its proposed role in executive function and cognition34, decline of which is a cardinal symptom of DLB and PDD, BA24 was selected for the early development of pathology encountered in this region in DLB and PDD35 whilst BA40 was selected because of its pathological predominance in AD as opposed to DLB and PDD36.\n\nValues are mean ± SEM. DLB: Dementia with Lewy Body; PDD: Parkinson’s Disease Dementia; AD: Alzheimer’s Disease. PMD: post mortem delay. Age at death, PMD and pH are mean values, MMSE is the median score prior to death with range in brackets.\n\nSemi-quantitative assessments of Aβ, tau and α-synuclein pathology were conducted blind to clinical diagnosis, by neuropathologists, using a scale of 0 (none), 1 (sparse), 2 (mild) and 3 (severe/frequent) to score sections from BA9, BA24 and BA40 according to published criteria35,36. For detection of senile Aβ plaques sections were stained with an anti-Aβ 1E8 (gift from GSK) or 4G8 antibody (Covance SIG39220 mouse monoclonal) raised to Aβ17-24, at 1:1000. Tau immunohistochemistry (AT8 antibody (Innogenetics) at 1:200) and silver impregnation (Gallyas or modified Bielschowsky) were used to detect neurofibrillary tangles, neuritic plaques, dystrophic neurites and neuropil threads. α-Synuclein pathology was detected using NCL-SYN antibody (Novacastra Laboratories) at 1:200.\n\nPreparation of tissue for western blotting was as previously described37. Briefly, 500mg of frozen tissue was taken from each brain region. Meninges, white matter, blood vessels and clots were dissected from the frozen tissue to leave approximately 200mg of grey matter which was homogenised in ice cold buffer containing 50mM Tris-HCL, 5mM EGTA, 10mM EDTA, ‘complete protease inhibitor cocktail tablets’ (Roche, 1 tablet per 50ml of buffer), and 2μg/ml pepstatin A dissolved in ethanol:DMSO 2:1 (Sigma). Buffer was used at a ratio of 2ml to every 100mg of tissue and homogenisation performed using an IKA Ultra-Turrax mechanical probe (KIA Werke, Germany) until the liquid appeared homogenous.\n\nProtein concentration was established using the Coomassie (Bradford) Protein Assay Kit (Thermo Scientific), briefly 10μl of crude homogenate was diluted 1:50 and read in triplicate at 595nm using a FlexStation 3 (Molecular Devices). Concentration was calculated using a BSA standard curve run at the same time as samples.\n\nCrude brain homogenate was diluted 4:5 with 5× sample buffer (Genscript MB01015), boiled for 5 minutes then stored at -20°C. Samples were loaded at 20μg/ml total protein on 10% SDS-polyacrylamide gel for protein separation, transferred to nitrocellulose membrane (Hydrobond-C, Amersham) and probed with either anti-totalCaMKIIα (Santa Cruz sc-136212, 1:10000) or anti-phospho-CaMKII (Santa Cruz sc-12886-R, 1:200) and the relevant secondary antibody (IRDye from LI-COR, anti-mouse for total CaMKIIα and anti-rabbit for phospho-CaMKII). Bands were detected using an Odyssey infrared fluorescent scanner, the integral of intensity quantified using Odyssey infrared imaging systems application software version 3.0.16 and expressed as ratios to rat cortex in arbitrary units.\n\nUsing the crude brain homogenates already prepared, tissue debris was then removed by centrifugation maintained at 15000× g for 15min at 4°C. The resultant supernatant was collected and the protein concentrations were determined using the Coomassie (Bradford) Protein Assay Kit as previously described. Dynamin1 concentration was measured using a commercial ELISA kit developed by USCNLIFE TM (Wuhan China). The microtiter plate was pre-coated with biotinylated polyclonal antibody specific to Dynamin1. 100μl standards or samples (10 × diluted) were added and incubated for two hours at 37°C. 100μl detection reagent A (avidin conjugated to Horseradish Peroxidase (HRP)) was added to each micro plate well and incubated for 1hr at 37°C. After washing 4 times, 100μl of a TMB (3,3’,5,5’ tetramethyl-benzidine) substrate solution was added to each well and incubated for 1hr at 37°C. Colour developed in proportion to the amount of bound analyte. Finally, the enzyme-substrate reaction was terminated by adding 1N sulphuric acid. Quantification of Dynamin1 was achieved measuring colour changes using a spectrophotometer at a wavelength of 450nm. The concentration of Dynamin1 in the samples was determined by comparing the optical density (OD) of the samples to the standard curve. Within-assay precision for a replicated sample on the same plate (%CV of intra-assay variation) for a selected sample was < 6%. The inter-assay variability, (for same sample analyzed on three different plates) was < 15%.\n\nThe normality of the data for each protein was determined using the Shapiro-Wilk test and normalised where necessary. In each case, the protein values were subsequently expressed as residuals (unstandardised) created from the multivariable regression analysis, to eliminate the confounding effect of the demographic variables (gender, post mortem delay (PMD), age at death, length of brain storage) on the protein values. Unstandardised residuals were used in all subsequent analyses. We tested for differences in protein levels between groups using one-way ANOVA and Bonferroni post-hoc test or Kruskall-Wallis ANOVA followed by Mann Whitney U test as appropriate Intercorrelations of neurochemical variable and correlations with demographic and clinical features were examined using Pearson product moment (r) or Spearman rank (Rs) correlation as appropriate. Statistical analyses were conducted using SPSS version 20.\n\n\nResults\n\nDemographics of the cohort used are summarised in Table 1. AD patients were significantly older at death (one-way ANOVA F(3;126)=6.044, p=0.001) than controls (p=0.001) or patients with DLB (p=0.008) or PDD (p=0.001). There were no significant differences in PMD, tissue pH or gender between diagnostic groups.\n\nThe results for protein expression in the prefrontal cortex, anterior cingulate and parietal cortex are shown in Figure 1, Figure 2, and in Table 2. Dynamin1 protein levels were not significantly different between groups (Kruskall-Wallis p=0.682 for BA9; p=0.120 for BA24, Figure 1 and dataset 3 for BA40). On the other hand, changes were found for the expression of CaMKII and the fraction of activated form, i.e. phospho-CaMKII (Table 2). Indeed, the phospho-/total CaMKII ratio was significantly lower in the AD group compared to control, PDD and DLB groups in the prefrontal cortex (one-way ANOVA F(3,115)=7.129, p<0.001; Figure 2). In the anterior cingulate cortex, this ratio was decreased in all three dementia groups compared to controls (Kruskall-Wallis χ2(3)=14.44, p=0.003; Figure 2). In the same brain region, although the level of Dynamin1 was decreased in the AD group, there was no significant difference between groups. In the parietal cortex, expression of phospho-CaMKII was significantly lower in PDD, DLB and AD compared to controls, with a stronger decrease in the AD group (Kruskall-Wallis χ2(3)=35.942, p<0.001; Table 2). The level of the ratio phospho-/total CaMKII in the DLB group was decreased compared to other groups (one-way ANOVA F(3,114)=3.45, p=0.019; Figure 2).\n\n(A) BA9, (B) BA24, PDD: Parkinson’s Disease Dementia; DLB: Dementia with Lewy Body; AD: Alzheimer’s Disease. Bars represent mean and error bars SEM.\n\n(A) BA9, (B) BA24 and (C) BA40. Bars represent mean of ratio phospho/totalCaMKII and error bars SEM. ** (p<0.05) and ** (p<0.01) compared to controls, # (p<0.05), ## (p<0.01) and ### (p<0.001) between dementia groups.\n\nValues represent the means of relative intensity band measurements ± SEM. Number in brackets is the number of cases. * (p<0.05), ** (p<0.01) and *** (p<0.001) compared to controls.\n\nFigure 3 summarises the relationships found between synaptic markers and semi-quantitative scores of AD pathology in BA9 and BA40. The ratio phospho-/total CaMKII was decreased, with a high score of plaques (Kruskall-Wallis χ2(3)=8.549, p=0.036), and medium and high scores of tangles (one-way ANOVA F(3,111)=5.375, p=0.002) in BA9. On the other hand, phospho-CaMKII was significantly decreased with high scores of plaques and tangles in BA40 (one-way ANOVA F(3,111)=5.227, p=0.002 for plaques; one-way ANOVA F(3,112)=9.282, p<0.001 for tangles). There was no correlation between Dynamin1 concentration and neuropathological scores in BA9 or BA40 (one-way ANOVA, p>0.05, see data sets). No significant relationships were found between any neurochemical variable and pathological features in the anterior cingulate cortex (one-way ANOVA, p>0.05, see data sets).\n\n(A) ratio phospho/totalCaMKII level with plaques scores in BA9, (B) ratio phospho/totalCaMKII with plaques scores in BA9, (C) phosphoCaMKII with plaques scores in BA40 and (D) phosphoCaMKII with tangles scores in BA40. Scatter plots represent values and bars the mean for each group. º (p<0.05), ºº (p<0.01) and ººº (p<0.001).\n\nA significant association between MMSE decline per year and the level of Dynamin1 was observed in BA9, with a decrease in Dynamin1 level with the rate of cognitive decline (r=-0.280, p=0.019, n=70; Figure 4). This correlation is also significant when the analysis was restricted to the PDD and DLB cases (r=-0.327, p=0.014, n=56). In the same brain region, the ratio phospho/totalCaMKII was positively correlated to the MMSE scores before death (r=0.256, p=0.024, n=78), highlighting a decrease in the fraction of activated CaMKII protein with the cognitive deficit. However, when the AD group was excluded from the analysis, there was no correlation between phospho-CaMKII and MMSE.\n\nCorrelation between values of Dynamin1 level in BA9 in dementia cases and the MMSE decline per year.\n\n\nDiscussion\n\nThe main findings of this study are that the Dynamin1 level in prefrontal cortex, while unaltered between diagnostic groups, was related to the rate of cognitive decline observed in our cohort of people with Lewy body dementia (DLB and PDD) and AD; this relationship remained in the cohort of LBD cases only (when the AD group was excluded from the analysis). On the other hand, altered p(Thr286)CaMKII levels in AD, PDD and DLB parietal cortex, and in AD prefrontal cortex, were associated with high scores of plaques and tangles.\n\nPrevious studies have identified decreased expression of Dynamin1 in AD cases, showing particularly a degradation of Dynamin1 at early phase of AD38–40. A decrease in Dynamin1 expression could create defects in synaptic vesicle recycling, neurotransmitter reuptake and receptor endocytosis18. This would lead to a diminished ability of the neuron to regulate synaptic transmission and may prove to be an example of one of the processes that initiate synaptic dysfunction in dementia. However, Dynamin1 has not previously been implicated in LBD. In our cohort, there was no significant difference in the level of Dynamin1 between diagnosis groups, neither was there a correlation between Dynamin1 and neuropathological features. This is particularly surprising for AD pathology, as some potential links between Dynamin1 expression and Aβ have been reported41,42. However, these differences could relate to the relatively small comparison group used for this study. Nevertheless the progressive decrease in Dynamin1 in BA9 with the rate of MMSE decline provides new evidence for a role of this protein in cognitive dysfunction in DLB and PDD.\n\nThe essential role of CaMKII in long-term synaptic plasticity and cognitive function is well documented22–24. Here we confirmed this finding with a decreased level of phospho-/total CaMKII, specifically in prefrontal cortex of the AD group. In addition, the level was significantly lower in the prefrontal cortex of dementia patients with a high score of plaques. Moreover, a lower expression of p(Thr286)CaMKII was observed in parietal cortex of patients with a high score of plaques. An interesting finding in another study was the co-localization of CaMKII-α with senile plaques (SPs)43. With respect to the AD-related neuropathology, in vitro experiments indicated that CaMKII might participate in the tau protein phosphorylation44,45. Simonian et al.46 found that the majority of tangle-bearing neurons in AD brain expressed CaMKII. We also found an association between the level of p(Thr286)CaMKII and phosphoTau, with a decrease in p/totalCaMKII in prefrontal cortex and phospho-CaMKII in parietal cortex of dementia patients with a moderate or severe spread of plaques. However, when the AD group is excluded from the analysis, there were no correlations between phospho-CaMKII and MMSE or pathological features in any brain region. This is a key finding, as it may rule out the potential role of this kinase in molecular mechanisms leading to the development of cognitive decline in Lewy body dementia. However, it does appear to confirm previous reports of impaired phosphorylation CaMKII in AD47,48.\n\nTaken together, previous studies and the findings reported here suggest that Ca2+ dysregulation in AD, and LBD within selected brain regions, may be sufficient to initiate a deregulation in CaMKII- and Dynamin1-dependent molecular pathways, and that this, in turn, may contribute to cognitive decline.\n\n\nData availability\n\nfigshare: Data for synaptic proteins expression levels and pathological scores in prefrontal cortex, anterior cingulate gyrus and parietal cortex, http://dx.doi.org/10.6084/m9.figshare.98708749",
"appendix": "Author contributions\n\n\n\nPTF, TH, JA, JOB, DA conceived the study and obtained funding. JV and PTF designed the experiments. JV and SR carried out the research. DRW and AA contributed to the design of experiments and provided expertise in western blotting. JV and PTF prepared the first draft of the manuscript. DH contributed to the experimental design and preparation of the manuscript. TH, MJ and JA contributed to the collection of Neuropathological scores, CA, AJT, JOB and DA contributed to the collection of Clinical information. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe authors would like to express their gratitude to the Alzheimer’s Society who were the principal funders and to the BUPA Foundation for additional funding for this study. Grant reference number 109 to Pr P. Francis as lead investigator. Mary Johnson is funded by a grant from the Dunhill Medical Trust (R173/1110).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nHuman brain tissue was supplied by MRC London Neurodegenerative Diseases Brain Bank, The Thomas Willis Oxford Brain Collection and the Newcastle Brain Tissue Resource, which are all part of the Brains for Dementia Research Network. In particular, we thank Dr. Claire Troakes at the MRC London Neurodegenerative Diseases Brain Bank, and Dr Catharine Joachim at the Thomas Willis Oxford Brain Collection for assistance in obtaining relevant clinical information. We would like to gratefully acknowledge all the donors of the tissue used in this study.\n\nThis Newcastle Brain Tissue Resource is supported by the National Institute for Health Research (NIHR) Newcastle Biomedical Research Unit based at Newcastle upon Tyne Hospitals NHS Foundation Trust and Newcastle University and the Medical Research Council and Brains for Dementia Research. The MRC London Neurodegenerative Diseases Brain Bank is funded by the Medical Research Council and Brains for Dementia Research. We would like to thank Dr Stephen Newhouse at the Institute for Psychiatry, KCL, London, for advice on the statistical analysis and the NIHR Biomedical Research Unit for Dementia at King’s College London for supporting Clive Ballard’s involvement.\n\n\nReferences\n\nAarsland D, Rongve A, Nore SP, et al.: Frequency and case identification of dementia with Lewy bodies using the revised consensus criteria. Dement Geriatr Cogn Disord. 2008; 26(5): 445–52. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nThal DR, Rüb U, Orantes M, et al.: Phases of A beta-deposition in the human brain and its relevance for the development of AD. Neurology. 2002; 58(12): 1791–1800. PubMed Abstract\n\nFolstein MF, Folstein SE, McHugh PR: “Mini-mental state”. A practical method for grading the cognitive state of patients for the clinician. J Psychiatr Res. 1975; 12(3): 189–98. PubMed Abstract | Publisher Full Text\n\nFuster JM: The prefrontal cortex--an update: time is of the essence. Neuron. 2001; 30(2): 319–33. PubMed Abstract | Publisher Full Text\n\nAlafuzoff I, Ince PG, Arzberger T, et al.: Staging/typing of Lewy body related alpha-synuclein pathology: a study of the BrainNet Europe Consortium. Acta Neuropathol. 2009; 117(6): 635–52. PubMed Abstract | Publisher Full Text\n\nAlafuzoff I, Arzberger T, Al-Sarraj S, et al.: Staging of neurofibrillary pathology in Alzheimer’s disease: a study of the BrainNet Europe Consortium. Brain Pathol. 2008; 18(4): 484–96. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKirvell SL, Esiri M, Francis PT: Down-regulation of vesicular glutamate transporters precedes cell loss and pathology in Alzheimer’s disease. J Neurochem. 2006; 98(3): 939–950. PubMed Abstract | Publisher Full Text\n\nColeman PD, Yao PJ: Synaptic slaughter in Alzheimer’s disease. Neurobiol Aging. 2003; 24(8): 1023–1027. PubMed Abstract | Publisher Full Text\n\nKelly BL, Vassar R, Ferreira A: Beta-amyloid-induced dynamin 1 depletion in hippocampal neurons. A potential mechanism for early cognitive decline in Alzheimer disease. J Biol Chem. 2005; 280(36): 31746–53. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWanatabe T, Iwasaki K, Takasaki K, et al.: Dynamin1 depletion and memory deficits in rats treated with Abeta and cerebral ischemia. J Neurosci Res. 2010; 88(9): 1908–17. PubMed Abstract | Publisher Full Text\n\nKelly BL, Ferreira A: beta-Amyloid-induced dynamin 1 degradation is mediated by N-methyl-D-aspartate receptors in hippocampal neurons. J Biol Chem. 2006; 281(38): 28079–89. PubMed Abstract | Publisher Full Text\n\nZhu L, Su M, Lucast L, et al.: Dynamin 1 Regulates Amyloid Generation through Modulation of BACE-1. PLoS One. 2012; 7(9): e45033. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang YJ, Chen GH, Hu XY, et al.: The expression of calcium/calmodulin-dependent protein kinase II-alpha in the hippocampus of patients with Alzheimer’s disease and its links with AD-related pathology. Brain Res. 2005; 1031(1): 101–8. PubMed Abstract | Publisher Full Text\n\nBaudier J, Cole RD: Phosphorylation of tau proteins to a state like that in Alzheimer’s brain is catalyzed by a calcium/calmodulin-dependent kinase and modulated by phospholipids. J Biol Chem. 1987; 262(36): 17577–83. PubMed Abstract\n\nSingh TJ, Grundke-Iqbal I, Wu WQ, et al.: Protein kinase C and calcium/calmodulin-dependent protein kinase II phosphorylate three-repeat and four-repeat tau isoforms at different rates. Mol Cell Biochem. 1997; 168(1–2): 141–8. PubMed Abstract | Publisher Full Text\n\nSimonian NA, Elvhage T, Czernik AJ, et al.: Calcium/calmodulin-dependent protein kinase II immunostaining is preserved in Alzheimer’s disease hippocampal neurons. Brain Res. 1994; 657(1–2): 294–9. PubMed Abstract | Publisher Full Text\n\nAmada N, Aihara K, Ravid R, et al.: Reduction of NR1 and phosphorylated Ca2+/calmodulin-dependent protein kinase II levels in Alzheimer’s disease. Neuroreport. 2005; 16(16): 1809–13. PubMed Abstract\n\nReese LC, Laezza F, Woltjer R, et al.: Dysregulated phosphorylation of Ca(2+)/calmodulin-dependent protein kinase II-α in the hippocampus of subjects with mild cognitive impairment and Alzheimer’s disease. J Neurochem. 2011; 119(4): 791–804. PubMed Abstract | Publisher Full Text\n\nVallortigara J, Rangarajan S, Whitfield DR, et al.: Data for synaptic proteins expression levels and pathological scores in prefrontal cortex, anterior cingulate gyrus and parietal cortex. Figshare. 2014. Data Source"
}
|
[
{
"id": "5607",
"date": "30 Sep 2014",
"name": "Stephen M. Gentleman",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is well -presented study reporting an association between cognitive change and levels of the synaptic protein Dynamin1 in post-mortem tissue from the prefrontal cortex of Lewy body dementia patients. There is ongoing debate in the literature as to the respective roles of alpha synuclein, A-beta peptide and tau pathologies in the pathogenesis of Lewy body dementia so it is interesting to see a change of focus to the presumed anatomical basis of breakdown in inter-neuronal communication i.e. synapse loss or dysfunction.The title is appropriate, the abstract provides a fair summary of the study and the methodology is described in sufficient detail. The discussion is well written and the conclusion regarding possible links between deregulation of synaptic proteins and cognitive decline is compelling. Just a couple of comments:In this study DLB and PDD are grouped together under the heading of Lewy body dementias. For those outside the field it would perhaps be useful to provide more detail on the clinical and neuropathological (?) differences between the two conditions.A simple diagram showing the possible functional inter-relationship between Dynamin1 and CaMKII might be useful to help justify the choice of these two markers to look at synaptic function.MinorIn the abstract \"cingulated\" needs changing to cingulate",
"responses": []
},
{
"id": "7429",
"date": "23 Jan 2015",
"name": "Tim Anderson",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThere remains uncertainty regarding the relative contributions of alpha-synuclein, tau and amyloid pathology to the cognitive impairment in the Lewy body dementias (LBDs) - Parkinson’s disease dementia (PDD) and Dementia with Lewy bodies (DLB). On the justifiable preposition that whatever the underling pathology, synaptic dysfunction is a key causative mechanism of cognitive change in PDD and DLB (as it is in Alzheimer’s disease, AD), the authors in this excellent study on postmortem brain tissue examined expression levels and ratios of two synaptic proteins, Dynamin1 and CaMKII, in PDD and DLB in comparison with controls and AD, and correlated them with rate of MMSE decline. The key findings were firstly, that Dynamin1 levels in prefrontal cortex were correlated with rate of cognitive (MMSE) decline in all three dementia groups despite mean concentrations being no different from controls, and secondly, that CaMKII levels in parietal cortex were correlated with degree of plaque and tangle formation. Thus, the authors reasonably conclude that that (calcium-related) synaptic protein pathways are altered in the LBDs and likely contribute to cognitive impairment. Specific comments:The authors chose cortical regions according to known affectation in the three dementias. It might have been of interest to have included the striatum as well given that striatal amyloid changes have been reported in the LBDs. The AD group comprised greater than 2:1 females to males, whereas the control and LBD groups comprised more males than females. Is there (and was there) a gender difference in the levels of the two synaptic proteins examined? If so, could that have had any influence on the results? There is inconsistency in the concentration units of Dynamin1 in figures 1 and 4 (μgm/g protein and ng/g protein respectively). Figure 4 depicts the significant relationship between Dynamin1 levels and MMSE progression. Three patients had a decline of around 15 MMSE points per year, a remarkably rapid progression and considerably greater than the remainder of the dementia patients. Is there something different about these patients and does the significant relationship still hold if these three are omitted from the analysis?The association of Dynamin1 levels with rate of cognitive decline in the face of normal Dynamin1 levels seems surprising and a clearer explanation of this apparent ambiguity would be welcome.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-108
|
https://f1000research.com/articles/3-107/v1
|
13 May 14
|
{
"type": "Research Article",
"title": "Colonoscopic polyp detection rate is stable throughout the workday including evening colonoscopy sessions",
"authors": [
"David Thurtle",
"Michael Pullinger",
"Jordan Tsigarides",
"Iris McIntosh",
"Carla Steytler",
"Ian Beales",
"David Thurtle",
"Michael Pullinger",
"Jordan Tsigarides",
"Iris McIntosh",
"Carla Steytler"
],
"abstract": "Objective: Polyp detection rate (PDR) is an accepted measure of colonoscopy quality. Several factors may influence PDR including time of procedure and order of colonoscopy within a session. Our unit provides evening colonoscopy lists (6-9 pm). We examined whether colonoscopy performance declines in the evening. Design: Data for all National Health Service (NHS) outpatient colonoscopies performed at Norfolk and Norwich University Hospital in 2011 were examined. Timing, demographics, indication and colonoscopy findings were recorded. Statistical analysis was performed using multivariate regression. Results: Data from 2576 colonoscopies were included: 1163 (45.1%) in the morning, 1123 (43.6%) in the afternoon and 290 (11.3%) in the evening. Overall PDR was 40.80%. Males, increasing age and successful caecal intubation were all significantly associated with higher polyp detection. The indications ‘faecal occult blood screening’ (p<0.001) and ‘polyp surveillance’ (p<0.001) were strongly positively associated and ‘anaemia’ (p=0.01) was negatively associated with PDR. Following adjustment for covariates, there was no significant difference in PDR between sessions. With the morning as the reference value, the odds ratio for polyp detection in the afternoon and evening were 0.93 (95% CI = 0.72-1.18) and 1.15 (95%CI = 0.82-1.61) respectively. PDR was not affected by rank of colonoscopy within a list, sedation dose or trainee-involvement. Conclusions: Time of day did not affect polyp detection rate in clinical practice. Evening colonoscopy had equivalent efficacy and is an effective tool in meeting increasing demands for endoscopy. Standardisation was shown to have a considerable effect as demographics, indication and endoscopist varied substantially between sessions. Evening sessions were popular with a younger population",
"keywords": [
"Adenomas",
"cancer",
"endoscopy"
],
"content": "Introduction\n\nColonoscopy is an important screening and surveillance tool. Much attention has focussed on optimising its effectiveness at diagnosing pre-malignant lesions. Polyp detection rate (PDR) or adenoma detection rate (ADR) can be used as markers for colonoscopy quality. The implication is that careful and optimal mucosal visualisation enhances polyp detection and is a reliable marker of a colonoscopist performing appropriately and effectively1,2. Recent data have demonstrated the importance of adenoma detection rate as not only a metric of colonosocopy performance but as an independent predictor of protection against subsequent colorectal cancer3. PDR is defined as the detection of one or more polyps at colonoscopy. Several factors may influence PDR, obviously including patient selection and case mix, but recently it has been suggested that the time the procedure is performed and the order of colonoscopy within a session also influence colonoscopy performance.\n\nInconsistent results examining the PDR of colonoscopies throughout the workday have been reported. A number of studies have reported that PDRs decrease as the day4,5 or endoscopy list6 progress, and that PDRs in morning lists are higher than afternoon lists7. However this effect does not appear to be universal across endoscopy units with other studies reporting no such reduction in polyp detection overall8 or when colonoscopies are performed in half day blocks9 or 3-hour shifts10.\n\nThis effect, where shown, has been generally attributed to endoscopist fatigue4,11 although this was not independently assessed in those studies. This effect appears logical and correlates with increasing errors in other repetitive or tiring vocations such as errors by house officers related to time in hospital12, inadvertent errors at simulated laparoscopy as mental and physical workload increases13 and impairment in perceptual and motor ability in nurses toward the end of nightshifts14. Queue position has been suggested as a novel surrogate measure for operator fatigue6. There has also been a suggestion that endoscopists may rush procedures later in the list15. Another possible hypothesis is that bowel preparation may be a factor, the inference being that bowel preparation is often prescribed appropriately for morning endoscopies but the regimen is not appropriately altered for optimum bowel preparation in the afternoon.\n\nThis apparent effect of reduced PDR throughout the work day has been superimposed on top of the difference in performance between endoscopists, which has been reported on multiple occasions; even within a group of fully competent endoscopists there appears to be spectrum of polyp detection rates3,16,17. This endoscopist effect has not been consistently accounted for in the published data examining the effect of timing on PDR7.\n\nThe optimisation of colonoscopy will impact upon its safety and cost-effectiveness. Increasing numbers of endoscopies are being performed both in the UK18 and USA19, partly as a result of successful screening programmes18. This has led to higher patient flow though endoscopy units. To accommodate this increase in demand, units have had to expand their provision of colonoscopies by number of endoscopy rooms, length of lists or extra lists.\n\nTo accommodate this extra-workload, our unit has run regular evening lists (6–9pm) for over two years to meet this increasing demand and improve patient convenience and we are aware of other endoscopy units developing similar strategies. However it is not known whether evening colonoscopy is as effective as traditional day-time colonoscopy. As evening colonoscopy makes up a significant and increasing amount of our overall workload, it is important to establish that the service in terms of performance metrics is appropriate. It is also unknown whether colonoscopy performance declines in the evening; this specific effect, as opposed to morning or afternoon colonoscopy has not been examined in the previous studies. This study aims to review polyp detection rates in our routine clinical practice, with particular reference to this evening session. We aimed to test the hypothesis that polyp detection would be reduced in the evening sessions. The majority of evening colonoscopists will have already worked a full working day. Although not specifically assessed, they may be assumed to be more fatigued than those working in the morning or afternoon lists. We also aim to review PDRs in relation to other factors in routine clinical practice.\n\n\nMethods\n\nEndoscopy session data was collected retrospectively from the Hospital’s booking system for all NHS outpatient colonoscopies booked for weekdays in the Norfolk and Norwich University Hospital in 2011. Endoscopy sessions were classified as: ‘Morning’ 08.00 to 12.00, ‘Afternoon’ 13.00 to 17.00 and evening 17.30 to 21.00 with colonoscopies usually afforded 30–45 minutes per slot. The times of colonoscopies were recorded; each colonoscopy was grouped by session (morning, afternoon, evening) and ranked by order within their session. Lists sometimes included other endoscopic procedures which were each assigned a value of 1 in the ranking. For example a colonoscopy performed after two oesophago-gastro-duodenoscopies would be ranked number 3 in its session although it was the 1st colonoscopy. Private patients were excluded.\n\nThe Gastroenterology Endoscopy Unit at the Norfolk and Norwich University Hospital provides a full diagnostic and therapeutic endoscopy service to the local population and tertiary referral services in advanced colonoscopic polypectomy, small bowel enteroscopy, endoscopic ultrasound and upper GI endoscopic mucosal resection. The unit is multidisciplinary with colonoscopy performed by medical gastroenterologists, colorectal surgeons and independent nurse endoscopists, with patients booked to most procedures from a common waiting list, although the specific bowel cancer screening lists are only performed by 6 of the endoscopists. The majority of colonoscopy procedures are performed on colonoscopy-only lists, a smaller number are on mixed lists. It is usual practice for our endoscopists to only perform one colonoscopy session (morning, afternoon or evening) per day; in the relatively rare occurrence of an endoscopist providing two sessions within the same day, our usual policy is to have one as a predominantly gastroscopy session.\n\nThe unit is the regional endoscopic training centre and an accredited bowel cancer screening centre. The study was approved by and registered with the Audit and Clinical Governance Department at Norfolk and Norwich University Hospital, (project code 34810) the project was regarded as service evaluation and approval from a Research Ethics Committee was not required.\n\nComputerised records were retrieved individually for each colonoscopy from the endoscopy reporting system, Scribes® (iSoft Health, Banbury UK). Patient demographics, indication, endoscopist, trainee-involvement and sedation information were recorded retrospectively along with information specific to the procedure itself, namely bowel prep quality, diagnosis and polyps detected.\n\nColonoscopy patients received 2 sachets of Picolax® (Sodium picosulfate with magnesium citrate, Ferring Pharmaceuticals Ltd, West Drayton UK) bowel preparation, 1 sachet at 6pm the day prior to procedure and 1 sachet at 6am on the day of procedure, for morning and afternoon endoscopies and at 6am and 2pm on the day of the procedure for evening colonoscopies. Quality of bowel prep was subjectively recorded as poor, satisfactory or good by the endoscopist at the time of the procedure. Throughout the study period Olympus-240 and -260 series video-endoscopes were used. Endoscopists recorded their findings themselves immediately after each procedure with polyps qualitatively described and site recorded as accurately as possible.\n\nThe indication for colonoscopy was recorded and later simplified into 6 categories for numerical analysis, such that each category contained had at least 10 colonoscopies in each of morning, afternoon and evening sessions and at least 50 procedures overall. Categories with fewer than this were grouped under ‘other’. Thirty different endoscopists performed colonoscopies throughout the year. Those performing over 30 colonoscopies were treated as independent variables and those with fewer than 30 to their name were grouped under ‘other’ for analysis.\n\nSample size calculations were not formally performed as the intention was to review one year’s colonoscopy practice; the final number of procedures included exceeds those calculated to be required by other similar studies8.\n\nPDR was defined as the detection of one or more polyps at colonoscopy. Unadjusted PDRs were calculated and demographic information, indication and endoscopists assessed for each colonoscopy session. Due to the considerable variability in these factors between these sessions multivariate regression analysis was performed to adjust for differences. Stata® 12 (StataCorp LP, Texas USA) was used to perform multivariate regression analysis with the variables outlined in the results section below Results are expresses as adjusted odds ratio (OR) for polyp detection with 95% confidence intervals. Differences between groups were considered statistically significant if the “p” value was < 0.05.\n\n\nResults\n\nData from 2576 colonoscopies were included: 1163 (45.1%) were performed in the morning, 1123 (43.6%) in the afternoon and 290 (11.3%) in the evening.\n\nMean age was lower in the evening sessions (58.2) compared to morning (64.7) and afternoon (62.3). Table 1 demonstrates the breakdown of demographic and endoscopic information between the three sessions. Trainee-involvement was higher in the evening (17.9%) and afternoon (14.6%) compared to the morning sessions (5.5%). Caecal intubation rates (uncorrected for bowel preparation and impassable strictures) were above 94% in each of the three sessions without any significant difference between sessions. Full data on all procedures included are included in the associated data files.\n\nUnadjusted PDR in the morning, afternoon and evening session were 46.4%, 35.9% and 37.2% respectively.\n\nFactors associated with polyp detection were assessed by multivariate logistic regression (Table 2). Male gender (odds ratio (OR) = 1.76, 95% confidence intervals (CI) 1.48 - 2.11, p<0.001), increasing age (OR = 1.04, 95%CI = 1.03 - 1.06, p<0.001) and successful caecal intubation (OR = 2.48, 95%CI = 1.53 - 4.01, p<0.001) were all significantly independently associated with higher polyp detection. The indications ‘faecal occult blood (FOB) screening’ (those subjects in the UK national bowel cancer screening programme) (p<0.001) and ‘polyp surveillance’ (p<0.001) were strongly positively associated and ‘anaemia’ (p=0.01) negatively associated with PDR. Subjectively ‘Good’ bowel preparation was significantly associated with a higher PDR as compared to ‘Poor’ bowel prep. As Table 1 demonstrates, a considerable proportion of the morning endoscopies were performed for FOB screening. Indeed 54.90% of all FOB screening colonoscopies at our unit were performed in the morning sessions. This correlates with the increased mean age in the morning session and high unadjusted PDR in the morning.\n\nResults expresses as adjusted odds ratio for detection of any polyp. CI, confidence interval: NS, not significant\n\nFollowing standardisation of covariates, there was no significant difference in PDR between sessions. With the morning as the reference value, the odds ratio for polyp detection in the afternoon and evening were 0.93 (95%CI = 0.72 - 1.18) and 1.15 (95%CI = 0.82 - 1.61) respectively. PDR was not shown to be affected by rank order of colonoscopy within a list (p=0.904), sedation type or trainee-involvement.\n\n\nDiscussion\n\nOur data correlate with much larger cohorts that have purely analysed colonoscopic diagnoses without any reference to time of procedure in demonstrating a higher PDR in males than females and with increasing age20. Overall, the PDR and caecal intubation rates are comparable to, and in fact slightly better, than those reported in the recent UK-wide audit of colonoscopy practice (which reported an uncorrected caecal intubation rate of 92.3% and polyp detection rate of 32.1%)21. In our study PDR was significantly higher with complete caecal intubation demonstrating the validity of this as a performance metric of colonoscopy quality. When more of the colon is visualised, more polyps will be detected and it is also likely that the most skilled and careful colonoscopists are both more likely to complete a colonoscopy to the caecum and visualise the mucosal completely.\n\nOur data do not support our original hypothesis that evening PDR might be lower than other sessions in the day. Our data suggest there is no significant difference in detection of polyps in either the evening compared to the morning, or based on an endoscopy’s rank order within a session. This is contrary to some previous reports in the literature4–6. The reasons for this are unclear. Our unit aims to minimise pressures on endoscopists by allowing 30 minutes per colonoscopy (45 minutes for bowel cancer screening colonoscopy) and by strictly avoiding more than 5 colonoscopy procedures per endoscopy list. Also, as demonstrated by our large number of endoscopists (30 in this study), we believe our staff are less likely to fatigue. Endoscopy remains part of a varied working life for both physicians and surgeons within our hospital, rather than operators performing solely endoscopy. Equally apart from the bowel cancer screening specific lists, the majority of colonoscopy lists include a variety of patients with different indications. During the course of this study evening colonoscopy was performed by some consultant staff either working the evening as a planned session within their job plan or by other consultant staff or nurse endoscopists working and remunerated for extra sessions above their full job plan. Arguably the latter group may be more fatigued, but again our data show no deterioration of colonoscopy performance when this group was analysed separately.\n\nWe would therefore recommend evening colonoscopy sessions as a potential solution to safely and effectively meet increasing endoscopy demand. We feel it is important that any unit instigating evening colonoscopy carefully audits performance-metrics to ensure that colonoscopy performance is satisfactory in these later sessions. It may be that factors related to individual endoscopy units are important in explaining the differences in polyp detection reported in relation to endoscopy timings. The setting was an endoscopy training centre with an internal high priority on quality of endoscopy and well-established practices of governance, peer-review and teaching and it is possible these are drivers that help maintain PDR.\n\nAnecdotal evidence within our patient group suggests these sessions are popular with patients also. The evening sessions were popular with a considerably younger demographic (mean age 58.2) which may be assumed to represent more convenience for working individuals.\n\nOur data suggest evening lists allowed a higher degree of trainee-involvement. This may be due to ward or clinic commitments of trainees in the morning, indeed some registrars chose to stay later for evening colonoscopy lists, when they have no other distractions, in order to gain further experience and enhance their portfolio of procedures.\n\nIt is interesting to note that type of sedation made no difference to PDR, however, patient comfort or length of procedure was not measured (although ongoing audits separate to this study show low and appropriate pain scores and high patient satisfaction). A large Italian prospective study showed caecal intubation rates of only 81.6% of unsedated patients22, although data from the UK bowel cancer screening programme suggests that, as in the current study, colonoscopy without intravenous analgesia and/or sedation is not associated with impaired caecal intubation or adenoma detection23. In our study it is also unknown whether a procedure was attempted without sedation and sedation later given (although in practice the later addition of sedation seems unusual).\n\nOne of the flaws in this dataset is the reliance on accurate documentation by endoscopists on each endoscopy report. Parts of their report, such as bowel preparation, are easily open to subjectivity. Equally, we used PDR as our performance metric: at present the information technology available is not suitable for readily determining the ADR, but we believe that the PDR is a useful surrogate marker of the care taken during colonoscopy and has the advantage that this can be readily compared to other large datasets such as the UK colonoscopy audit21. That the PDR was higher when bowel preparation was recorded as ‘good’ compared to ‘poor’ (OR=1.46, 95%CI = 1.03-2.07) may indeed reflect this subjectivity when endoscopists blame the quality of images for the inability to visualise any polyps.\n\nAlthough it was not a primary outcome measure, we noted that the proportion of colonoscopies recorded as having ‘good’ bowel preparation was much higher in the afternoon (82.4%) compared to the morning (59.0%) sessions. We had not assessed patients’ adherence to laxative dosing protocols but if adherence were equivalent this may suggest the protocols we use require adjustment. Previous recommendations have proposed split-dose bowel preparation as the standard of care24, which we adhered to.\n\nA validated bowel preparation scale used prospectively may improve the reliability of these data and it is recommended that this be included in future evaluations.\n\nOther limitations of this study are inherent within its design. It was retrospective, without any randomisation to different sessions. The external generalizability of the data may also be questioned although we believe we have a balanced workforce of medical, surgical and specialist-nurse endoscopists that is fairly representative of a modern UK NHS unit. We did not record data on withdrawal time on the non-bowel cancer screening colonoscopies. This would have been useful in comparing these data to other datasets which provide this information.\n\nIn conclusion, this study shows that the detection of polyps at colonoscopy is independent of the time of day that the procedure is performed. Specifically, evening colonoscopy is no less effective at detecting polyps than morning colonoscopy. We attribute this consistency to appropriate colonoscopy scheduling and an interested, engaged workforce with varied working patterns, both of which could be directly tested in prospective studies. Evening colonoscopy is a suitable initiative to increase procedure numbers but we recommend continued governance of performance metrics to ensure that timing of procedures does not adversely influence outcomes.\n\n\nData availability\n\nfigshare: Dataset showing all procedures included in analysis of polyp detection study, http://dx.doi.org/10.6084/m9.figshare.101698225",
"appendix": "Author contributions\n\n\n\nDT and ILPB jointly conceived the study. DT, MP, JT, IM, CS collectively designed the study, performed the data collection and jointly analysed the data. DT wrote the original draft of the manuscript and ILPB revised and wrote the final draft and is the guarantor of the paper. All authors agree with the final draft.\n\n\nCompeting interests\n\n\n\nILPB is Chair of the Trainee’s Section of the Joint Advisory Group (JAG) Quality Assurance Working Group, on Endoscopy Training and Co-director of the Norwich Endoscopy Training Centre and is interested in the adoption of safe and effective endoscopy training and performance, and the application of formal validated assessment tools to assess competence and performance in endoscopy.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe authors would like to thank the medical, nursing and administrative staff in the endoscopy unit for support during the performance of this study. This study was presented in abstract form at the British Society of Gastroenterology Meeting, Liverpool, 2013 and published in abstract form in Gut (2013); 62(Suppl 1): A59).\n\n\nReferences\n\nKaminski MF, Regula J, Kraszewska E, et al.: Quality indicators for colonoscopy and the risk of interval cancer. N Engl J Med. 2010; 362(19): 1795–803. PubMed Abstract | Publisher Full Text\n\nBarclay RL, Vicari JJ, Doughty AS, et al.: Colonoscopic withdrawal times and adenoma detection during screening colonoscopy. N Engl J Med. 2006; 355(24): 2533–41. PubMed Abstract | Publisher Full Text\n\nCorley DA, Jensen CD, Marks AR, et al.: Adenoma detection rate and risk of colorectal cancer and death. N Engl J Med. 2014; 370(14): 1298–306. PubMed Abstract | Publisher Full Text\n\nChan MY, Cohen H, Spiegel BM: Fewer polyps detected by colonoscopy as the day progresses at a Veteran's Administration teaching hospital. Clin Gastroenterol Hepatol. 2009; 7(11): 1217–23. PubMed Abstract | Publisher Full Text\n\nLong MD, Martin C, Sandler RS, et al.: Reduced polyp detection as endoscopy shift progresses: experience with screening colonoscopy at a tertiary-care hospital. J Clin Gastroenterol. 2011; 45(3): 253–8. PubMed Abstract | Publisher Full Text\n\nLee A, Iskander JM, Gupta N, et al.: Queue position in the endoscopic schedule impacts effectiveness of colonoscopy. Am J Gastroenterol. 2011; 106(8): 1457–65. PubMed Abstract | Publisher Full Text\n\nSanaka MR, Deepinder F, Thota PN, et al.: Adenomas are detected more often in morning than in afternoon colonoscopy. Am J Gastroenterol. 2009; 104(7): 1659–64. PubMed Abstract | Publisher Full Text\n\nFreedman JS, Harari DY, Bamji ND, et al.: The detection of premalignant colon polyps during colonoscopy is stable throughout the workday. Gastrointest Endosc. 2011; 73(6): 1197–206. PubMed Abstract | Publisher Full Text\n\nGurudu SR, Ratuapli SK, Leighton JA, et al.: Adenoma detection rate is not influenced by the timing of colonoscopy when performed in half-day blocks. Am J Gastroenterol. 2011; 106(8): 1466–71. PubMed Abstract | Publisher Full Text\n\nMunson GW, Harewood GC, Francis DL: Time of day variation in polyp detection rate for colonoscopies performed on a 3-hour shift schedule. Gastrointest Endosc. 2011; 73(3): 467–75. PubMed Abstract | Publisher Full Text\n\nKaneshiro M, Ho A, Chan M, et al.: Colonoscopy yields fewer polyps as the day progresses despite using social influence theory to reverse the trend. Gastrointest Endosc. 2010; 72(6): 1233–40. PubMed Abstract | Publisher Full Text\n\nGaba DM, Howard SK: Patient safety: fatigue among clinicians and the safety of patients. N Engl J Med. 2002; 347(16): 1249–55. PubMed Abstract | Publisher Full Text\n\nYurko YY, Scerbo MW, Prabhu AS, et al.: Higher mental workload is associated with poorer laparoscopic performance as measured by the NASA-TLX tool. Simul Healthc. 2010; 5(5): 267–71. PubMed Abstract | Publisher Full Text\n\nChang YS, Wu YH, Hsu CY, et al.: Impairment of perceptual and motor abilities at the end of a night shift is greater in nurses working fast rotating shifts. Sleep Med. 2011; 12(9): 866–9. PubMed Abstract | Publisher Full Text\n\nRex DK: Improving detection during colonoscopy: multiple pathways for investigation. J Clin Gastroenterol. 2011; 45(3): 207–9. PubMed Abstract | Publisher Full Text\n\nChen SC, Rex DK: Endoscopist can be more powerful than age and male gender in predicting adenoma detection at colonoscopy. Am J Gastroenterol. 2007; 102(4): 856–61. PubMed Abstract | Publisher Full Text\n\nImperiale TF, Glowinski EA, Juliar BE, et al.: Variation in polyp detection rates at screening colonoscopy. Gastrointest Endosc. 2009; 69(7): 1288–95. PubMed Abstract | Publisher Full Text\n\nRees CJ, Bevan R: The National Health Service Bowel Cancer Screening Program: the early years. Expert Rev Gastroenterol Hepatol. 2013; 7(5): 421–37. PubMed Abstract | Publisher Full Text\n\nLieberman DA, Williams JL, Holub JL, et al.: Colonoscopy utilization and outcomes 2000 to 2011. Gastrointest Endosc. 2014. pii: S0016-5107(14)00038-8. [Epub ahead of print]. PubMed Abstract | Publisher Full Text\n\nBarret M, Boustiere C, Canard JM, et al.: Factors associated with adenoma detection rate and diagnosis of polyps and colorectal cancer during colonoscopy in France: results of a prospective, nationwide survey. PLoS One. 2013; 8(7): e68947. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGavin DR, Valori RM, Anderson JT, et al.: The national colonoscopy audit: a nationwide assessment of the quality and safety of colonoscopy in the UK. Gut. 2013; 62(2): 242–9. PubMed Abstract | Publisher Full Text\n\nPaggi S, Radaelli F, Amato A, et al.: Unsedated colonoscopy: an option for some but not for all. Gastrointest Endosc. 2012; 75(2): 392–8. PubMed Abstract | Publisher Full Text\n\nLee TJ, Rees CJ, Blanks RG, et al.: Colonoscopic factors associated with adenoma detection in a national colorectal cancer screening program. Endoscopy. 2014; 46(3): 203–11. PubMed Abstract | Publisher Full Text\n\nCohen LB: Split dosing of bowel preparations for colonoscopy: an analysis of its efficacy, safety, and tolerability. Gastrointest Endosc. 2010; 72(2): 406–12. PubMed Abstract | Publisher Full Text\n\nDavid T, Michael P, Jordan T, et al.: Dataset showing all procedures included in analysis of polyp detection study. figshare. 2014. Data Source"
}
|
[
{
"id": "5014",
"date": "19 Jun 2014",
"name": "Adam Haycock",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper is timely and addresses a problem that will be of increasing importance as units struggle to meet waiting time targets. This is the first paper as far as I am aware to look at the quality of colonoscopy done on evening lists, although there is literature supporting evidence of fatigue for colonoscopy elsewhere.The paper is well written, with clear title, methodology, statistics, results and discussion, including limitations. I have no additions or amendments to suggest.",
"responses": [
{
"c_id": "934",
"date": "07 Aug 2014",
"name": "Ian Beales",
"role": "Author Response F1000Research Advisory Board Member",
"response": "Thank you for the reviewer's comments."
}
]
},
{
"id": "5380",
"date": "17 Jul 2014",
"name": "Christopher Wells",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe the practice of performing colonoscopy during an evening session. With increasing demand on endoscopic services in the UK the move towards 7 day & 3 lists per day working will become more commonplace. Given the concerns about deterioration in performance it is important to know that the quality of the procedure is not affected by when the procedure is performed. The authors describe the polyp detection rate, a marker of quality of colonoscopy, in a retrospective dataset gathered from their hospital records. The data are used to construct a model which predicts changes in PDR. The data suggests that the quality of colonoscopy is maintained in evening sessions. This provides evidence supporting this evolving change of practice that will provide units with the assurances that this change in work pattern is a viable option. The paper is therefore useful and timely.One possible confounder that the authors do not describe is the number of colonoscopists that perform evening endoscopies - if it is just a few enthusiasts then they may be more vigilant, compared with the daytime regular endoscopists. It would be useful to know that the proportion of procedures performed by each endoscopist was the same throughout the 3 sessions as any differences may be due to the technical skills of these endoscopists.",
"responses": [
{
"c_id": "933",
"date": "07 Aug 2014",
"name": "Ian Beales",
"role": "Author Response F1000Research Advisory Board Member",
"response": "We thank the reviewer for his very pertinent comments. We entirely agree that colonoscopist enthusiasm and interest are important variables that could influence polyp detection rates in evening sessions. All the relevant data are contained in the data file, but in total 26 different colonoscopists did at least one evening colonoscopy but in fact only 10 colonoscopists performed more than 10 evening procedures in the study period. So it is possible that this is a self-selecting group of more vigilant colonoscopists and indeed our data may not be more generalisable if fixed evening endoscopy becomes a routine part of most endoscopists' job plans and on the volunteer basis that we currently utilise.We agree that the ideal analysis would be to examine the variability of polyp-detection for each endoscopist individually through all 3 sessions of the day. At present, it is not possible to draw any reliable conclusions from these data as the numbers in each subgroup are small. As might be expected from working practices in the United Kingdom NHS, where endoscopy (including gastroscopy, colonoscopy and often ERCP) generally fits into a working pattern with inpatient and outpatient work, very few of our endoscopists performed an even distribution of colonoscopy throughout all 3 sessions. In fact only two of the colonoscopists performed more than 40 procedures in each of the 3 sessions: their crude polyp detection rates, uncorrected for case mix, were 28%, 23%, 38% and 59%, 44% 59% in morning, afternoon and evening respectively.We are continuing to collect data on this important area and hope that a larger cohort (or combination of data, possibly using the data accessibility inherent with F1000Research publications) will enable analyses with sufficient power to examine this further.We agree with the reviewer that the individual technical skill and interest of the colonoscopist is possibly the most important variable, but do not feel this alters our overall conclusion that evening colonoscopy is both feasible and effective. We feel it is important to stress that ongoing quality assurance for performance metrics (including polyp detection) is essential and that time of day should be assessed in such assessments."
}
]
}
] | 1
|
https://f1000research.com/articles/3-107
|
https://f1000research.com/articles/3-106/v1
|
13 May 14
|
{
"type": "Research Article",
"title": "Thiolation-enhanced substrate recognition by D-alanyl carrier protein ligase DltA from Bacillus cereus",
"authors": [
"Liqin Du",
"Yu Luo",
"Liqin Du"
],
"abstract": "D-alanylation of the lipoteichoic acid on Gram-positive cell wall is dependent on dlt gene-encoded proteins DltA, DltB, DltC and DltD. The D-alanyl carrier protein ligase DltA, as a remote homolog of acyl-(coenzyme A) (CoA) synthetase, cycles through two active conformations for the catalysis of adenylation and subsequent thiolation of D-alanine (D-Ala). The crystal structure of DltA in the absence of any substrate was observed to have a noticeably more disordered pocket for ATP which would explain why DltA has relatively low affinity for ATP in the absence of any D-alanyl carrier. We have previously enabled the thiolation of D-alanine in the presence of CoA as the mimic of D-alanyl carrier protein DltC which carries a 4’-phosphopantetheine group on a serine residue. Here we show that the resulting Michaelis constants in the presence of saturating CoA for both ATP and D-alanine were reduced more than 10 fold as compared to the values obtained in the absence of CoA. The presence of CoA also made DltA ~100-fold more selective on D-alanine over L-alanine. The CoA-enhanced substrate recognition further implies that the ATP and D-alanine substrates of the adenylation reaction are incorporated when the DltA enzyme cycles through its thiolation conformation.",
"keywords": [
"The cell surface of most Gram-positive bacteria contains wall teichoic acid and lipoteichoic acid with a poly-alditol phosphate backbone. The remaining hydroxyls of the alditol moiety are ubiquitously modified by D-alanyl esterification or glycosylation1–3. A dlt operon",
"which typically codes for DltA",
"DltB",
"DltC and DltD proteins",
"is required for the D-alanylation of lipoteichoic acids4. D-alanylation brings in positively charged amino-groups and partially neutralizes the net negative charges of the phosphate groups on the lipoteichoic acid backbone. The reduction of D-alanyl content on the cell surface has been found to be associated with increased autolysis3",
"5 and susceptibility to host defense peptides and other antibiotics6",
"7. Impaired D-alanylation of lipoteichoic acid also reduces the ability of bacteria to colonize any surface8 and form antibiotics-resistant biofilms9. Therefore",
"the dlt-encoded proteins required for the lipoteichoic acid D-alanylation pathway could serve as novel targets for fighting emerging infectious diseases caused by Gram-positive pathogens10. The DltA inhibitor 5’-O-[N-(D-alanyl)-sulfamoyl]adenosine for example",
"designed by analogy to D-alanyl adenylate (D-Ala-AMP)",
"has been shown to significantly suppress the growth of B. subtilis when used in combination with vancomycin11."
],
"content": "Introduction\n\nThe cell surface of most Gram-positive bacteria contains wall teichoic acid and lipoteichoic acid with a poly-alditol phosphate backbone. The remaining hydroxyls of the alditol moiety are ubiquitously modified by D-alanyl esterification or glycosylation1–3. A dlt operon, which typically codes for DltA, DltB, DltC and DltD proteins, is required for the D-alanylation of lipoteichoic acids4. D-alanylation brings in positively charged amino-groups and partially neutralizes the net negative charges of the phosphate groups on the lipoteichoic acid backbone. The reduction of D-alanyl content on the cell surface has been found to be associated with increased autolysis3,5 and susceptibility to host defense peptides and other antibiotics6,7. Impaired D-alanylation of lipoteichoic acid also reduces the ability of bacteria to colonize any surface8 and form antibiotics-resistant biofilms9. Therefore, the dlt-encoded proteins required for the lipoteichoic acid D-alanylation pathway could serve as novel targets for fighting emerging infectious diseases caused by Gram-positive pathogens10. The DltA inhibitor 5’-O-[N-(D-alanyl)-sulfamoyl]adenosine for example, designed by analogy to D-alanyl adenylate (D-Ala-AMP), has been shown to significantly suppress the growth of B. subtilis when used in combination with vancomycin11.\n\nThe D-alanyl carrier protein ligase DltA (formed by ~500 amino acid residues)4 closely resembles (~30% sequence identity) the adenylation domains (also called adenosine monophosphate (AMP)-forming domains) found in bacterial non-ribosomal peptide synthetases12. Its remote homologs include the acyl- and aryl-(coenzyme A) CoA synthetases and firefly luciferases13. As shown in Figure 1, DltA catalyzes the ATP-driven adenylation of D-alanine and the subsequent transfer of the activated D-alanyl group to the thiol group of 4’-phosphopantetheine, which is covalently bound to a serine side chain of D-alanyl carrier protein DltC (~80 amino acid residues)4. As previously shown for such one-protein-two-enzymes homologs14,15, crystal structures of two DltA proteins have also been observed in adenylation and thiolation conformations, respectively16,17 (Figure 1). The thiolation conformation of B. subtilis DltA (BsDltA)18 resembles previously determined structures of a bacterial acetate-CoA synthetase (ACS) and 4-chlorobenzoate-CoA ligase crystallized in their respective thiolation state15,19. On the other hand, the adenylation conformation of B. cereus DltA (BcDltA)17 resembles those observed in the crystal structures of the firefly luciferase PheA (phenylalanine-activating domain of the first module of the Bacillus brevis gramicidin S synthetase I), DhbE (2,3-dihydroxybenzoate activation domain from B. subtilis), a yeast acetyl-CoA synthetase and 4-chlorobenzoate-CoA ligase13,14,20–22. The two conformations captured in the crystal structures of the two closely related DltA proteins (56% sequence identity) are related by a ~146º rotation around a hinge residue Asp-399 in BcDltA (Asp-398 in BsDltA), which is a invariable residue equivalent to Asp-517 in Salmonella typhimurium acetyl-CoA synthetase23. The region surrounding this hinge residue (residues Arg-397 to Glu-413 of BcDltA green in Figure 1B) contains an important triphosphate-interacting residue Arg-39724 and a β-hairpin which serves as part of the pantetheine channel as observed in the bacterial acetyl-CoA synthetase23.\n\nA. A two-step reaction catalyzed by DltA. B. The adenylation and thiolation conformations of DltA. In the first adenylation reaction, D-alanine is converted into DltA-bound D-alanine adenylate (D-Ala-AMP). This reaction is catalyzed by the adenylation conformation of DltA shown as DltA/AMP complex. In the subsequent thiolation reaction, the activated alanyl group in the intermediate is transferred to the thiol group of DltC-linked 4’-phosphopantetheine (DltC-SH). The second reaction is catalyzed by the thiolation conformation of DltA shown also as DltA/AMP complex. Most part of the major N-terminal domain of DltA (residues 2 - 399) is shown in gray. The majority of the minor C-terminal domain of DltA (residues 400–504) is shown in salmon. Residues between Arg-397 and Glu-413 are highlighted in green.\n\nWe previously studied the adenylation reaction of BcDltA in the absence of any D-alanyl carrier to enable the thiolation reaction17. The resulting specificity constants (kcat/KM) for D- and L-alanine differed by merely ~3-fold, which could not explain the fact that teichoic acid is overwhelmingly modified by D- but not L-alanine25. As suggested by a study on a spectrum of DltA homologs which have residual aminoacyl-CoA synthetase activity42, CoA has been confirmed to be a substitute for the D-alanyl carrier protein DltC as the thiolation substrate of BcDltA24. Here we report further biochemical analysis of BcDltA. Noticeable differences were observed in Michaelis constants KM and turnover rates kcat. The presence of CoA, hence the enabled adenylation and thiolation cycle, enhanced the enzyme’s apparent affinities to its cognitive substrate ATP and D-alanine by approximately an order of magnitude. Since BcDltA is a slow enzyme with turnover rate less than 1 s-1, much slower than bacterial Acyl-CoA synthetases (~102 s-1)23 and 4-chlorobenzoate-CoA ligase (~10 s-1)26, the observed Michaelis constants should closely approximate the corresponding dissociation constants, and therefore provide some insight into the stability of short-lived enzyme-substrate complexes. We also determined the structure of BcDltA in the absence of any substrate. This structure is noticeably more disordered than previously reported DltA structures16,17,24, which may explain the enzyme’s lower affinity to ATP in the absence of the other two substrates. Interestingly, CoA-enhanced affinities to ATP and D-alanine imply that the thiolation substrate CoA-bound BcDltA has higher affinity to both adenylation substrates as compared to CoA-free BcDltA.\n\n\nMethods\n\nAll reagents were from VWR unless specified otherwise. The wild-type as well as the C269A mutant of DltA from B. cereus was cloned for over-expression of BcDltA as described previously17. The pET28-BcDltA construct carries an Ala-1 mutation at the N-terminus and eight extra residues at the C-terminus (LEHHHHHH). The soluble fraction of BcDltA was purified by nickel-affinity chromatography followed by gel filtration. BcDltA was concentrated to ~20 mg/mL by ultra-filtration.\n\nThe concentrated BcDltA protein was crystallized using the hanging drop crystallization method at a room temperature of 21°C. The optimal well solution for crystallization contained 0.1 M MgCl2, 0.5 M KCl, 16% polyethylene glycol (PEG) 3,350 (Sigma-Aldrich) and 0.05 M Hepes-NaOH buffer at pH 7.2. Each drop was composed of 1 μL of protein and 1 μL of well solution. The plate-shaped crystals grew to a maximal size of 0.4 mm × 0.3 mm × 0.05 mm in three days. Crystals were gradually transferred to stabilizing solutions composed of the crystallization well solution supplemented with 8%, 16% and 24% glycerol, soaked for 1 minute, then flash-cooled to - 173.15°C in a nitrogen stream generated by an Oxford CryoSystems device. A total of 400 0.4-degree oscillation images were acquired and processed using a Brukers Proteum-R system as already described27. The previously solved BcDltA model (PDB code 3DHV)17 was used as the starting model for two-domain rigid body refinement followed by positional refinement using Crystallography & NMR System (CNS)28. This resulting model was subjected to ten cycles of rebuilding and refinement using Arp/Warp29. The rebuilt model was iteratively rebuilt using XtalView30 and then refined using CNS28. The final model had 90.8% of the residues in the most favored regions on a Ramachandran plot. Val-301 with clear electron density and Asp-336 with blurry electron density were the only two residues found in the disfavored region. Statistics of the diffraction data, refinement and geometry are listed in Table 1. The molecular figures were generated using Molscript31 and rendered using Raster3D32. The coordinates and structure factors have been deposited in the Protein Data Bank33–35 (entry code 4PZP).\n\nNotes:\n\na Values in parentheses refer to values in the highest resolution shell.\n\nb Rsym = Σ| Ih - <I>h |/ΣIh, where <I>h is average intensity over symmetry equivalents, h is reflection index. The summation is over all measured reflections.\n\nc Rwork = Σ| Fcalc - Fobs |/ΣFobs. The summation is over all reflections used in refinement. Rfree is calculated using a randomly selected 5% of the reflections set aside throughout the refinement.\n\nThe intrinsic tryptophan fluorescence of 1.0 ml 0.4 uM BcDltA solution with 0 to 2 mM ATP was acquired at a room temperature of 21°C using a PerkinElmer LS-55 fluorescence spectrometer. The excitation wavelength was 305 nm and a fluorescent emission in the 310 nm to 390 nm range was recorded. The relative fluorescence increase at 345 nm was used to quantify the ATP-bound fraction of BcDltA. Assuming that the fluorescence gain is proportional to [ATP]/(KD + [ATP]), Prism software (GraphPad Software) was used to derive the dissociation constant of ATP.\n\nAs previously described, pyrophosphate released from the adenylation reaction is broken down into phosphate by pyrophosphatase17. The resulting phosphate was quantified by a dye solution containing 0.033% w/v Malachite Green, 1.3% w/v ammonium molybdate and 1.0 M HCl36. The 200 μL reaction solutions contained 5 μM BcDltA, 0.1 M KCl, 0.01 M MgCl2, 0.05 M Tris-Hepes buffer at pH 7.2, 5 unit/mL of inorganic pyrophosphatase from baker’s yeast (Sigma-Aldrich) and specified concentrations of D-alanine, ATP and CoA (Sigma-Aldrich). A volume of 25 μL of reaction solution was retrieved every 3 or 5 minutes and mixed thoroughly with 475 μL of the dye solution. The absorption at a wavelength of 620 nm was recorded after 90 seconds. The initial rates (1/2 of the phosphate concentration increase per minute) of the adenylation reaction were derived from the time courses of phosphate accumulation. The correlation between initial reaction rate and substrate concentration was fitted with Michaelis-Menten equation using the Prism software (GraphPad Software).\n\nThe free thiol group of CoA was quantified as described previously24 by a dye solution composed of 1 mM 5,5’-dithio-bis (2-nitrobenzoic acid) (DTBN), or Ellman’s reagent (Sigma-Aldrich) and 50 mM Tris-EDTA solution at pH 8.0. Absorption at a wavelength of 412 nm was used to quantify the concentration of free thiol group. The reaction rate was derived by the rate of thiol depletion. The correlation between initial reaction rate and substrate concentration was also fitted with Michaelis-Menten equation using the Prism software.\n\n\nResults\n\nThe same DltA protein from B. cereus with a C-terminal hexahistidyl fusion tag used in our previous crystallographic studies on BcDltA17,24 was crystallized in the absence of ATP, D-alanine or CoA. One crystal diffracted to 1.9 Å resolution and belonged to space group P21 (Table 1), the same space group as in previously reported crystals of BcDltA in complex with D-alanine adenylate17 and with ATP24. Despite having 8 Å shorter crystallographic a axis, 5 Å shorter b axis and 5° smaller β angle than the previously reported crystal of DltA/D-alanine adenylate complex, the structure was successfully solved by rigid-body refinement using the previously determined BcDltA structure17 (PDB code 3DHV) as the starting model. The 504-residue BcDltA structure can be divided into two domains (Figure 2): an N-terminal major domain from the N-terminus to Asp-399, and a C-terminal minor domain from residue 400 to the C-terminus. The disposition of the two domains in the substrate-free BcDltA structure remains similar to that of the starting model (Figure 2A). The electron density map indicated several disordered regions (Ser-153 to Pro-159, Pro-363 to Glu-367, Arg-397 to Glu-413, Lys-433 to Tyr-440) with the corresponding regions in the starting model highlighted in magenta in Figure 2B. The first disordered region is part of a highly conserved P-loop (Thr-152 to Lys-160) found in homologous AMP-forming proteins37–39. Due to its similar amino acid composition (glycine, serine, threonine and lysine) to that of P-loop or Walker A motif found in ATPases and GTPases40, this loop has long been thought to catalyze the adenylation reaction. In the crystal structure of human medium-chain acyl-CoA synthetase in complex with ATP, this loop intimately interacts with the β- and γ-phosphates of the ATP substrate41. Functional relevance of regions 363–367 and 433–440 are unknown. The former is located at a 2-turn helix at the surface of the N-terminal domain (Figure 2B). The latter is interacting with the longest disordered inter-domain region in this structure (397–413) which contains several key elements of DltA. Arg-397 has been observed to interact with the β-phosphate of ATP and to play an important role in catalysis24. Asp-399, equivalent to Asp-398 in BsDltA, serves as the hinge residue for domain rotation. As observed for the equivalent Asp-402 of 4-Chlorobenzoate-CoA ligase15, the rotation around main-chain single bonds in this hinge residue could account for a 146° swing of the C-terminal domain as we compared the crystallized adenylation conformation of BcDltA and thiolation domain of BsDltA (Figure 1, bottom with the disordered inter-domain linker in green). The equivalent main-chain atoms in the N-terminal domains of BcDltA in adenylation conformation (PDB entry 3DHV) and BsDltA in thiolation conformation (PDB entry 3E7W) are superposed with a root-mean-deviation of 0.97 Å, and the deviation for the C-terminal domain was 1.00 Å. These values indicate that there is no dramatic conformational change within each domain in addition to the 146° rotation around the hinge aspartate residue. The C-terminal part of this flexible inter-domain region also contains a β-hairpin which has been observed to interact with CoA in homologous acetyl-CoA synthetase23.\n\nThe ribbons representation of previously reported BcDltA structure (PDB entry: 3DHV) is shown with D-alanine-adenylate and surrounding side-chains in ball-and-stick model. The N-terminal domain is shown in gray, and the C-terminal domain in gold. A. The substrate-free structure of BcDltA is superimposed on the BcDltA/D-Ala-AMP complex. The N- and C-terminal domains are colored in cyan and magenta, respectively. B. The four corresponding regions of the BcDltA/D-Ala-AMP complex, which are disordered in the substrate-free form of BcDltA, are highlighted in magenta.\n\nIn our previous study, we have verified that CoA can mimic D-alanyl carrier protein DltC24, as also discovered for DltA homologs42. In that study, we have observed that the reaction rate is increased by nearly an order of magnitude by the presence of saturating concentration of CoA, which is explained by the faster release of the thiolation product rather than by release of the adenylation intermediate. In order to get a comprehensive understanding of the effects by CoA as the DltC mimic, we further studied the enzymatic properties of BcDltA in the presence of a saturating concentration (5 mM) of ATP or D-alanine, and in the absence of CoA or in the presence of a saturating concentration (5 mM) of CoA. The reaction rates derived from the pyrophosphate accumulation assay and the thiol depletion assay were similar (Figure 4). The thiolation assay was noticeably noisier than the pyrophosphate assay and we therefore limit the discussion to KM and kcat values derived from the pyrophosphate assay. Somewhat unexpectedly, BcDltA showed much higher apparent affinity, or decreased KM value, towards ATP (0.46 mM to 0.01 mM) and D-alanine (1.1 mM to 0.03 mM) in the presence of 5 mM CoA (Figure 3 and Figure 4, Table 2). On the contrary, the apparent affinity towards L-alanine decreased in the presence of CoA, with KM increased from 14.4 mM to 109 mM (Figure 4 and Table 2).\n\nThe reaction solutions contained 0.005 mM wild-type BcDltA, specified concentrations of ATP and CoA, 5 mM D-alanine, 0.1 M KCl, 0.01 M MgCl2, 0.05 M Hepes-NaOH buffer at pH 7.2, 5 unit/mL of yeast inorganic pyrophosphatase. A. The initial rates of pyrophosphate accumulation divided by the BcDltA concentration are shown. The reaction rates in the presence of CoA are taken from a previous study24. B. Relative fluorescence gains as a fraction of the fluorescence intensity in the absence of ATP are shown.\n\nThe reaction solutions contained 0.005 mM wild-type BcDltA, 5 mM D-alanine, 0.1 M KCl, 0.01 M MgCl2, 0.05 M Hepes-NaOH buffer at pH 7.2, 5 unit/mL of yeast inorganic pyrophosphatase and specified concentrations of ATP, alanine and CoA. Reaction rates for wild-type BcDltA are shown on the left, and those for the C269A mutant protein are shown on the right. The reaction rates in the absence of CoA are taken from a previous study17.\n\nThe side-chain of Cys-269 sits at the bottom of the D-alanine-binding pocket which may make VDW clash with the methyl side-chain of L-Alanine16,17. We also studied the effect of CoA on D- and L-alanine preference of the C269A mutant of BcDltA (Figure 4 and Table 2). In the presence and absence of CoA, the C269A protein showed relaxed preference for D-alanine over L-alanine. As observed for the wild-type protein, CoA also enhances the D-alanine preference of this BcDltA mutant protein. However, the CoA-induced changes in Michaelis constant KM were less dramatic (3.1 mM to 0.50 mM for D-alanine, 6.6 mM to 8.8 mM for L-alanine) than those observed for the wild-type protein.\n\nChange in tryptophan fluorescence of BcDltA was minimal in the presence of ATP in the 10 micromolar range. We then found that at an excitation wavelength of 305 nm, the absorption of up to 2 mM of ATP was negligible and there was fluorescence gain associated with increasing concentration of ATP. The fluorescence gain-derived dissociation constant KD for ATP (0.43 mM) was similar to the Michaelis constant KM (0.46 mM) derived in the absence of CoA (Figure 3). There was no detectable fluorescence change to derive KD values for D-alanine. For CoA, we were not able to isolate fluorescence change from absorption by CoA in millimolar concentration.\n\n\nDiscussion\n\nBacteria selectively incorporate D- over L-alanine in cell wall components. There appears to be no exception in ubiquitous esterification of lipoteichoic acids by alanine25. The enantiomer selectivity of BcDltA observed in the absence of any D-alanyl carrier17, however, has been intriguingly mediocre. The newly acquired kinetic data in the presence of saturating CoA, a DltC mimic, shows that the kcat values are less than 2-fold different for D- and L-alanine (10.9 and 19.3 min-1) while the KM values are more than 1000-fold different favouring D-alanine over L-alanine (0.03 and 109 mM). Adding the fact that the intracellular concentration of D-alanine (in the order of 102 μM) is approximately 10-fold more abundant than the L-enantiomer (in the order of 101 μM)43, DltA functioning at a saturating concentration of the D-alanyl carrier protein DltC would favor the ligation of the D-enantiomer by approximately 4 orders of magnitude. Such striking enantiomer selectivity is consistent with the much lower L-alanine content found in lipoteichoic acid25. A recent study has shown that the dlt operon is induced by cell envelope stress such as acidic pH and antibiotics44. It is possible that stress-induced expression of DltC may reach a saturating concentration for interacting with DltA and therefore ensure the almost exclusive enantiomer selectivity as observed in the presence of the DltC mimic. The comparison between the kinetic properties of the wild-type and the C269A mutant proteins also supports the notion that Cys-269, and its equivalence in other DltA proteins, contributes to the enantiomer selectivity of DltA16,17.\n\nIt is satisfying to observe the stringent D-alanine preference. At the same time, it is also puzzling to appreciate that such selectivity on the chirality of alanine, a substrate of the adenylation reaction, can only be achieved in the presence of CoA, a substrate of the thiolation reaction. The intrinsic fluorescence of tryptophan in BcDltA enabled us to derive the dissociation constant KD for ATP in the absence of other substrates. The new form of crystal structure in the absence of any substrate is noticeably more disordered than the previously observed adenylation conformation of BcDltA17 and thiolation conformation of BsDltA16. The longest such disordered region is between Arg-397 and Glu-413, which contains the inter-domain hinge residue Asp-399, interacts with β-phosphate of ATP, and forms the pantetheine channel. Possibly, this important region remains disordered in the presence of saturating D-alanine, therefore providing an explanation for the similar values between the above mentioned KD (0.43 mM) and the KM (0.46 mM) for ATP, in the presence of saturating D-alanine but in absence of CoA. The more disordered nature of the substrate-free conformation of BcDltA also implies that the previously observed adenylation and thiolation conformations are intrinsically unstable unless stabilized by the interaction with one or more substrates. This structural feature of BcDltA likely explains the relatively low sub-millimolar affinity for ATP, since part of the stabilizing BcDltA-ATP interactions would be used to compensate the cost of establishing the adenylation conformation of the protein.\n\nBcDltA is a very slow enzyme. Unless the substrate dissociation step happens to be extremely slow as well, we could approximate the observed KM values to the KD values of corresponding BcDltA-substrate intermediates, and therefore enable reasoning in the context of structural stability of such intermediates. As such, we reasoned that the approximately one order of magnitude difference in KM values for the adenylation substrates ATP and D-alanine observed in the presence and absence of the thiolation substrate may imply the existence of a quadruple intermediate of the DltA enzyme in complex with all three substrates, which may be markedly different from a ternary intermediate of BcDltA with the two adenylation substrates.\n\nWe then resorted to three-dimensional model building so as to answer the question on which of the adenylation and thiolation conformations may be compatible with binding to all three substrates. The BcDltA/D-alanine-adenylate complex (PDB entry 3DHV)17 was chosen as the adenylation conformation and as the reference set of atomic coordinates. The adenylated intermediate was dissected to generate the D-alanine model. The N-terminal domain of the BcDltA/ATP complex (PDB entry 3FCE)24, which is also in the adenylation conformation, was superposed on the reference set to orient the ATP substrate. The N-terminal domain of the BsDltA/AMP complex (PDB entry 3E7W) was also superposed on the reference set to derive the re-oriented thiolation conformation. Main-chain atoms equivalent to those interacting with AMP in BcDltA (270–272, 292–299) in the quadruple complex of acetyl-CoA synthetase in its thiolation conformation (PDB entry 2P2F)23 were superimposed on the reference set to orient the CoA model. In the adenylation conformation (Figure 5A, the pantetheine channel is apparently blocked by the main-chain atoms immediately preceding the catalytic Lys-492 of BcDltA (Lys-491 of BsDltA). Although we could not completely rule out the possibility that an allosteric site for CoA exists, no such site has ever been observed for this superfamily of enzymes. Therefore, a quadruple complex in the adenylation conformation is unlikely. The thiolation conformation (Figure 5B), on the other hand, appears to be compatible with binding ATP so long as Arg-408 of BcDltA (Arg-407 of BsDltA) adopts another rotamer. It has been previously observed that the homologous acetyl-CoA synthetase in its thiolation conformation binds AMP, acetate and CoA23, and that BsDltA in its thiolation conformation binds AMP and appears to have a well-formed D-alanine-binding pocket16. These structural evidences seem to suggest that a quadruple intermediate may form with the enzyme in its thiolation conformation but not in the adenylation conformation. Since the thiolation conformation has an AMP-binding site and its N-terminal domain, which provides most of the ATP-interacting residues such as the P-loop and Arg-397, remains essentially identical to that that in the adenylation conformation, it is not surprising that only one arginine side-chain is required to adopt another rotamer to accommodate ATP. This arginine residue (Arg-408 of BcDltA) forms a salt bridge with the divalent cation-anchoring side-chain of Glu-298 in BcDltA (Glu-297 in BsDltA)24, which appears to be an important structural feature to modulate the conformational change16. Another arginine residue (Arg-397 of BcDltA, Arg-396 of BsDltA) also seems to facilitate the conformational change. It forms a salt bridge with the hinge aspartate residue in the thiolation conformation. In the adenylation conformation, this arginine side-chain adopts a more extended rotamer and forms a salt bridge with the β-phosphate group of ATP (Figure 5A). ATP binding would result in re-orientation of both arginine residues and disruption of two salt bridges, thus mobilizing the thiolation conformation.\n\nCα traces of the two modelled quadruple complexes are shown in stereo. Except for the green-colored region between Arg-397 and Glu-413, the N- and C-terminal domains are colored in gray and salmon, respectively. The three substrates and selected side-chains are shown in ball-and-stick model. A. The quadruple complex in adenylation conformation. Residue numbers in BcDltA are shown. B. The quadruple complex in thiolation conformation. Residue numbers in BsDltA are shown.\n\nThe majority of ATP-binding elements lie in the N-terminal domain which remains similar in both adenylation and thiolation conformations. The most significant structural feature in the C-terminal domain for binding ATP is the catalytic Lys-492 of BcDltA (Lys-491 of BsDltA) in the adenylation conformation. In the thiolation conformation, the catalytic residue is replaced by Lys-403 (Lys-402 of BsDltA) (Figure 5). In addition, the thiolation conformation is more ordered than the substrate-free conformation. The favourable DltA/ATP interactions may no longer be used to compensate the energetic cost of stabilizing the disordered hinge region. Therefore the affinity for ATP by the CoA-bound BcDltA in its thiolation conformation should be higher than substrate-free BcDltA.\n\nFor both the wild-type and C269A mutant BcDltA, the presence of saturating CoA increases the apparent affinity for D-alanine while decreasing affinity for L-alanine. The majority of alanine-interacting residues lie in the N-terminal domain. The amino group of D-alanine is stabilized by Asp-197 of BcDltA (Asp-196 of BsDltA). Cys-269 of BcDltA (Cys-268 of BsDltA) lies close to Cα of D-alanine and serves as one major determinant of enantiomer selectivity. The carboxylate group of D-alanine is stabilized by Lys-492 in the adenylation conformation or by Lys-403 in the thiolation conformation. At the end of the pantetheine channel, Phe-196 of BcDltA (Phe-195 of BsDltA) adopts different rotamers in the two conformations. In the adenylation conformation of BcDltA/D-alanine adenylate, the shortest distance from Phe-196 side-chain to methyl group of D-alanine is 4.15 Å. In the modelled quadruple complex in the thiolation conformation, the Phe-196 side-chain does not contact D-alanine but the thiol group of CoA lies in closer proximity of D-alanine side-chain (3.34 Å), as expected from the pending thiolation reaction between the thiol group of CoA and the carbonyl group of D-alanine-adenylate. On the other hand, the tighter alanine-binding pocket may exert stronger VDW repulsion toward L-alanine, thus lowering further the affinity for the wrong enantiomer. Interestingly, Cys-269 also has a thiol group. The removal of this group in the C269A mutant of BcDltA reduces the affinity for D-alanine and increases the affinity for L-alanine by approximately one order of magnitude, which is almost exactly the opposite to the effect of introducing the thiol group of CoA in the alanine-binding pocket. Since sulphur atoms are larger and more inducible than the second-period elements carbon and oxygen, it is not surprising that the VDW interaction involving a thiol group makes a significant impact on the stability of enzyme/substrate complex.\n\nIntracellular concentration of D-alanine43 generally exceeds the KM value in the presence of saturating ATP and CoA. Typical intracellular concentration of ATP lies in the millimolar range45, exceeding the KM value for ATP as well. The concentration of the possibly stress-induced DltC44 may also reach saturating level in bacteria when such stress is present. As implied by the CoA-triggered dramatic change in KM values for ATP and D-alanine, the two adenylation substrates are likely incorporated by the enzyme with pre-bound CoA rather than with merely the other adenylation substrate. In addition, three-dimensional modelling suggests the CoA-bound state of the enzyme can only exist in its thiolation conformation. We therefore hypothesize a two-conformation model for the enzymatic cycle catalyzed by DltA (Figure 6) in the presence of saturating concentration of the three substrates. Our model differs from the three-conformation model proposed for DltA and adenylation domains in non-ribosomal peptide synthetase16 which includes a third substrate-free conformation. This model also differs from the two-conformation model proposed for and for 4-chlorobenzoate-CoA ligase26,46 which includes substrate-free state of the enzyme. The two previously proposed models are consistent with a typical Ping-Pong mechanism. Neither a substrate-free conformation nor a substrate-free state of the enzyme is required in our model for the enzymatic cycle of DltA once the protein enters the reaction cycle. The adenylation reaction starts from a ternary complex with ATP and D-alanine and proceeds with the release of pyrophosphate. A domain rotation around the aspartate hinge residue follows transforming into the thiolation conformation which binds CoA, or DltC in bacteria, and catalyzes the thiolation reaction with D-alanine by displacing AMP. In our model, the resulting complex with the two thiolation products proceeds with AMP release, D-alanine-CoA/CoA exchange, D-alanine binding, and ATP binding. In the quadruple complex, the ATP-bound magnesium ion would replace Arg-408 of BcDltA in forming a bridge with Glu-298, as observed in the crystal structure BcDltA/ATP complex24. The disruption of the Arg-408 to Glu-298 salt bridge would destabilize the thiolation conformation and facilitate a reverse domain rotation and release of CoA which is not compatible with the adenylation conformation. The hypothesized D-alanine-CoA/CoA exchange step is the central piece of this enzymatic cycle, which reflects the finding that CoA affects DltA’s apparent affinities with D-alanine and ATP. If DltA were to become substrate-free for a long enough period of time, it would form a ternary complex with ATP and D-alanine, which contradicts the observed effect by the thiolation substrate CoA. It is worth noting that the sequence of D-alanine and ATP binding is uncertain. Similarly, AMP release has to occur before ATP binding but not necessarily before exchange with CoA or D-alanine binding.\n\nNon-covalent DltA complexes are shown in text. Forward reaction, substrate binding and release steps are shown by arrows. Steps fulfilled by the adenylation conformation are shown in the green box, and those by the thiolation conformation in brown. The thioester between D-Ala and CoA (D-Ala-CoA) is the final product.\n\nThe hypothesized enzymatic cycle involves a second CoA binding step, and a CoA release step in addition to the Ping-Pong mechanism previously proposed for DltA and its homologs. Both additional steps seem unnecessary for the enzymatic reaction itself, but are required to explain our enzymatic data and are consistent with the three-dimensional models of BcDltA and BsDltA. A Ping-Pong mechanism would require that the binding of CoA and the binding of either adenylation substrate be uncompetitive, and therefore the apparent KM and kcat values for either adenylation substrate both would become larger at higher CoA concentration. While the kcat values for both ATP and D-alanine did become larger at saturating CoA concentration, the KM values actually became smaller, therefore contradicting the typical Ping-Pong mechanism. Another property of aminoacyl-CoA synthetases including DltA is the inhibitory effect of CoA at high concentration42, which is difficult to explain by a typical Ping-Pong mechanism unless the enzymatic cycle includes an additional CoA-dissociation step as in our model. The closest homologs of DltA are amino acid-activation domains found in non-ribosomal peptide synthetases42. Similar to DltA, these homologs also pass the adenylated intermediate to the 4’-phosphopantetheine group attached to a serine residue on a peptide carrier domain. It is possible that such amino acid activation domains may also act as DltA.\n\nThe extra binding step for 4’-phosphopantetheine D-alanyl carrier could serve as the sensor for the availability of the carrier. The maximum rate catalyzed by BcDltA is approximately seven times faster in the presence of CoA than in its absence, with respective kcat values 10.9 min-1 and 1.5 min-1. Moreover, the intracellular concentration of D-alanine is typically found in the 100 micromolar range43, which lies above the KM value for D-alanine in the presence of CoA (~30 µM) but below the KM value in the absence of CoA (1100 µM) (Table 2). The reaction rate should become slower by approximately 100 fold when the thiol carrier is absent. It is worth noting that the adenylated D-alanine intermediate generated by the adenylation reaction is not covalently attached to the enzyme and could be released and wasted when the thiolation substrate is absent. The significant slowing down of the adenylation reaction in the absence of the 4’-phosphopantetheine carrier therefore provides a biological advantage.\n\n\nData availability\n\nfigshare: Data of pyrophosphate release, ATP-induced fluorescence and thiol depletion for DltA in Bacillus cereus, http://dx.doi.org/10.6084/m9.figshare.101848947",
"appendix": "Author contributions\n\n\n\nYL conceived the study, designed the experiments, determined the crystal structure, and wrote the manuscript. LD carried out cloning, expression, crystallization and biochemical assays, and approved the manuscript for publication.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work is supported by a Saskatchewan Health Research Foundation Phase 3 Team Grant to the Molecular Design Research Group at University of Saskatchewan, and by a Natural Sciences and Engineering Research Council Discovery Grant 261981-2010 to YL.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe thank Drs. Gabriele Schatte and Wilson Quail for assistance with the X-ray facility at the Saskatchewan Structural Sciences Centre.\n\n\nSupplementary file\n\nPDB validation report\n\n\nReferences\n\nArmstrong JJ, Baddiley J, Buchanan JG, et al.: Composition of teichoic acids from a number of bacterial walls. Nature. 1959; 184: 247–248. PubMed Abstract | Publisher Full Text\n\nHyyrylainen HL, Vitikainen M, Thwaite J, et al.: D-Alanine substitution of teichoic acids as a modulator of protein folding and stability at the cytoplasmic membrane/cell wall interface of Bacillus subtilis. J Biol Chem. 2000; 275(35): 26696–26703. PubMed Abstract | Publisher Full Text\n\nFischer W: Physiology of lipoteichoic acids in bacteria. Adv Microb Physiol. 1988; 29: 233–302. PubMed Abstract\n\nPerego M, Glaser P, Minutello A, et al.: Incorporation of D-alanine into lipoteichoic acid and wall teichoic acid in Bacillus subtilis. Identification of genes and regulation. J Biol Chem. 1995; 270(26): 15598–15606. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiqin Du, Yu Luo: Data of pyrophosphate release, ATP-induced fluorescence and thiol depletion for DltA in Bacillus cereus. Figshare. 2014. Data Source"
}
|
[
{
"id": "5123",
"date": "16 Jun 2014",
"name": "Ting Fang Wang",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper provides an intriguing two-conformation hypothesis for the enzymatic cycle of B. cereus D-alanyl carrier protein ligase DltA. Overall, both structural determination and biochemical assays were well done.A minor point: The two-conformation hypothesis (Figure 6) suggests that the inclusion of ATP to the DltA-CoA-Ala complex functions includes CoA release and a large domain rotation. It is important to show that AMP-CPP, a non- hydrolyzable analog of ATP, can indeed induce CoA release.",
"responses": [
{
"c_id": "875",
"date": "23 Jun 2014",
"name": "Yu Luo",
"role": "Author Response",
"response": "It would be interesting to carry out an experiment to study ATP-stimulated release of CoA or DltC. One plan is to use a surface plasmon resonance-based technique, or similar techniques, to study the association and dissociation of DltA with DltC under various conditions."
}
]
},
{
"id": "5026",
"date": "17 Jun 2014",
"name": "Charles Bell",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nltA catalyzes two reactions: (1) an adenylation reaction in which ATP is joined to D-Ala to form D-Ala-AMP (PPi is released), and (2) a thiolation reaction in which D-Ala is transferred from D-Ala-AMP to a phosphopantetheine group of D-alanyl carrier protein (DltC). The reaction can be mimicked in vitro by substituting CoA for DltC (does this also happen in vivo?). Previous crystal structures have indicated that the two reactions are promoted by two distinct conformational states of the enzyme, which differ by a large rearrangement of a C-terminal domain that closes over the active site. In this work, Du & Luo provide kinetic data to suggest that binding of the two substrates for the adenylation reaction (ATP and D-Ala) is promoted by the presence of the substrate (CoA) for the second reaction. The selectivity for binding of D-Ala as opposed to L-Ala is also enhanced significantly by the presence of CoA. A new scheme for the reaction cycle is proposed in which instead of forming an empty enzyme when the final product D-Ala-CoA is released, free CoA is rapidly “exchanged” in for D-Ala-CoA to keep the enzyme in its thiolation conformation. Then new D-Ala and ATP substrates bind (with the enzyme still in its CoA-bound thiolation conformation), and CoA is released to allow the enzyme to adopt the adenylation conformation, which is required for ATP and D-Ala to react. Binding of CoA to the newly formed D-Ala-AMP complex then puts the enzyme back in the thiolation conformation. The new reaction scheme is considerably more complicated than one would expect, but seems to account for the observed kinetic data. The authors propose that the biological purpose for this type of mechanism is to limit binding (and reaction) of ATP and D-Ala to conditions in which the 2nd substrate (presumably DltC in vivo) is available, so as to avoid non-productive formation and release D-Ala-AMP. A crystal structure of the apo form of DltA is also determined, which shows that in the absence of substrates the enzyme adopts the adenylation conformation, and that several loops on the enzyme are considerably more disordered than in previous structures of various substrate or product-bound states. The new structure does not add a lot, but is meant to support the concept that ATP and D-Ala do not bind tightly to the enzyme unless CoA is around to reduce the overall flexibility. The manuscript is reasonably well written, scientifically sound, and should be of fairly broad interest, particularly for those studying other types of two-reaction enzymes, such as in non-ribosomal peptide synthesis, and possibly even enzymes like amino-acyl tRNA synthetases. I found the manuscript to be rather uninteresting, until I got to the end of the discussion, where the newly proposed reaction scheme of Figure 6 is presented, and a biological purpose of the observed phenomena is proposed. If there is some way to bring these aspects into the Abstract or the Introduction, a potential reader might be drawn in a little bit earlier than I was. The proposed scheme is quite unusual in that CoA supposedly promotes binding of ATP and D-Ala, but subsequent release of CoA is required for the two to react. Presumably this means that the rate of the catalytic step for the adenylation reaction must be considerably faster than the rates of ATP and D-Ala dissociation, or else the enzyme could return to the apo state, and presumably the adenylation conformation, when CoA is released. Are these parameters known? Also, is the physiological concentration of DltC acceptor protein, which presumably takes over the role of CoA in vivo, high enough to be consistent with the proposed reaction cycle? It would need to rebind to the enzyme in the exchange step before the enzyme reverts to the adenylation conformation. Presumably the rate of DltC binding is faster than the rate of reversion back to the adenylation conformation? The authors should discuss/clarify these points. Minor points: The data in this paper demonstrate the effect on the enzyme by CoA, a mimic of the in vivo substrate DltC, but not for DltC itself. Is there some reason why this is difficult to demonstrate with DltC experimentally? Was it previously known from other structures that the apo form of the enzyme is in the adenylation conformation, or is this a new insight? In general the authors could do a little bit of a better job describing the previous structures in the Introduction. Why were the previous structures of the B. subtilis enzyme found to be in the thiolation conformation despite the absence of CoA (or DltC)? Is there no structure available with CoA? References 16 and 18 are the same.",
"responses": [
{
"c_id": "876",
"date": "23 Jun 2014",
"name": "Yu Luo",
"role": "Author Response",
"response": "As far as we know, the rate constants of substrate binding, reaction and product dissociation steps have not been measured for DltA or its closely related homologs that transfer the activated amino acyl or peptidyl group to a carrier protein. We do not have expertise in measuring such rate constants.It is generally accepted that DltC is the D-alanine carrier in vivo. CoA, however, is a more feasible analog of DltC for enzymatic assays. If DltC were used, the assay would require either millimolar concentration of DltC or addition of a thioesterase to regenerate DltC, for which we haven't resolved the technical difficulties. The B. subtilis DltA in thiolation conformation has been co-crystallized with DltC, although DltC is disordered or absent from the crystal structure. Homologous acyl and aryl CoA synthetases have been known to crystallize in either adenylation or thiolation conformation. Remotely related Japanese firefly luciferase has been crystallized in an apo-form dissimilar to its adenylation conformation. However, such luciferases do not catalyze the second thiolation reaction and recently, DltA from S. pyogenes has been crystallized in the absence of any substrate. The structure (PDB entry 3L8C, to be published) resembles the adenylation conformation of B. cereus DltA."
}
]
}
] | 1
|
https://f1000research.com/articles/3-106
|
https://f1000research.com/articles/3-105/v1
|
12 May 14
|
{
"type": "Research Article",
"title": "Exercise-induced arterial hypertension - an independent factor for hypertrophy and a ticking clock for cardiac fatigue or atrial fibrillation in athletes?",
"authors": [
"Roman Leischik",
"Norman Spelsberg",
"Hiltrud Niggemann",
"Birgit Dworrak",
"Klaus Tiroch",
"Norman Spelsberg",
"Hiltrud Niggemann",
"Birgit Dworrak",
"Klaus Tiroch"
],
"abstract": "Background: Exercise-induced arterial hypertension (EIAH) leads to myocardial hypertrophy and is associated with a poor prognosis. EIAH might be related to the “cardiac fatigue” caused by endurance training. The goal of this study was to examine whether there is any relationship between EIAH and left ventricular hypertrophy in Ironman-triathletes.Methods: We used echocardiography and spiroergometry to determine the left ventricular mass (LVM), the aerobic/anaerobic thresholds and the steady-state blood pressure of 51 healthy male triathletes. The main inclusion criterion was the participation in at least one middle or long distance triathlon.Results: When comparing triathletes with LVM <220g and athletes with LVM >220g there was a significant difference between blood pressure values (BP) at the anaerobic threshold (185.2± 21.5 mmHg vs. 198.8 ±22.3 mmHg, p=0.037). The spiroergometric results were: maximum oxygen uptake (relative VO2max) 57.3 ±7.5ml/min/kg vs. 59.8±9.5ml/min/kg (p=ns). Cut-point analysis for the relationship of BP >170 mmHg at the aerobic threshold and the probability of LVM >220g showed a sensitivity of 95.8%, a specificity of 33.3%, with a positive predictive value of 56.8 %, a good negative predictive value of 90%. The probability of LVM >220g increased with higher BP during exercise (OR: 1.027, 95% CI 1.002-1.052, p= 0.034) or with higher training volume (OR: 1.23, 95% CI 1.04 -1.47, p = 0.019). Echocardiography showed predominantly concentric remodelling, followed by concentric hypertrophy.Conclusion: Significant left ventricular hypertrophy with LVM >220g is associated with higher arterial blood pressure at the aerobic or anaerobic threshold. The endurance athletes with EIAH may require a therapeutic intervention to at least prevent extensive stiffening of the heart muscle and exercise-induced cardiac fatigue.",
"keywords": [
"Myocardial hypertrophy in hypertensive patients has a negative influence on long-term prognosis1",
"cardiac arrhythmias and mortality2",
"3. Myocardial hypertrophy in otherwise healthy",
"non-hypertensive individuals can be caused by exercise-induced arterial hypertension (EIAH)4–6 and may also result in poor prognosis7. Moreover",
"exercise-induced hypertrophy may cause sudden cardiac death in athletes8. However",
"myocardial hypertrophy induced by extensive exercise presents so called “normal diastolic” function",
"which might be a result of “physiological” adaptation9",
"10. EIAH or elevated blood pressure values during exercise might have a “negative” impact on cardiac function in athletes and might be one of the important factors causing “exercise-induced” cardiac fatigue (Figure 1). Our hypothesis is provocative",
"but this suggestion might become important for many professional and leisure athletes (Figure 2)."
],
"content": "Introduction\n\nMyocardial hypertrophy in hypertensive patients has a negative influence on long-term prognosis1, cardiac arrhythmias and mortality2,3. Myocardial hypertrophy in otherwise healthy, non-hypertensive individuals can be caused by exercise-induced arterial hypertension (EIAH)4–6 and may also result in poor prognosis7. Moreover, exercise-induced hypertrophy may cause sudden cardiac death in athletes8. However, myocardial hypertrophy induced by extensive exercise presents so called “normal diastolic” function, which might be a result of “physiological” adaptation9,10. EIAH or elevated blood pressure values during exercise might have a “negative” impact on cardiac function in athletes and might be one of the important factors causing “exercise-induced” cardiac fatigue (Figure 1). Our hypothesis is provocative, but this suggestion might become important for many professional and leisure athletes (Figure 2).\n\nThis figure shows the factors with possible negative influence on myocardium like inflammation, fibrosis etc. It demonstrates the possible complexity of different actions.\n\nRight and left atrium have more connective tissue construct as muscular ventricular chambers and more affinity for pathological enlargement in case of pressure overload.\n\nPhysical activity in the general population is of fundamental importance11,12. The role of EIAH in normotensive adult athletes13 or healthy men is currently under discussion6,14. It is unclear how far endurance sport can influence a “negative remodelling” of the athlete’s heart15. The dosage of exercise bouts which causes cardiac injury18,19, and the “true pathologic values” of EIAH are unknown or under debate6.\n\nEndurance sport is linked to cardiac injury20. In individual cases, long term training might lead to arrhythmias21, atrial fibrillation22,23 or myocardial fibrosis16,24 and early sudden cardiac death24–26, female athletes are less commonly affected26,27.\n\nThe type of sport discipline has also an influence on the type of hypertrophy. Some authors distinguish the strength-trained heart and an endurance-trained heart10,28. Further factors that might influence exercise-induced hypertrophy are genetic factors29,30, gender31, environmental factors32, endocrine factors33 and arterial hypertension34.\n\nIn this study, we examined the impact of EIAH on cardiac hypertrophy in 51 normotensive (at rest) healthy Ironman athletes with long daily training times.\n\n\nMaterials and methods\n\nThe influence of EIAH on cardiac hypertrophy was examined in 51 male triathletes (mean age 37.2, Table 1) who finished an Ironman 70.3 (n=17, 1.9km swim, 90km bicycle ride and 21,1km run) or Ironman full distance (n=34/3.8km swim, 180km bicycle ride and/42.2km run). The training habits were similar for both the 70.3 and long distance Ironman. The minimum training-time was two years. All triathletes have been examined by spiroergometry and echocardiography. There is no consensus about the value of systolic BP that determines EIAH6. According to the literature, EIAH is described as systolic BP >210mmHg for males and >190mmHg for females, as maximal values during exercise5. In our study, the absolute values primarily were not defined. Odds ratios analysis was used to calculate the probability of elevated blood pressure and hypertrophy. The estimation of sensitivity/specificity to detect the boundaries of the EIAH and the positive or negative predictive values for the blood pressure boundaries should be performed during the study. Our interest was directed to the aerobic and anaerobic threshold, because this is a constant level of blood pressure maintained during training or competitions.\n\nThe blue coloured area shows the anthropometric data of the two groups with different LVM. The orange coloured data are echocardiographic data of the left ventricle. The green coloured data are the Doppler-flow data. The last two lines are the data of the right ventricle.\n\nMean = mean value. sd = standard deviation. BMI = body mass index. BSA = body surface area. LAESV = left atrial endsystolic volume. IVS = interventricular septum. PWD = diastolic left ventricular posterior wall thickness. RWT: relative wall thickness: (2xPWD/LVEDD). LVEDD = left ventricular end-diastolic diameter. LVM = left ventricular mass. LVEDV: left ventricular enddiastolic volume. LVESV: left ventricular endsystolic volume. SV: Stroke volume. EF: Ejection fraction in %. LVOT = left ventricular outflow tract. MV: Mitral valve. parasternal: right ventricular diameter in 2D parasternal view. RV AFC%: right ventricular area fractional change.\n\nA Vivid 7 model echocardiograph manufactured by general Electric was used for the examinations. The Ergobike 8I manufactured by Daum and the Metalizer 3B produced by Cortex were used for the spiroergometric examination35.\n\nThe assessment of each triathlete was performed in 2011 and 2012 on the same day with the echocardiography first followed by spiroergometry. The spiroergometry was performed as follows: the stress test (exercise bike) was conducted in stages after successful gas and volume calibration: 50W for 3 minutes, 100W for further 3 minutes and thereafter increased by another 30W for 3 minutes (ramp-test). The test ended when the subject could no longer maintain the predefined rpm of 90 or if the subject was exhausted.\n\nThe echocardiographic analysis was conducted according to general recommendations36,37. The formula recommended by the American Society of Echocardiography (ASE) was used for calculate the muscle mass. Enddiastolic LV-volume (EDV) and Endsystolic LV-volume (ESV) were determined monoplane after the modified Simpson method36.\n\nThe spiroergometric analyses were conducted according to previously published protocols38,39: VAT (ventilatory aerobic threshold) was determined as the first non-linear increase of the ventilatory equivalent for oxygen without simultaneous increase of the ventilatory equivalent for CO2, and RCP (respiratory compensation point: anaerobic threshold) was determined as simultaneous non-linear increase of both ventilatory equivalents according to previous recommendations38,39.\n\nVO2max was registered as the highest average value of oxygen absorption over 30 seconds.\n\nThe entire statistical analysis plan was designed as follows: Stata/IC 13.1 for Windows was used for data preparation and statistical analysis. The Mann-Whitney-U-Test was used to compare the groups with LVM >220g and LVM <220g. Odds Ratios were calculated to measure the association between blood pressure, training habits and the probability of LVM >220g. Since these exposure variables are quantitative variables, an approximate estimate of the log odds-ratio for a one-unit increase in exposure and a 1-degree-of-freedom test for trend were calculated. All statistical tests were two-sided with a signficance level of 0.05.\n\nIn addition, sensitivity, specificity, positive and negative predictive values as well as the proportion of correctly classified participants were calculated for each possible cut-point of blood pressure to describe the performance of blood pressure as a “diagnostic test” for LVM >220g.\n\n\nResults\n\nAnthropometric baseline data of triathletes are listed in Table 1. The blood pressure values of the two groups (LVM <220g and LVM >220g) are visualized in Figure 3 (for exact values see Table 2). In Figure 3 one can see that triathletes with LVM >220g have higher blood pressure values at ventilatory aerobic threshold (VAT) and anaerobic threshold (RCP) and at maximum achieved Watt-level (Wattmax). The results showed myocardial hypertrophy in most participants and were classified as according to Lang et al.36. Normal morphology was found in three triathletes, eccentric hypertrophy was shown in one athlete, concentric remodelling was observed in 26 triathletes and concentric hypertrophy in 21. Right ventricular remodelling or other pathological findings of the right ventricle were not found in any of the athletes. Left ventricular function was good in all triathletes (EF >55%). All relevant echocardiographic values are shown in Table 1. All further parameters are shown in Table 2, sorted according to the p-value.\n\nTriathletes in the group with LVM >220g have significant longer training times and distances on bike, longer overall training times (Mann-Whitney-U-Test).\n\nTr = training. BP = Blood Pressure\n\nBPsAnaerobicThreshold = systolic blood pressure at the anaerobic threshold\n\nBPsWattmax = systolic blood pressure at the maximum power output time\n\nrel. VO2RCP = relative oxygen uptake at the anaerobic threshold\n\nrel. VO2max ml/min/kg = relative maximal oxygen uptake\n\nIVRT = Isovolumetric relaxation time\n\nWattmax = maximum power output\n\nThe group with LVM >220 shows significant higher systolic blood pressure (BP) values at the aerobic threshold (VAT), anaerobic threshold (RCP) and at the maximum exercise-level (Wattmax).\n\nBP = Blood pressure. LVM = left ventricular mass. VAT = ventilatore aerobic threshold.\n\nRCP = respiratory compensation point. Wattmax = Maximum exercise-level.\n\nOxygen uptake, ergometer performance and heart rate with VAT, RCP and at peak capacity are shown in Table 3. Participants with LVM >220g achieved at all thresholds and at maximum level higher power output values. Relative oxygen uptake values were slightly higher in the group with LVM >220g but not significant different at the maximum stage of loading. Spiroergometric maximum oxygen uptake (relVO2max) was 57.3±7.5 ml/min/kg vs. 59.8±9.5 ml/min/kg (p=n.s.) for LVM <220g vs. >220g, respectively.\n\nThe table is divided in three main blocks: the first block reflects data at the aerobic threshold, the second one at the anaerobic threshold and the last one at the maximum exercise stage.\n\nMv = Mean value;\n\nsd = standard deviation,\n\naVO2 = absolute oxygen uptake in L/min,\n\nrVO2 = relative oxygen uptake in ml/min/kg,\n\n% point of the overall exercise-test\n\nHR = heart rate,\n\nWatt = power output\n\nTable 4 shows the cut-point analysis for blood pressure values and the probability of development of LVM >220g. BP values over 180mmHg at the aerobic threshold might define the athletes at risk of developing LVM >220g and a possible further cardiac fatigue.\n\nAccording to the values reported by Devereux et al.40 and Bove et al.41, we divided the triathletes in two groups: group 1 (LVM >220g) and group 2 (LVM <220g) to assess the possible reasons for left ventricular hypertrophy. The significant differences between the two groups are shown in Table 1 and Table 2. In summary, left ventricular mass (<220g vs. >220g) is associated with significantly different blood pressure values at the anaerobic threshold (185.2±21.5mmHg vs. 198.8±22.3mmHg, p=0.037).\n\nThe probability of dependent factors for LV-hypertrophy was calculated by odds ratios (Table 5). Odds ratios analysis showed a significant relationship between the arterial pressure values during exercise (significant p-values at the aerobic and anaerobic threshold in Table 5). The significant values are bold in Table 5. A further relationship was found between bike training times and overall training times and LVM >220g (Figure 4). Values above 1.0 show this significant relationship.\n\nThe significant p-values at the aerobic and anaerobic threshold are in bold.\n\nmean = mean value.\n\nBSA = Body Surface Area\n\nTr = Training\n\nBPs = systolic blood pressure\n\nAerobicT = Aerobic threshold\n\nAnaerobicT = Anaerobic Threshold\n\nThis figure is based on the values reported in Table 5. Values above 1.0 show a significant relationship between bike training times and overall training times and LVM >220g.\n\n\nDiscussion\n\nThe most interesting finding of this study is that myocardial hypertrophy depends on exercise-induced arterial hypertension. This confirms the results described by Douglas et al.13 and Longas-Tejero et al.42, who found a hypertensive response to exercise in eight of 37 healthy athletes (18 soccer players, 12 mountain climbers and seven canoeists). In this cited study, athletes with EIAH showed higher LVM (205g/m2) compared to those without exaggerated blood pressure response to exercise (143g/m2). There is no consensus about the value of systolic blood pressure that constitutes EIAH6. According to our study, it seems that a systolic BP value >180mmHg at the aerobic threshold indicates exercise-induced arterial hypertension. So far, the boundaries for EIAH have never been estimated. In this study, we have chosen the aerobic threshold as the measuring point because the majority of the triathlete’s training is carried out at this level. When the hypertensive BP value is reached, we should analyse whether a careful low dosage treatment might be beneficial (for example with ACE inhibitors or AT1-blockers). The goal of such therapy would be to cut the blood pressure peaks (bouts) during training or competitions and avoid an increase of stiffness of the aorta43 or LV-hypertrophy in people at risk. Raised BP bouts can lead to pathological enlargement of atrial dimensions in athletes (Figure 2 and Figure 5). Enlargement of the left atrium may lead to atrial fibrillation and higher activity of electric circuits. There are no clear statements or guidelines regarding the role of EIAH in the daily practice of sports medicine44. This manuscript may encourage a discussion about this important issue. The possible impact of EIAH on cardiac structures in triathletes is shown in Figure 2. A specific case of cardiac remodelling is shown in Figure 5. In this Figure are shown normal heart cavities of a triathlete with EIAH in 2011 and massive atrial enlargement in 2014. Exercise-induced hypertension was often discussed in the 1990s4,5,7 reflecting the results of the Framingham Study5. The negative role of EIAH in non-athletic men is relatively clear6, but the impact on athletes needs to be discussed and the “pathological range” of EIAH should be evaluated. Exercise-induced hypertension promotes myocardial hypertrophy4 and increases cardiovascular risks7 in normotensive men. Athletes with EIAH are in similar way “persons at risk” and may develop a pathological cardiac chamber enlargement and atrial fibrillation, but have less “cardiovascular risk” because of the healthier life style and the positive impact of sport in the development of arteriosclerotic complications.\n\nIn 2011, the participant showed a normal size of the right and left atrium. An atrial enlaragement occured 2014 (arrows) after a period of high intensity training (echocardiography in 4 chamber view during atrial fibrillation). Even after cardioversion (2014) into sinus rhythm (7 days in sinus rhythm) he retains larger atrial cavities as in 2011.\n\nThe specific endurance training of triathletes leads to physiological changes of performance parameters45 and also results in changes in cardiac function or heart structures46. This adaptation is linked to the nature and magnitude of the physical exercise31. The physiological adaptation is a “harmonic increase in size” of a healthy heart caused by physical activity47. The term “athlete’s heart”9,31 has been known since 189948. Cardiovascular adaptations to exercise have been systematically defined according to the type of endurance training.\n\nConcentric hypertrophy in triathletes has already been described49,50. Douglas et al.50 suggested that athletes develop hypertrophy possibly due to the systolic blood pressure increase under exercise, which could be explained by the frequency of the training. Diastolic function was shown to be normal under those conditions.\n\nIn the present study, odds ratio analysis showed a significant relationship of myocardial thickening to exercise-induced blood pressure. It can be assumed that training over an extended period with exercise-induced blood pressure elevation can lead to hypertrophy in a similar way to that found in pathological conditions with arterial hypertension. Concentric remodelling was found in 26 cases and concentric hypertrophy in 21 cases. Concentric remodelling and concentric hypertrophy occurs more often in male athletes31. Different authors have concluded that strength training predominantly leads to concentric hypertrophy and endurance training to eccentric hypertrophy28. In this study concentric remodelling was observed most frequently. George et al.31 reported that the expected pattern of eccentric enlargement was replaced by a pattern of concentric or symmetric enlargement in groups of highly trained athletes. Generally, the adaptation of the cardiac mass seems not to depend on the type of conditioning31. In 1989, Douglas et al.13 published a comparison of 36 triathletes with 17 normal controls and 15 arterial hypertension patients. They determined that triathletes undergo cardiac adaptations similar to pressure overload of the left ventricle and they described a relative wall thickness (RWT) of 0.41. The authors concluded that the relation of myocardial hypertrophy to exercise training is strengthened further by exercise blood pressure. However, unlike the pathologic hypertrophy resulting from hypertension, the triathlete’s heart would show “normal” diastolic LV-function.\n\nThe difference between triathletes and racing cyclists is that the thriathlete’s training does not only take place under strength/endurance conditions, but also under running conditions. Modified strength training also results in different changes in the cardiac structures of triathletes in comparison to professional racing cyclists51. In a study including 14 professional cycle racers it was shown that the left ventricular muscle mass resulted in eccentric hypertrophy compared to concentric hypertrophy as also shown in our study. Therefore, the functional changes found in the cardiac structures for triathletes resemble the changes in runners52. Sudden cardiac death of athletes is more common in male athletes27,53. The different causes of sudden cardiac death are silent coronary disease54, hypertrophic cardiomyopathy55 and arrhythmogenic right ventricular cardiomyopathy56 (Table 6). Maron et al.55 described hypertrophic cardiomyopathy as common cause of sudden cardiac death (36%) in young athletes and 8% were presented with indeterminate LV-hypertrophy (possible HCM). The probability of the negative role of hypertrophy in athletes10 and the problem of qualitative and quantitative relevance are under discussion57,58.\n\ncong. HD = Congenital Heart Disease. ARVC = Arrhythmogenic Right Ventricular Cardiomyopathy. QT = QT-Syndrome: Romano-Ward Syndrome, and Jervell-Lange-Nielsen-Syndrome. WPW: Wolff-Parkinson-White Syndrome. CM = Cardiomyopathy. HCM = hypertrophic Cardiomyopathy. MVP = Mitral Valve Prolapse.\n\nSome papers have reported that excessive endurance training may cause negative remodelling of cardiac structures15,59. Predominantly marathons and Ironman-distance triathlons can cause a transient overload of the right ventricle59. Fibrosis of the left ventricle in older runners was described as a possible cause of death24,25. Numerous investigations regarding the increase in bio-markers (mainly Troponin cTnI and NTproBnP) in runners of marathons60 as well as triathlon61 competitions have been conducted. A significant increase in bio-markers after the race was found in all those studies. Uniformly, this was considered as a proof of possible injuries to the heart muscle62. Overall, the increase in bio-markers in athletes with intensive muscle work should not necessarily be interpreted as heart specific63, because it also depends on the athlete’s weight64 and may be associated with the myolysis (creatine kinase up to 10000 U/l after long-term running)65. The discussion on this issue is ongoing17,20,66.\n\nThe cross-sectional design of this study does not allow a causality regarding the negative role of EIAH in athletes to be established. Although our data suggest that left ventricular hypertrophy might be related to EIAH beyond the normal exercise-induced adaptation, confirmatory longitudinal work is necessary.\n\nThe results of this study support the authors’ subjective impression of daily practice and engagement in sports medicine over 15 years. We observe rhythm disorders in many cyclists and triathletes around the age of 50, and many of them have elevated blood pressure values during exercise. The probability of increasing stiffness of the aorta as an aging process supported by EIAH remains to be discussed. The present study attempts to analyse the probability of LVM and EIAH and should stimulate further follow-up investigations. It is a very important aim to prevent a potential fibrosis of the left atrium67 or left ventricular myocardium in athletes16 in order to avoid “negative cardiac remodelling” induced by exercise and to preserve the positive effects of physical activity12. Approximately two million people participate in long-distance races in the United States annually68 and there are only limited data regarding their exercise-induced blood pressure, which might be one of the main factors triggering cardiac events69,70.\n\n\nConclusions\n\nThe relationship between myocardial hypertrophy and arterial blood pressure during exercise remains an open issue. The literature13,42 seems to suggest a clear relationship. The relevance of EIAH has to be examined in the future in consideration of serious reports8,57,58. The cited authors suggested the isolated (without EIAH) exercise-induced hypertrophy as a substrate for sudden cardiac death or rhythm disorders. EIAH may enhance the “physiological” exercise-induced hypertrophy in a pathological way. Accordingly, the blood pressure values or EIAH should be thoroughly examined during routine or pre-event check-up.\n\nThe long training-times for Ironman-distances of triathletes with EIAH can lead to additional enlargement of the heart cavities (Figure 2) and may trigger possible sudden cardiac death during triathlon competitions71.\n\nThere is strong evidence that athletes have higher incidence of atrial fibrillation and bradyarrhythmias increasing with age21–23. We don’t know the definitive reasons for this, but EIAH and LVM might be one of the factors. Cases of early death in individual cases due by myocardial fibrosis are possible24,25. However, the general prevalence or incidence of EIAH in athletes is unknown. The problem of EIAH seems to be linked more to competitive athletes with vigorous training and mainly to males. It is known that low-intensity training72 and aerobic exercise have a positive impact on blood pressure lowering73–75. The hypertensive or non-hypertensive response to exercise seems to be related to hereditary factors76, to aging or to the individual arterial stiffness43. It is crucial to define the people at risk and possibly start therapy77. In our daily practice we treat the athletes at risk with low-dose ACE-inhibitors or AT1-blockers before training or competition. The dosage should be tested using an exercise test. Possible therapies for the prevention of fibrosis or atrial fibrillation have already been discussed23,78.\n\nFurther international, prospective, longitudinal studies on possible negative cardiac remodelling caused by EIAH and sport should be conducted. These studies could help to avoid the adverse effects of sport in people at risk. The overlap of EIAH and exercise-induced hypertrophy has the potential for increased QT-dispersion79 and is a ticking clock for cardiac fatigue especially for middle aged men. Independent of all the competitive sporting activities with an enormous importance for hobby-athletes, media and industry, physical activity in general population is of fundamental importance11,12.\n\n\nConsent\n\nAll athletes provided written informed consent to voluntary testing of the performance and using the data for the study. Triathletes underwent their annual medical check-up or examination for planning their training, which would have been carried out in clinical routine in any case. A special approval by an ethics committee was not mandatory because of the study independent character of the examinations. The examinations were a part of clinical routine support of the triathletes. Pharmaceutical interventions in the triathletes were not affected by the study.\n\n\nData availability\n\nfigshare: Data of exercise-induced arterial hypertension in triathletes, doi: http://dx.doi.org/10.6084/m9.figshare.101016080",
"appendix": "Author contributions\n\n\n\nRoman Leischik: designed, analyzed and performed the examinations, wrote the manuscript, reviewed the literature and prepared tables and figures (schematic design).\n\nNorman Spelsberg: analyzed and performed the examinations, provided part of the literature, prepared parts of the tables.\n\nHiltrud Niggemann: statistical analysis, prepared parts of the tables.\n\nBirgit Dworrak: critically revised the manuscript at all stages and reviewed the literature.\n\nKlaus Tiroch: critically revised the manuscript at all stages and reviewed the literature.\n\nAll authors revised the manuscript and agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThanks Frank Blumberg for the graphical preparation of Figure 1 and Figure 2.\n\n\nReferences\n\nVerdecchia P, Schillaci G, Borgioni C, et al.: Prognostic significance of serial changes in left ventricular mass in essential hypertension. Circulation. 1998; 97(1): 48–54. PubMed Abstract | Publisher Full Text\n\nMensah GA, Pappas TW, Koren MJ, et al.: Comparison of classification of the severity of hypertension by blood pressure level and by World Health Organization criteria in the prediction of concurrent cardiac abnormalities and subsequent complications in essential hypertension. J Hypertens. 1993; 11(12): 1429–1440. PubMed Abstract\n\nMcLenachan JM, Henderson E, Morris KI, et al.: Ventricular arrhythmias in patients with hypertensive left ventricular hypertrophy. N Engl J Med. 1987; 317(13): 787–792. PubMed Abstract | Publisher Full Text\n\nGottdiener JS, Brown J, Zoltick J, et al.: Left ventricular hypertrophy in men with normal blood pressure: relation to exaggerated blood pressure response to exercise. Ann Intern Med. 1990; 112(3): 161–166. PubMed Abstract | Publisher Full Text\n\nLauer MS, Levy D, Anderson KM, et al.: Is there a relationship between exercise systolic blood pressure response and left ventricular mass? The Framingham Heart Study. Ann Intern Med. 1992; 116(3): 203–210. PubMed Abstract | Publisher Full Text\n\nSchultz MG, Otahal P, Cleland VJ, et al.: Exercise-induced hypertension, cardiovascular events, and mortality in patients undergoing exercise stress testing: a systematic review and meta-analysis. Am J Hypertens. 2013; 26(3): 357–366. PubMed Abstract | Publisher Full Text\n\nAllison TG, Cordeiro MA, Miller TD, et al.: Prognostic significance of exercise-induced systemic hypertension in healthy subjects. Am J Cardiol. 1999; 83(3): 371–375. PubMed Abstract | Publisher Full Text\n\nHart G: Exercise-induced cardiac hypertrophy: a substrate for sudden death in athletes? Exp Physiol. 2003; 88(5): 639–644. PubMed Abstract | Publisher Full Text\n\nLauschke J, Maisch B: Athlete’s heart or hypertrophic cardiomyopathy? Clin Res Cardiol. 2009; 98(2): 80–88. PubMed Abstract | Publisher Full Text\n\nFagard R: Athlete’s heart. Heart. 2003; 89(12): 1455–1461. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWarburton DE, Nicol CW, Bredin SS: Health benefits of physical activity: the evidence. CMAJ. 2006; 174(6): 801–809. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDanaei G, Ding EL, Mozaffarian D, et al.: The preventable causes of death in the United States: comparative risk assessment of dietary, lifestyle, and metabolic risk factors. PLoS Med. 2009; 6(4): e1000058. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDouglas PS: Cardiac considerations in the triathlete. Med Sci Sports Exerc. 1989; 21(5 Suppl): S214–218. PubMed Abstract\n\nMichelsen S, Knutsen KM, Stugaard M, et al.: Is left ventricular mass in apparently healthy, normotensive men correlated to maximal blood pressure during exercise? Eur Heart J. 1990; 11(3): 241–248. PubMed Abstract\n\nOxborough D, Birch K, Shave R, et al.: “Exercise-induced cardiac fatigue”--a review of the echocardiographic literature. Echocardiography. 2010; 27: 1130–1140.\n\nWilson M, O’Hanlon R, Prasad S, et al.: Diverse patterns of myocardial fibrosis in lifelong, veteran endurance athletes. J Appl Physiol (1985). 2011; 110(6): 1622–1626. PubMed Abstract | Publisher Full Text | Free Full Text\n\nO’Keefe JH, Harshal RP, Lavie CJ, et al.: Potential adverse cardiovascular effects from excessive endurance exercise. Mayo Clin Proc. 2012; 87(6): 587–595. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBlair SN, Kohl HW, Gordon NF, et al.: How much physical activity is good for health? Annu Rev Public Health. 1992; 13: 99–126. PubMed Abstract | Publisher Full Text\n\nEaton SB, Eaton Iii SB: An evolutionary perspective on human physical activity: implications for health. Comp Biochem Physiol A Mol Integr Physiol. 2003; 136(1): 153–159. PubMed Abstract | Publisher Full Text\n\nLeischik R: Endurance Sport and Cardiac Injury. Polish Heart Journal, Kardiologia Polska. Ahead of Print. 2014–04–08. 2014. Reference Source\n\nAndersen K, Farahmand B, Ahlbom A, et al.: Risk of arrhythmias in 52 755 long-distance cross-country skiers: a cohort study. Eur Heart J. 2013; 34(47): 3624–3631. PubMed Abstract | Publisher Full Text\n\nCalvo N, Brugada J, Sitges M, et al.: Atrial fibrillation and atrial flutter in athletes. Br J Sports Med. 2012; 46(Suppl 1): i37–43. PubMed Abstract | Publisher Full Text\n\nLeischik R, Littwitz H: Slow recovery of the right and left ventricular deformation after conversion from atrial fibrillation. American Journal of Sports Science. 2014; 2: 13–16. Reference Source\n\nWhyte G, Sheppard M, George K, et al.: Post-mortem evidence of idiopathic left ventricular hypertrophy and idiopathic interstitial myocardial fibrosis: is exercise the cause? BMJ Case Rep. 2009; 2009: bcr08.2008.0758. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRowe WJ: A world record marathon runner with silent ischemia without coronary atherosclerosis. Chest. 1991; 99(5): 1306–1308. PubMed Abstract | Publisher Full Text\n\nLeischik R, Spelsberg N, Littwitz H, et al.: Physiological performance and cardiac function in female ironman-triathletes. American Journal of Sports Science. 2014; 2: 41–47. Reference Source\n\nMarijon E, Tafflet M, Celermajer DS, et al.: Sports-related sudden death in the general population. Circulation. 2011; 124(6): 672–681. PubMed Abstract | Publisher Full Text\n\nPluim BM, Zwinderman AH, van der Laarse A, et al.: The athlete’s heart a meta-analysis of cardiac structure and function. Circulation. 2000; 101(3): 336–344. PubMed Abstract | Publisher Full Text\n\nBray MS, Hagberg JM, Perusse L, et al.: The human gene map for performance and health-related fitness phenotypes: the 2006–2007 update. Med Sci Sports Exerc. 2009; 41(1): 35–73. PubMed Abstract | Publisher Full Text\n\nAlvarez R, Terrados N, Ortolano R, et al.: Genetic variation in the renin-angiotensin system and athletic performance. Eur J Appl Physiol. 2000; 82(1–2): 117–120. PubMed Abstract | Publisher Full Text\n\nGeorge KP, Wolfe LA, Burggraf GW: The ‘athletic heart syndrome’. A critical review. Sports Med. 1991; 11(5): 300–330. PubMed Abstract | Publisher Full Text\n\nAdams TD, Yanowitz FG, Fisher AG, et al.: Heritability of cardiac size: an echocardiographic and electrocardiographic study of monozygotic and dizygotic twins. Circulation. 1985; 71(1): 39–44. PubMed Abstract | Publisher Full Text\n\nCumming DC, Wall SR, Galbraith MA, et al.: Reproductive hormone responses to resistance exercise. Med Sci Sports Exerc. 1987; 19(3): 234–238. PubMed Abstract | Publisher Full Text\n\nKaplan NM, Deveraux RB, Miller HS Jr: 26th Bethesda conference: recommendations for determining eligibility for competition in athletes with cardiovascular abnormalities. Task Force4: systemic hypertension. J Am Coll Cardiol. 1994; 24(4): 885–888. PubMed Abstract\n\nRoalstad MS: Physiologic testing of the ultraendurance triathlete. Med Sci Sports Exerc. 1989; 21(5 Suppl): S200–204. PubMed Abstract | Publisher Full Text\n\nLang RM, Bierig M, Devereux RB, et al.: Recommendations for chamber quantification. Eur J Echocardiogr. 2006; 7(2): 79–108. PubMed Abstract | Publisher Full Text\n\nNagueh SF, Appleton CP, Gillebert TC, et al.: Recommendations for the evaluation of left ventricular diastolic function by echocardiography. Eur J Echocardiogr. 2009; 10(2): 165–193. PubMed Abstract | Publisher Full Text\n\nBeaver WL, Wasserman K, Whipp BJ: A new method for detecting anaerobic threshold by gas exchange. J Appl Physiol (1985). 1986; 60(6): 2020–2027. PubMed Abstract\n\nWasserman K, Whipp BJ, Koyl SN, et al.: Anaerobic threshold and respiratory gas exchange during exercise. J Appl Physiol. 1973; 35(2): 236–243. PubMed Abstract\n\nDevereux RB, Alonso DR, Lutas EM, et al.: Echocardiographic assessment of left ventricular hypertrophy: comparison to necropsy findings. Am J Cardiol. 1986; 57(6): 450–458. PubMed Abstract | Publisher Full Text\n\nBove KE, Rowlands DT, Scott RC: Observations on the assessment of cardiac hypertrophy utilizing a chamber partition technique. Circulation. 1966; 33(4): 558–568. PubMed Abstract | Publisher Full Text\n\nLongas Tejero MA, Casanovas Lenguas JA: [Prevalence of hypertensive response to exercise in a group of healthy young male athletes. Relationship with left ventricular mass and prospective clinical implications]. Rev Esp Cardiol. 1996; 49(2): 104–110. PubMed Abstract\n\nLaurent S, Cockcroft J, Van Bortel L, et al.: Expert consensus document on arterial stiffness: methodological issues and clinical applications. Eur Heart J. 2006; 27(21): 2588–2605. PubMed Abstract | Publisher Full Text\n\nPelliccia A, Fagard R, Bjørnstad HH, et al.: Recommendations for competitive sports participation in athletes with cardiovascular disease: a consensus document from the Study group of Sports Cardiology of the Working group of Cardiac Rehabilitation and Exercise Physiology and the Working group of Myocardial and Pericardial Diseases of the European Society of Cardiology. Eur Heart J. 2005; 26(14): 1422–1445. PubMed Abstract | Publisher Full Text\n\nSuriano R, Bishop D: Physiological attributes of triathletes. J Sci Med Sport. 2010; 13(3): 340–347. PubMed Abstract | Publisher Full Text\n\nAtchley AE Jr, Douglas PS: Left ventricular hypertrophy in athletes: morphologic features and clinical correlates. Cardiol Clin. 2007; 25(3): 371–382, v. PubMed Abstract | Publisher Full Text\n\nDickhuth H, Hipp A, Niess A, et al.: Differenzialdiagnostik der physiologischen Herzhypertrophie (Sportherz). Deutsche Zeitschrift für Sportmedizin. 2001; 52: 205–210. Reference Source\n\nHenschen S: Skilanglauf und Skiwettlauf. Eine medizinische Sportstudie. Mitt Med Klin Upsala. 1899; 2: 74.\n\nDouglas PS, O'Toole ML, Katz SE, et al.: Left ventricular hypertrophy in athletes. Am J Cardiol. 1997; 80(10): 1384–1388. PubMed Abstract\n\nDouglas PS, O'Toole ML, Hiller WD, et al.: Left ventricular structure and function by echocardiography in ultraendurance athletes. Am J Cardiol. 1986; 58(9): 805–809. PubMed Abstract | Publisher Full Text\n\nBekaert I, Pannier JL, Van de Weghe C, et al.: Non-invasive evaluation of cardiac function in professional cyclists. Br Heart J. 1981; 45(2): 213–218. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFagard R, Aubert A, Staessen J, et al.: Cardiac structure and function in cyclists and runners. Comparative echocardiographic study. Br Heart J. 1984; 52(2): 124–129. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMaron BJ, Pelliccia A: The heart of trained athletes: cardiac remodeling and the risks of sports, including sudden death. Circulation. 2006; 114(15): 1633–1644. PubMed Abstract | Publisher Full Text\n\nSolberg EE, Gjertsen F, Haugstad E, et al.: Sudden death in sports among young adults in Norway. Eur J Cardiovasc Prev Rehabil. 2010; 17(3): 337–341. PubMed Abstract | Publisher Full Text\n\nMaron BJ, Thompson PD, Ackerman MJ, et al.: Recommendations and considerations related to preparticipation screening for cardiovascular abnormalities in competitive athletes: 2007 update: a scientific statement from the American Heart Association Council on Nutrition, Physical Activity, and Metabolism: endorsed by the American College of Cardiology Foundation. Circulation. 2007; 115(12): 1643–1655. PubMed Abstract | Publisher Full Text\n\nCorrado D, Basso C, Rizzoli G, et al.: Does sports activity enhance the risk of sudden death in adolescents and young adults? J Am Coll Cardiol. 2003; 42(11): 1959–1963. PubMed Abstract | Publisher Full Text\n\nMcCann GP, Muir DF, Hillis WS: Athletic left ventricular hypertrophy: long-term studies are required. Eur Heart J. 2000; 21(5): 351–353. PubMed Abstract | Publisher Full Text\n\nShephard RJ: The athlete's heart: is big beautiful? Br J Sports Med. 1996; 30(1): 5–10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLa gerche A, Burns AT, Mooney DJ, et al.: Exercise-induced right ventricular dysfunction and structural remodelling in endurance athletes. Eur Heart J. 2012; 33(8): 998–1006. PubMed Abstract | Publisher Full Text\n\nNeilan TG, Januzzi JL, Lee-Lewandrowski E, et al.: Myocardial injury and ventricular dysfunction related to training levels among nonelite participants in the Boston marathon. Circulation. 2006; 114(22): 2325–2333. PubMed Abstract | Publisher Full Text\n\nRifai N, Douglas PS, O'Toole M, et al.: Cardiac troponin T and I, echocardiographic [correction of electrocardiographic] wall motion analyses, and ejection fractions in athletes participating in the Hawaii Ironman Triathlon. Am J Cardiol. 1999; 83(7): 1085–1089. PubMed Abstract\n\nShave RE, Dawson E, Whyte G, et al.: Evidence of exercise-induced cardiac dysfunction and elevated cTnT in separate cohorts competing in an ultra-endurance mountain marathon race. Int J Sports Med. 2002; 23(7): 489–494. PubMed Abstract | Publisher Full Text\n\nTanindi A, Cemri M: Troponin elevation in conditions other than acute coronary syndromes. Vasc Health Risk Manag. 2011; 7: 597–603. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShave R, George KP, Atkinson G, et al.: Exercise-induced cardiac troponin T release: a meta-analysis. Med Sci Sports Exerc. 2007; 39(12): 2099–2106. PubMed Abstract | Publisher Full Text\n\nKim YJ, Shin YO, Lee JB, et al.: The effects of running a 308km ultra-marathon on cardiac markers. Eur J Sport Sci. 2014; 14 Suppl 1: S92–97. PubMed Abstract | Publisher Full Text\n\nLeischik R: Ugly duckling or Nosferatu? - marathon running and cardiovascular function How to screen the athletes? 2014. Reference Source\n\nBurstein B, Nattel S: Atrial fibrosis: mechanisms and clinical relevance in atrial fibrillation. J Am Coll Cardiol. 2008; 51(8): 802–809. PubMed Abstract | Publisher Full Text\n\nKim JH, Malhotra R, Chiampas G, et al.: Cardiac arrest during long-distance running races. N Engl J Med. 2012; 366(2): 130–140. PubMed Abstract | Publisher Full Text\n\nShaper AG, Wannamethee G, Walker M: Physical activity, hypertension and risk of heart attack in men without evidence of ischaemic heart disease. J Hum Hypertens. 1994; 8(1): 3–10. PubMed Abstract\n\nKurl S, Laukkanen JA, Rauramaa R, et al.: Systolic blood pressure response to exercise stress test and risk of stroke. Stroke. 2001; 32(9): 2036–2041. PubMed Abstract | Publisher Full Text\n\nHarris KM, Henry JT, Rohman E, et al.: Sudden death during the triathlon. Jama. 2010; 303(13): 1255–7. PubMed Abstract | Publisher Full Text\n\nRogers MW, Probst MM, Gruber JJ, et al.: Differential effects of exercise training intensity on blood pressure and cardiovascular responses to stress in borderline hypertensive humans. J Hypertens. 1996; 14(11): 1369–1375. PubMed Abstract\n\nKelley GA, Sharpe Kelley K: Aerobic exercise and resting blood pressure in older adults: a meta-analytic review of randomized controlled trials. J Gerontol A Biol Sci Med Sci. 2001; 56(5): M298–303. PubMed Abstract | Publisher Full Text\n\nWhelton SP, Chin A, Xin X, et al.: Effect of aerobic exercise on blood pressure: a meta-analysis of randomized, controlled trials. 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PubMed Abstract | Publisher Full Text\n\nZoghi M, Gurgun C, Yavuzgil O, et al.: QT dispersion in patients with different etiologies of left ventricular hypertrophy: the significance of QT dispersion in endurance athletes. Int J Cardiol. 2002; 84(2–3): 153–9. PubMed Abstract\n\nLeischik R, Spelsberg N, Niggermann H: Data of exercise-induced arterial hypertension in triathletes. Figshare. 2014. Data Source"
}
|
[
{
"id": "4745",
"date": "17 Jun 2014",
"name": "Céline Fiset",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study examined the impact of exercise-induced arterial hypertension (EIAH) on cardiac hypertrophy in normotensive healthy Ironman athletes. The hypothesis they wish to test is that elevated blood pressure values during exercise promote left ventricular hypertrophy in athletes and is one important factor causing “exercise-induced” cardiac fatigue. This is an interesting and current research topic considering that more and more evidence suggest that intense exercise training can lead to harmful cardiac remodelling in athletes who are otherwise healthy. In support of a negative role for EIAH, findings reported in this cross-sectional study indicate that left ventricular hypertrophy is associated with higher arterial blood pressure in the athletes examined. This paper provided interesting information; however, as acknowledged by the authors further research is required to clearly establish the causal relationship between EIAH and left ventricular hypertrophy. Moreover, based on the results presented in this study, it seems precipitate to suggest that endurance athletes with EIAH may require a therapeutic intervention such as low-dose ACE-inhibitors or AT1-blockers before training or competition to prevent exercise-induced cardiac hypertrophy. The authors report that the problem of EIAH is mostly encountered in men. The authors may want to consider that this apparent sex-specific regulation of EIAH is presumably a “gender”-related differences rather than a “sex”-related difference; considering that it is likely that less women will be inclined to enrol in vigorous training such as Ironman-distance triathlon, suggesting that biological and/or physiological differences are probably not involved in the male prevalence of EIAH. The title of the paper is somewhat misleading as the data reported in this study is not directly related to atrial fibrillation. Similarly, the study does not address the relationship between EIAH and bradycardia or QT dispersion. Furthermore, given that no data directly addressed the link between EIAH and AF, bradycardia and QT dispersion, the authors should consider revising their text to not over extrapolate their findings.",
"responses": []
},
{
"id": "5134",
"date": "01 Jul 2014",
"name": "Marta Sitges",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study examined the potential relationship between exercise-induced arterial hypertension (EIAH) and left ventricular hypertrophy in 51 normotensive healthy male triathletes. For that purpose, they divided the study population into two groups according to left ventricular mass (LVM), choosing as cut-off point of significance left ventricular hypertrophy: 220g and analyzed the systolic blood pressure (BPs) in a cardiopulmonary exercise test on a cycle-ergometer. Triathletes with LVM> 220g showed significantly higher BPs at the anaerobic threshold and a trend to higher BPs values at the aerobic threshold and at maximum exercise level. They concluded that significant left ventricular hypertrophy is associated with higher blood pressure at the anaerobic and aerobic threshold and suggest that endurance athletes with EIAH could benefit from preventive antihypertensive treatment. Comments:The assessment of blood pressure response to exercise has been typically limited to systolic values. Classic studies such as the Framingham study and preliminary studies from our group 1 2 have identified the diastolic pressure response to exercise as the best predictor of late development of hypertension. It would have been interesting to perform a complete analysis of the blood pressure response during exercise, including both systolic and diastolic values. The authors relate EIAH to the development of atrial fibrillation and in fact, this association is used in the title. The study analysis though, did not demonstrate this association, as clinical or electric data related to atrial fibrillation are not analyzed. Athletes with LV mass >220g showed a significantly higher left atrial diameter and a tendency to a higher left atrial volume but all values are within normal limits. Moreover, the author connects EIAH with aorta stiffness but no differences between groups were documented in LVOT and all values are also within normal limits. The authors claim that they did not find any particular right ventricular remodeling, but the only reported data concerning RV analysis are insufficient: fractional RV area and RV diameter measurement in the parasternal view. On the other hand, this later measurement was significantly larger in the group with LV> 220g. We would like to point out two potential typographic errors in Table 1: the data on weight and height seem to be exchanged and the data concerning the systolic diameter of the left ventricle seems indeed to be related to diastolic diameter. How was the cut-off point of LV mass 220g selected? Why didn’t the authors choose an indexed value of LV mass by body size? At least, this should be acknowledged as a limitation of the study. It is well known that measuring blood pressure at high loads of physical exercise might be challenging. The methodology of the determination of blood pressure during exercise should be better described. Overall a state of the art and an interesting line of investigation, with illustrative results but it may be still preliminary to claim an association with atrial fibrillation and advise preventive antihypertensive treatment in athletes.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-105
|
https://f1000research.com/articles/3-73/v1
|
17 Mar 14
|
{
"type": "Case Report",
"title": "Case report: Heterotopic intrarenally located adrenocortical oncocytoma",
"authors": [
"Konstantin Godin",
"Nicole Bang",
"Yuri Tolkach",
"Nicole Bang",
"Yuri Tolkach"
],
"abstract": "The clinical case of a 65-year-old woman with an incidentally detected left-sided mass in the upper renal pole is presented. A functional adrenal tumor was excluded. The mass was removed retroperitoneoscopically. The perioperative period was uneventful. The histopathological examination revealed a heterotopic intrarenal adrenocortical oncocytoma.Adrenal oncocytic neoplasms are very rare, with, to the authors’ knowledge, only 159 described cases so far. Most cases are non-functioning adenomas that can reach a considerable size. Only 10 heterotopic adrenal oncocytomas have been described (three retroperitoneal and seven intraspinal cases). Although the intrarenal adrenal rest is the most frequently appearing variant of adrenal heterotopia, to the best of our knowledge, this report is the first description of an intrarenally growing adrenocortical oncocytic adenoma.In addition to retroperitoneally located oncocytomas, this case could be interesting for urological practice because there are no diagnostic features which could provide a secure preoperative diagnosis of an adrenal oncocytic neoplasm and its malignant variant. Generally accepted indications for surgery of adrenal masses have to be respected. The definitive pathologic diagnosis is in most cases surprising because of its rarity. Benign adrenal oncocytic neoplasms do not require any adjuvant treatment. The oncocytic variant of adrenocortical carcinoma generally has a poor prognosis.",
"keywords": [
"Oncocytoma",
"Oncocytic neoplasm",
"Adrenal neoplasm"
],
"content": "Case report\n\nA 65-year-old Caucasian woman with a history of arterial hypertension underwent an ultrasound examination of the kidneys performed by her general practitioner in 2012. A left-sided suprarenal mass measuring about 5 cm was detected. The physical examination showed no palpable masses in the abdomen, and the peripheral lymph nodes were not enlarged. A 3-T magnetic resonance imaging (MRI) of the adrenal glands presented a heterogeneously enhancing mass measuring 48×48×33 mm in the left apical renal pole in contact with the adrenal gland (Figure 1). The findings seemed to suggest the presence of a renal cell carcinoma. A differential diagnosis of pheochromocytoma had also been considered. Enlarged intra- or retroperitoneal lymph nodes were not detectable in the MRI. Catecholamines and metanephrines, aldosterone-renin ratio and serum cortisol before and after inhibition were within normal range in a 24 hour-urine sample analysis. Thus, a functional adrenal tumor could be excluded.\n\nThe patient underwent a retroperitoneoscopic exploration. A round shaped 4.5 cm exophytic mass of the upper renal pole was excised retroperitoneoscopically in the fashion of a renal mass enucleation applying the zero-ischemia technique. Intraoperatively, the mass had a very thin, poorly defined pseudo-capsule which was adhering to the renal parenchyma caudally and to the macroscopically inconspicuous adrenal gland medially. The adrenal gland was removed en bloc with the mass (Figure 2). No judgement could be made regarding the origin of the tumor. The postoperative recovery period was uneventful. Histopathological examination (haematoxyline-eosin) of the specimen revealed an oncocytic adrenocortical adenoma that arose from the heterotopic intrarenal adrenal tissue (Figure 3).\n\nThe follow-up of this patient (approximately 2 years by the date of article submission) was uneventful. No additional treatment was necessary.\n\n\nDiscussion\n\nOncocytic neoplasms of the adrenal glands are extremely rare. A PUBMED search up to December 2013 uncovered only 159 cases of adrenocortical oncocytomas since the first description of this tumor in adrenal in 198617. Most of the papers describe only single patient case reports.\n\nOncocytic neoplasms or oncocytomas are mostly benign tumors which commonly occur in the kidneys, thyroid, parathyroid, pituitary and salivary glands1,2 and rarely in the respiratory tract3,4, choroid plexus5 and adrenal glands. Oncocytic neoplasms of the adrenal glands, with, to the authors’ knowledge, only 159 previously described cases, are an extremely rare phenomenon. Most of these tumors were discovered as incidental findings on CT or MRI as non-functional adrenal masses. Various malignant potential can be determined in 20% of adrenocortical oncocytic neoplasms6. Oncocytomas arising from the heterotopic adrenocortical tissue have been described in only 10 case reports, seven cases were located intraspinally7–13 and three cases in the retroperitoneum14–16. An oncocytic adenoma arising from heterotopic intrarenal adrenocortical tissue was not suspected in the preoperative assessment of this case because this entity has not been described previously. However, in addition to retroperitoneally located oncocytomas, this should be considered in the future as a differential diagnosis in cases presenting with an intrarenal or retroperitoneal mass.\n\nThis paper contains the first report on an oncocytic adenoma arising from the heterotopic intrarenal located adrenal tissue. Heterotopic adrenal tissue or adrenal rest presented mostly by cortical structures is more frequently located in the kidney. Other sites such as the celiac trunk, epididymis, spermatic cord, ovary, broad ligament, liver capsule, gallbladder, pancreas and spleen are rare18.\n\nOncocytic neoplasms are mostly encapsulated masses with a brown or yellow surface on cut-section. The radial scar can be absent. Oncocytic neoplasms microscopically consist of so called oncocytes, large cells with rich eosinophilic granulations due to the high concentration of mitochondria. The Weiss criteria19, which are commonly used in the histological diagnosis of adrenocortical malignancies, are not applicable to adrenocortical oncocytic neoplasms because all tumors have eosinophilic tumor cytoplasm, diffuse architecture and nuclear atypia. The modified Lin-Weiss-Bisceglia system differentiates between major, minor and definitional criteria for malignancy6. None of these criteria is present in benign adrenal oncocytic neoplasms. Masses with uncertain malignant potential demonstrate the presence of one to four minor criteria (>10 cm or >200g, necrosis, capsular invasion or sinusoidal invasion) in absence of major criteria (mitotic rate >5 mitoses per 50 high-power fields, any atypical mitoses or venous invasion). In adrenal oncocytic carcinomas any of the major criteria could be present.\n\nThere are no specific criteria on both computed tomography and MRI and MRI with chemical shift subtraction for adrenal oncocytic neoplasm and its malignant variant20. The bulk size cannot be used as a reliable criterion to estimate the risk of malignancy.\n\nEighty-three percent of adrenal oncocytic neoplasms are non-functioning masses21. In rare cases, an adrenal oncocytic neoplasm can produce catecholamines, cortisol or testosterone21.\n\nThe therapeutic standard is a minimally invasive adrenalectomy. In cases of a large mass, infiltration of surrounding structures, and lymph node bulks, an open surgery approach should be chosen.\n\nToday, there are no recommendations for the follow-up of benign adrenal oncocytic neoplasms. Only one local recurrence of a neoplasm that was originally diagnosed as benign has been described and fulfilled the criteria of uncertain malignant potential21. The oncocytic variant of adrenocortical carcinoma has a poor prognosis, with a postoperative recurrence rate of 75%, and a tumour-related mortality of 40% in a small group of 24 patients with a median follow-up of 21 months (range: 1 – 180 months)21. An adjuvant or palliative chemotherapy with mitotane can be administered in patients with adrenocortical carcinomas with beneficial effects22, but there is no evidence of efficacy when applied to oncocytic variant.\n\n\nConclusion\n\nTo the authors’ knowledge, this case report presents the first description of a heterotopic intrarenally located adrenocortical oncocytoma. Although being a rare location, this case is worth mentioning, given the challenging situation with regard to the diagnostics and differentiation from the potentially aggressive malignant lesions. Taking in account the absence of radiological criteria of a benign character, this tumor should be considered and treated as a malignant lesion, although a minimally invasive approach should be chosen when possible. The definitive pathologic diagnosis is in most cases surprising because of the rarity of this type of neoplasm and radiological appearance mimicking renal cell carcinoma or adrenal carcinoma. Benign adrenal oncocytic neoplasms do not require any adjuvant treatment.\n\n\nInformed consent\n\nWritten informed consent for the publication of clinical details and clinical images was obtained from the patient.",
"appendix": "Author contributions\n\n\n\nKG was an operating surgeon, conceived the article, drafted the manuscript and reviewed it for critical intellectual content. NB performed the histological examination, acquired the data and critically reviewed the manuscript for important intellectual content. YT contributed to data acquisition, drafted the manuscript and reviewed it for critical intellectual content. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nChang A, Harawi SJ: Oncocytes, oncocytosis and oncocytic tumors. Pathol Annu. 1992; 27(Pt 1): 263–304. PubMed Abstract\n\nTallini G: Oncocytic tumours. Virchows Arch. 1998; 433(1): 5–12. PubMed Abstract\n\nTsuta K, Kalhor N, Raso MG, et al.: Oncocytic neuroendocrine tumors of the lung: histopathologic spectrum and immunohistochemical analysis of 15 cases. Hum Pathol. 2011; 42(4): 578–585. PubMed Abstract | Publisher Full Text\n\nKozakiewicz J, Teodorowicz E, Haczyńska-Partyka A, et al.: [Adenoma oxyphillicum, an extremely rare case of tumour of the larynx end cancer lungs which are going together]. Otolaryngol Pol. 2007; 61(3): 311–4. PubMed Abstract | Publisher Full Text\n\nSav A, Scheithauer BW, Mazzola CA, et al.: Oncocytic choroids plexus carcinoma: case report. Clin Neuropathol. 2010; 29(1): 14–20. PubMed Abstract | Publisher Full Text\n\nBisceglia M, Ludovico O, Di Mattia A, et al.: Adrenocortical oncocytic tumors: report of 10 cases and review of the literature. Int J Surg Pathol. 2004; 12(3): 231–243. PubMed Abstract | Publisher Full Text\n\nSchittenhelm J, Ebner FH, Harter P, et al.: Symptomatic intraspinal oncocytic adrenocortical adenoma. Endocr Pathol. 2009; 20(1): 73–77. PubMed Abstract | Publisher Full Text\n\nKepes JJ, O'Boynick P, Jones S, et al.: Adrenal cortical adenoma in the spinal canal of an 8-year-old girl. Am J Surg Pathol. 1990; 14(5): 481–484. PubMed Abstract\n\nCassarino DS, Santi M, Arruda A, et al.: Spinal adrenal cortical adenoma with oncocytic features: report of the first intramedullary case and review of the literature. Int J Surg Pathol. 2004; 12(3): 259–264. PubMed Abstract | Publisher Full Text\n\nKarikari IO, Uschold TD, Selznick LA, et al.: Primary spinal intramedullary adrenal cortical adenoma associated with spinal dysraphism: case report. Neurosurgery. 2006; 59(5): E1144. PubMed Abstract | Publisher Full Text\n\nKim SH, Paik S, Yoon DH, et al.: Oncocytoma of the spinal cord. Case report. J Neurosurg. 2001; 94(2 Suppl): 310–312. PubMed Abstract | Publisher Full Text\n\nPark DH, Park YK, Oh JI, et al.: Oncocytic paraganglioma of the cauda equina in child. Case report and review of the literature. Pediatr Neurosurg. 2002; 36(5): 260–265. PubMed Abstract | Publisher Full Text\n\nPark HS, Jang KY, Kang MJ, et al.: Oncocytoma of the spinal cord causing paraplegia - a case report. Spinal Cord. 2007; 45(2): 183–186. PubMed Abstract | Publisher Full Text\n\nCorsi A, Riminucci M, Petrozza V, et al.: Incidentally detected giant oncocytoma arising in retroperitoneal heterotopic adrenal tissue. Arch Pathol Lab Med. 2002; 126(9): 1118–1122. PubMed Abstract | Publisher Full Text\n\nNguyen GK, Vriend R, Ronaghan D, et al.: Heterotopic adrenocortical oncocytoma. A case report with light and electron microscopic studies. Cancer. 1992; 70(11): 2681–2684. PubMed Abstract | Publisher Full Text\n\nSurrey LF, Thaker AA, Zhang PJ, et al.: Ectopic functioning adrenocortical oncocytic adenoma (oncocytoma) with myelolipoma causing virilization. Case Rep Pathol. 2012; 2012: 326418. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSmirnova EA, Mikhailov IG: [Electron microscopic characteristics of oncocytoma of the lung, small intestine and adrenal gland]. Arkh Pathol. 1986; 48(6): 79–81. PubMed Abstract\n\nLloyd R: Endocrine Pathology. New York, Springer-Verlag, 1990. Publisher Full Text\n\nMedeiros LJ, Weiss LM: New developments in the pathologic diagnosis of adrenal cortical neoplasms. A review. Am J Clin Pathol. 1992; 97(1): 73–83. PubMed Abstract\n\nMonk IP, Lahiri R, Sivaprakasam R, et al.: Adrenocortical oncocytoma: Review of imaging and histopathological implications. Int J Surg Case Rep. 2010; 1(3): 30–32. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWong DD, Spagnolo DV, Bisceglia M, et al.: Oncocytic adrenocortical neoplasms--a clinicopathologic study of 13 new cases emphasizing the importance of their recognition. Hum Pathol. 2011; 42(4): 489–499. PubMed Abstract | Publisher Full Text\n\nKerkhofs TM, Baudin E, Terzolo M, et al.: Comparison of two mitotane starting dose regimens in patients with advanced adrenocortical carcinoma. J Clin Endocrinol Metab. 2013; 98(12): 4759–4767. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "4146",
"date": "07 Apr 2014",
"name": "M. Hammad Ather",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors report a heterotopic adrenal oncocytoma in a 65 year-old lady with essential hypertension. A routine ultrasound detected a 5 cm adrenal mass, however, MRI showed a 4.8 x 4.8 x 3.3 cm mixed intensity upper polar renal mass. Features were not suggestive of a malignant tumour. Urinary and blood work up excluded a functioning adrenal adenoma. The oncocytoma was removed retroperitoneoscopically with the zero-ischemia technique.It is a well written report, however, I have the following observations on the current submission:The sequence of the work up is not clear: was the MRI done first; or were urinary catecholamines and metanephrines, aldosterone-renin ratio and serum cortisol determined first? In the latter case, why was a pheochromocytoma suspected when neither the clinical manifestations nor imaging (MR) suggested a pheochromocytoma? The authors have rightly noted that the imaging characteristics of benign and malignant oncocytoma are not clearly described in the current literature - they were entertaining a diagnosis of an RCC pre-operative. Did the authors consider doing a biopsy prior to surgical intervention? It would be pertinent to mention indications for biopsy in such a situation, and discuss its merits and demerits.",
"responses": [
{
"c_id": "804",
"date": "07 May 2014",
"name": "Yuri Tolkach",
"role": "Author Response",
"response": "Dear Dr. Ather,Thank you for you valuable comments.With regard to the first point: hormonal evaluation was done after MRI investigation to exclude the probability of hormone-producing tumor, while the tumor was located very close to the adrenal, though the probability was indeed very small. It was done according to the local guidelines.Concerning the second point, based on contemporary evidence it is hard to discuss the necessity of biopsy both in adrenal and renal tumors. We did not consider this diagnostic modality. In our opinion, in this case there was no place for the biopsy.With kind regards,Konstantin Godin, Nicole Bang and Yuri Tolkach"
}
]
},
{
"id": "4152",
"date": "09 Apr 2014",
"name": "Levent Turkeri",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a report of a very interesting case of a heterotopic intrarenal adrenocortical oncocytoma. As the authors stated this may be the first reported case in the literature. The major concern is the appearance of the mass in relation to the kidney at figure 1, exactly at the location of left adrenal gland. The authors stated that they performed a retroperitoneoscopical enucleation and, intraoperatively, the mass had a very thin, poorly defined pseudo-capsule which was adhering to the renal parenchyma as well as to the macroscopically inconspicuous adrenal gland. These observations raise the possibility that this mass was in fact arising from the adrenal gland itself and not from a heterotopic location within the kidney. A tumor of 4.5 cm located intrarenally would be difficult to enucleate without interfering with any renal parenchyma and therefore vascular structures - causing bleeding. The provided histopathological figure displays only diffuse sheets of oncocytic cells without any evidence any surrounding renal tissue. I would recommend a repeat discussion with the pathologist to see if this not really an adrenal oncocytoma, which would be still a rare entity, albeit with more than 50 cases reported in the literature.",
"responses": [
{
"c_id": "805",
"date": "07 May 2014",
"name": "Yuri Tolkach",
"role": "Author Response",
"response": "Dear Professor Turkeri,Thank you for your evaluation.Indeed, this tumor arose from the renal parenchyma and in fact left adrenal was intact and had no continuous contact to the tumor.We have discussed your concerns with the pathologist and in the revised version of the article we will provide a pathology slice, where the nearby renal parenchyma and normal adrenal cells could be seen direct by the tumor, making the final diagnosis undoubtful.With best wishes,Konstantin Goding, Nicole Bang and Yuri Tolkach"
}
]
}
] | 1
|
https://f1000research.com/articles/3-73
|
https://f1000research.com/articles/2-262/v1
|
02 Dec 13
|
{
"type": "Research Article",
"title": "Pupil dilation prediction of random events",
"authors": [
"Patrizio E Tressoldi",
"Massimiliano Martinelli",
"Luca Semenzato",
"Massimiliano Martinelli",
"Luca Semenzato"
],
"abstract": "We report the results of a conceptual replication of a study that reported that pupil dilation can predict potentially threatening random events above chance level. In this study, participants’ pupil dilation was used to predict the appearance of a threatening or a neutral stimulus, presented randomly in a double sequence of ten trials with replacement, i.e. replacing the chosen trial for the future extractions.In the first experiment, with a sample of 100 participants, the average correct prediction was 55.9%, with a small difference between the two stimuli.This effect was further tested in an exact pre-registered study where the average correct prediction was 58.7%. The reliability of these findings was checked utilizing both a frequentist and a Bayesian statistical parameters estimate approach.These findings collectively support the hypothesis that pupil dilation can be used to anticipate random and therefore theoretically “unpredictable” events in an implicit unconscious way that is without a conscious awareness, and that this ability is another characteristic of the powerful anticipatory adaptive systems of our psychophysiological system.",
"keywords": [
"anticipation",
"random events",
"pupil dilation",
"emotion"
],
"content": "Introduction\n\nThe anticipation, or prediction, of future events is a fundamental activity of our conscious and implicit, (i.e. unconscious) cognitive abilities. To be able to predict, even approximately, future movements, perceptions and events, reduces the risks and harms of a future threatening situation and more importantly, optimizes the use of our limited cognitive and energetic resources (e.g. Friston1).\n\nIt is therefore not surprising that the study of anticipation has become an interdisciplinary line of research encompassing psychophysiology and neurophysiology (e.g. Van Boxtel and Böcker2), cognitive processes (Barcelo, Bestmann and Yu3) and artificial intelligence (Butz, Sigaud and Baldassarre4).\n\nAt the core of the anticipatory activity is an innate sensitivity to the structure and statistics of the environment in order to build up correct representations of each event in order to prepare the organism to perceive and act in a way that minimizes errors and reduces unnecessary adjustments (e.g. Clark5). But this raises the question of how it is possible for the organism to prepare for future events if they are unpredictable or equally probable?\n\nOur interest is focused on this more extreme form of anticipation, that of random events. Even if the study of predicting unpredictable events seems a paradox, there is cumulative evidence related to almost 15 years of investigation, that humans, and perhaps every other living organism, can predict some classes of events above the level expected by chance (see the meta-analysis by Mossbridge, Tressoldi, and Utts6). If proven, this phenomenon will further enhance our knowledge about innate predictive survival abilities.\n\nIn this study, we demonstrate that pupil dilation (PD) reactions differ before the random presentation of a neutral or a potentially threatening stimulus.\n\nSimilar to other psychophysiological variables (e.g. heart rate, skin conductance, etc.), PD reacts to different emotional states that are correlated with the arousal of the autonomic sensory system (e.g. Bradley et al.7). Because the pupil dilates with sympathetic activity and constricts with parasympathetic activity (Beatty and Lucero-Wagoner8; Steinhauer et al.9), it represents an accurate and unobtrusive measure of the cognitive resources invested in a task, for example in doing arithmetic tasks.\n\nAn interesting application of PD is shown in tasks where participants are not consciously aware of some information but their PD reveals that it is unconsciously available and is being processed by the cognitive system. For example Bijleveld, Custers and Aarts10, used PD to reveal the strategic recruitment of resources upon presentation of subliminal reward cues.\n\nHowever PD not only gives information about unconscious cognitive activity but can even anticipate future perceptual or cognitive activities. Einhäuser, Stout, Koch, and Carter11 used PD to reveal perceptual selection and its prediction of subsequent stability in perceptual rivalry and Einhäuser, Koch, and Carter12 employed PD to reveal decision making before a person voluntarily reported it.\n\nStarting from these studies and the cumulative evidence that the human psychophysiological and electrophysiological systems react differently before random presentation of two categories of emotional stimuli such as pictures, sounds, etc. (Mossbridge, Tressoldi, and Utts6), we aimed to further investigate whether PD can actually predict random events at the level of each single trial of each participant (see Procedure). An increase of approximately 20% of alerting sounds above the mean chance expected (MCE) has already been observed by Tressoldi et al.13 in the prediction of auditory alerting and neutral sounds presented randomly at the level of single trials (a summary of results obtained in that study is presented in Table S1 in the Supplementary Material of this paper).\n\nIn this paper, we will present the results of a conceptual replication using visual information contrasting a potentially “threatening” stimulus with a neutral one using an experimental procedure that simulate an ecological condition where a dangerous or a neutral event could happen randomly.\n\n\nMethods\n\nParticipants were recruited by advertisements mainly among students of Padova University. Exclusion criteria included uncorrected vision and use of drugs that could affect pupil size and pupil dilation. These characteristics were ascertained by asking each participant.\n\nEstimating an effect size of approximately 0.30, to achieve a statistical power above 0.80, setting α=0.05, an opportunity sample of 100 students and personnel from Padova University (Faul et al.14) were recruited by a research assistant to participate in an experiment on a gambling task. The final sample comprised 32 males and 68 females with a mean age of 29.3 and with a standard deviation of 3.8.\n\nParticipation inclusion followed the ethics guidelines in accordance with the Helsinki Declaration and the study was approved by the Ethics Committee of Dipartimento di Psicologia Generale, the hosting institution. Before taking part in the experiment, each participant provided written consent after reading a brief description of the experiment.\n\nThe experiment consisted of two different phases, a preliminary and a formal one. The preliminary phase aimed at familiarizing each participant with the procedure and testing if they reacted differently to the two stimuli.\n\nEach participant was seated in front of a 19 inch monitor in a sound and light attenuated lab of approximately 120 cd/m2 measured with a Minolta light meter.\n\nBefore the formal sessions, each participant was told: “Before the formal experiment, we must record your personal pupil dilation reactivity to the two types of stimuli you will see behind the door. You must simply watch what will happen on the screen without doing anything. When the door opens, you will see a gun shooting at you, hearing a shot, or you will see a smile. You will see the shooting gun and the smile ten times each, in random order”.\n\nWe chose to limit the test to 10 trials per stimulus to avoid boredom and reduce the possibility of using controlled strategies to predict the target.\n\nIf there was no need for further clarification, the task started with the calibration of the eye position for the Eye Tracker apparatus. This consisted of participants following a dot moving slowly in different positions on the monitor in a natural way without the need to fix the head position.\n\nOnce the calibration was completed, the task started with the stimuli presentation. The sequence of events is presented in Figure 1. The inter-item interval was randomly chosen between 2 and 4 sec.\n\nThe two target stimuli, the gun and the smile, and the door, were calibrated for luminance (300 × 471 pixel; 72 horizontal and vertical dpi). The door was colored in black similar to the video background to avoid PD modification consequent to differences in luminosity. Their luminance measured using cd/m2 with a Minolta® photometer was, for the gun: 15 center, 90 periphery, Smile: 73 center, 4 periphery, door: 48 center, 8 periphery. After the preliminary phase, the formal phase started.\n\nThe research assistant’s instruction to each participant was: “Now your task is to let your pupil dilation predict what you will see behind a closed door that will be shown in the center of the monitor. Behind the door you can see a gun shooting at you or a smile. The computer will monitor your pupil dilation and will predict for you what you will see. Remember that the choice of the shooting gun and the smile, is completely random and hence it is not possible to find an underlying rule to predict their sequence. The task consists in two sessions of 10 trials each. For each correct hit you will earn 0.5 euros”.\n\nThe sequence of events is presented in Figure 2. In this case pupil dilation was measured for 5 seconds during the fixation of the door.\n\nEye-Tracker Apparatus: The eye-tracker model Tobii T120®, Tobii, Stockholm, has the following technical characteristics: data rate, 120 Hz; accuracy, 0.5 degrees; freedom of head movements, 30 × 22 × 30 cm; monitor, 17 inch; 1280 × 1024 pixels; automatic optimization of bright-dark pupil tracking. PD is measured automatically in millimeters by the apparatus using the incorporated near infrared detectors and software. These data were fed to an original software for their storage. This program, created using E-Prime™ v.2.0, written by two of the authors (MM and LS) and interfaced with the eye tracker, controlled events presentation and pupil size automatic recording. The source code is available at: dx.doi.org/10.6084/m9.figshare.848604.\n\nThe sampling with replacement of the two stimuli in the two series of ten trials was randomized using E-Prime™ v2.0 randomized statement and Random function which was reset after every trial. This procedure guarantee against the possibility to guess the incoming stimulus by learning implicit and explicit rules.\n\nThe light in the laboratory was constantly dim, approximately 30 cd/m2 to avoid undesired or unrelated changes to the participants’ pupils.\n\nThe time necessary to complete the calibration, 2 min on average and give the instructions to participants was long enough to accommodate their pupils to the ambient light before starting the experiment.\n\nWe used both a frequentist parameters estimation and a Bayesian model comparison approach, according to the American Psychology Association (APA)15, Kruschke16 and Wagenmakers, Wetzels, Borsboom and van der Maas’s17 statistical recommendations.\n\nThis statistical approach is recommended to limit the shortcomings of the classical Null Hypothesis Significant Testing (e.g. Tressoldi et al.18). Basically, each parameter of interest (mean, correlation, etc.) is estimated for its precision by the confidence intervals, and its effect size or Bayes Factor. For those interested in the classical statistical significance with this approach, it sufficient to check if the confidence intervals include (not significant) or exclude (significant) zero.\n\nInferential frequentist estimates were applied both to the sum and the average of correct guesses (hits) using a binomial and a one-sample t-test statistical test respectively to take in account the sum and the percentages of hit responses. Confidence intervals were estimated using a bootstrap procedure based on 5000 samples.\n\nWe adopted a model comparison approach contrasting the alternative hypothesis of a higher difference with respect to MCE (H1) with the Null Hypothesis (H0) of a nil difference with respect to the MCE. We calculated the Bayes Factor (BFH1/H0) using the software implemented by Morey and Rouder19 for the comparison with the one-tailed one-sample t-test, applying Jeffreys, Zellner, Siow (JZS) prior (see Jeffreys20) setting an effect size of 0.3, as suggested by Rouder et al.21.\n\nBefore proceeding with the statistical analyses, the data for each participant were screened for artifacts. All artifacts, i.e. missing or anomalous (PD values close to/below 1, or above 10) data recordings related to PD easily detected by inspecting the raw scores saved in the individual files, were eliminated. If they exceeded the threshold of 60%, that is 12 out 20 trials, the entire participant was excluded and substituted to keep the total sample equal to 100. The overall percentage of artifacts was 4%. The full raw data and corrected for anomalous data are available at http://dx.doi.org/10.6084/m9.figshare.818978 (Tressoldi)22.\n\nIn order to take into account individual differences, we standardized the PD values related to the 20 trials measured in the anticipation phase to z scores for each participant. Next, the means associated with the two stimuli chosen by the software were calculated. In this way a mean was always above zero and the second one below zero unless of an identical mean between the two stimuli. The prediction for each trial was obtained simply by defining whether the value of PD, above or below zero corresponded to the stimulus that was chosen randomly. For example, if the PD standardized means associated with the smile and the gun were respectively 0.25 and -0.15, each PD value above zero predicted a smile and vice versa, each value below zero predicted a gun. At the end of the trial, the sum and the percentage of hits (correct predictions) were calculated for each participant.\n\n\nResults\n\nIn Table 1 we report the descriptive statistics, in Figure 3 the hits percentages with their 95% Confidence Intervals (CIs). and in Table 2 the effect sizes estimation and the BFH1/H0 of the two stimuli and overall with respect the MCE with the corresponding 95% CIs.\n\n(n=100).\n\nEffect sizes with 95% CIs and BFH1/H0 values of hits percentage for the two stimuli and overall with respect the MCE = 50%.\n\nES=effect size; BF=Bayes Factor\n\nThe means estimate related to both stimuli and overall, show clearly that the prediction accuracy is above the mean chance expected of 50%.\n\nThe estimates of all effect sizes, both those referred to the binomial test and to the one-sample t-test, are above zero and in the range of medium effects. Furthermore the BF values range from 6 for the smile to 284394 for the overall accuracy.\n\nIt is plausible to expect a correlation in the difference between the anticipatory PD associated with the two stimuli and their prediction accuracy. The variance explained by this correlation is R2=0.348, 95%CI: 0.20 to 0.49. This moderate correlation suggests that anticipatory PD differences between the two stimuli explain only a part of the hits or correct predictions. This finding will be commented further after the results of the exact replication.\n\nExpectation bias, arises when a random sequence including multiple repetitions of the same stimulus type (e.g., five non-arousing stimuli) produces an expectation in the participant that the next stimulus should be of another type (e.g., an arousing stimulus) and the contrary (the Gambler’s Fallacy). In the Figure S1 in the Supplementary Material we report the trend observed for the two types of targets. Ninety-eight percent of all series of identical stimuli were comprised between 1 and 5. The visual inspection supported by the estimate of the linear trend, -0.0071 for the smile and -0.0128 for the gun, excludes an expectation bias for both the stimuli.\n\nThe overall prediction accuracy turned out above 50%, the chance expected. Even if of small magnitude in absolute terms, approximately 5%, the parameter estimates suggest that this is quite substantial in term of effect size and BF.\n\nBefore commenting further on these findings we wanted to test their reliability in an exact replication of the experiment.\n\n\nExact replication method\n\nFollowing the suggestions of Wagenmakers et al.23 and of the Open Science Collaboration24, the experiment was registered on the site http://www.openscienceframework.org before data collection.\n\nWe preplanned to recruit the same number of participants as in the original study assuming a similar effect size and setting the statistical power to 0.80. The final sample recruited as in the first experiment, comprised 26 males and 74 females with a mean age of 23.02 with an associated standard deviation of 2.7.\n\nIdentical to the original study. The overall percentage of artifacts was 7.3%.\n\n\nResults\n\nIn Table 3 we report the descriptive statistics, in Figure 4 the hits percentages with their 95% CIs and in Table 4 the effect sizes estimation and the BFH1/H0 of the two stimuli and overall with respect the MCE with the corresponding 95% Cis.\n\nEffect sizes with 95% CIs and BFH1/H0 values of hits percentage for the two stimuli with respect the mean chance expected, 50%.\n\nThe means estimates related to both stimuli and overall, show clearly that the prediction accuracy is above the mean chance expected of 50%.\n\nThe estimates of all effect sizes, both those referred to the binomial test and to the one-sample t-test, are above zero and in the range of medium to large effects. Furthermore the BF values range from 2317 for the smile to 1.5 × 1013 for the overall accuracy.\n\nThe correlations between the difference between the anticipatory PD associated with the overall prediction accuracy was R2=0.42, 95%CI: 0.27,0.56, overlapping that observed in the original experiment.\n\nThe same analysis used in the original experiment yielded similar results (see Figure S2 in the Supplementary Material), showing no sign of expectation bias.\n\nIn this replication, the hit percentages of the two stimuli and the overall hit percentage are slightly larger, as well as the effect sizes and BFs estimates, confirming the results of the original study. The BFs in this case are superior to those observed in the original study and in the range of extreme evidence according to Jeffreys20 criteria.\n\nThe difference between the anticipatory PD associated with the two stimuli predicts approximately one third of the variance related to the overall accuracy. At present we do not have hypotheses about which other predictors can contribute to the remaining variance.\n\n\nGeneral discussion\n\nThe results of the two experiments support the idea that PD can predict future random stimuli therefore adding more evidence to the findings reported by Tressoldi et al.13 using auditory stimuli.\n\nEven if these results were due to unpredicted methodological or statistical artifacts, as in all experiments, we can rule out that in our case they are a due to an improper randomization algorithm, the characteristics of our participants or a fault detection of PD by our apparatus.\n\nThe observed estimated prediction accuracy is between 5 to 10% above the chance level expected. It remains to be explored whether the 5 to 10% above chance represents an upper limit of this prediction or whether it can be enhanced.\n\nIt seems then that PD can be implicitly (unconsciously) modulated to predict random and hence statistically “unpredictable” events. Even if of small magnitude, this predictive ability could have important adaptive consequences, for example in cases of serious threats to life, suggesting that this characteristic is another expression of the powerful adaptive functions of our psychophysiological system that can anticipate future events, for example by promoting advantageous decision-making (e.g. Denburg et al.25), anticipating a reward (e.g. Hackley et al.26) and the pain of others (e.g. Caes et al.27).\n\nIt seems that PD, like possibly all other apparatuses regulated by the psychophysiological system (i.e. heart rate, skin conductance level), has innate characteristics specifically dedicated to the anticipation of future events, no matter how predictable, extending the potentialities of survival mechanisms.\n\nThe investigation of the mechanisms at the base of this capacity is an open question. If random events cannot be predicted using previous experiences and information, we can argue that some “guessing” mechanisms based on probabilistic estimations are adopted. For example, we are currently testing if our results can be modeled using the Bayesian hierarchical generative model suggested by Mathys, Daunizeau, Friston and Stephan28 to investigate individual learning under uncertainty.\n\nAnother hypothesis is the possibility that our psychophysiological system can manifest a sort of temporal quantum-like entanglement, restoring brief time-symmetry situations, apparently violating the past to future flow of time, allowing a connection between present and immediately future events like those observed in quantum physics (e.g. Ma et al.29) and recently studied in the perception of ambiguous images by Atmanspacher and Filk30.\n\nThe fact that it is possible to study this characteristic at the level of a single trial taking into account individual differences, opens up the possibility of devising proof of concept experiments for potential possible applications to be adopted in real life. For example, it is not very complicated to devise technical devices to add to glasses or a smartphone for instance, that can amplify the subtle variations of PD at the level of a conscious overt detection in a way to be used intentionally by every person or to use these variations to activate automatically an alarm that could enhance personal safety when driving or walking.\n\nHowever only independent exact or conceptual replications can support our findings, for example changing the type of stimuli and/or the randomization algorithm.",
"appendix": "Author contributions\n\n\n\nAuthorship: P. Tressoldi developed the study concept. M. Martinelli and L. Semenzato, devised the software. All authors contributed to the study design. Testing and data collection were performed by two research assistants. P. Tressoldi performed the data analysis and interpretation. All authors approved the final version of the paper for submission.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nWe acknowledge the relevant advices offered by Dick Bierman, Eva Lobach, Dean Radin and Julia Mossbridge in particular on how to analyse the anticipatory signals and Maaike Pols for revising the English.\n\n\nSupplementary material\n\n*Using Rouder et al. (2009) method, setting the scale of effect size r=0.3.\n\n\nReferences\n\nFriston K: The free-energy principle: a unified brain theory? Nat Rev Neurosci. 2010; 11(2): 127–138. PubMed Abstract | Publisher Full Text\n\nvan Boxtel GJM, Böcker KBE: Cortical measures of anticipation. J Psychophysiol. 2004; 18(2–3): 61–76. Publisher Full Text\n\nBarcelo F, Bestmann S, Yu AJ: Cognitive control, uncertainty and the brain. Research Topic, Frontiers in Human Neuroscience. 2010. Reference Source\n\nButz MV, Sigaud O, Baldassarre G: Anticipatory Behavior in Adaptive Learning Systems: From Brain to Individual an Social Behavior. Berlin Springer. 2007; 4520: 379. Publisher Full Text\n\nClark A: Whatever next? Predictive brains, situated agents, and the future of cognitive science. Behav Brain Sci. 2013; 36(3): 181–204. PubMed Abstract | Publisher Full Text\n\nMossbridge J, Tressoldi P, Utts J: Predictive Physiological Anticipation Preceding Seemingly Unpredictable Stimuli: A Meta-Analysis. Front Psychol. 2012; 3: 390. PubMed Abstract | Publisher Full Text\n\nBradley MM, Miccoli L, Escrig MA, et al.: The pupil as a measure of emotional arousal and autonomic activation. Psychophysiology. 2008; 45(4): 602–607. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBeatty J, Lucero-Wagoner B: The pupillary system. In J.T. Cacioppo, L.G. Tassinary, & G.G. Berntson (Eds.), Handbook of psychophysiology (2nd ed.). New York: Cambridge University Press. 2000; 142–162. Reference Source\n\nSteinhauer SR, Siegle GJ, Condray R, et al.: Sympathetic and parasympathetic innervation of pupillary dilation during sustained processing. Int J Psychophysiol. 2004; 52(1): 77–86. PubMed Abstract | Publisher Full Text\n\nBijleveld E, Custers R, Aarts H: The unconscious eye opener: pupil dilation reveals strategic recruitment of resources upon presentation of subliminal reward cues. Psychol Sci. 2009; 20(11): 1313–1315. PubMed Abstract | Publisher Full Text\n\nEinhäuser W, Stout J, Koch C, et al.: Pupil dilation reflects perceptual selection and predicts subsequent stability in perceptual rivalry. Proc Natl Acad Sci U S A. 2008; 105(5): 1704–1709. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEinhäuser W, Koch C, Carter O, et al.: Pupil dilation betrays the timing of decisions. Front Hum Neurosci. 2010; 4(18): 1–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTressoldi PE, Martinelli M, Semenzato L, et al.: Let Your Eyes Predict: Prediction Accuracy of Pupillary Responses to Random Alerting and Neutral Sounds. SageOpen. 2011; 1(2): 1–8. Publisher Full Text\n\nFaul F, Erdfelder E, Lang AG, et al.: G*Power 3: A flexible statistical power analysis for the social, behavioral, and biomedical sciences. Behav Res Methods. 2007; 39(2): 175–191. PubMed Abstract | Publisher Full Text\n\nReporting standards for research in psychology: why do we need them? What might they be? APA Publications and Communications Board Working Group on Journal Article Reporting Standards. Am Psychol. 2008; 63(9): 839–851. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKruschke J: Introduction to Special Section on Bayesian Data Analysis. Perspect Psychol Sci. 2011; 6(3): 272–273. Publisher Full Text\n\nWagenmakers EJ, Wetzels R, Borsboom D, et al.: Why psychologists must change the way they analyze their data: the case of psi: comment on Bem (2011). J Pers Soc Psychol. 2011; 100(3): 426–432. PubMed Abstract | Publisher Full Text\n\nTressoldi PE, Giofré D, Sella F, et al.: High Impact= High Statistical Standards? Not Necessarily So. PLoS One. 2013; 8(2): e56180. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMorey RD, Rouder JN: Package BayesFactor. 2013. Reference Source\n\nJeffreys H: Theory of probability. Oxford, England. Oxford University Press. 1961. Reference Source\n\nRouder JN, Speckman PL, Sun D, et al.: Bayesian t-tests for accepting and rejecting the null hypothesis. Psychon Bull Rev. 2009; 16(2): 225–237. PubMed Abstract | Publisher Full Text\n\nTressoldi P: PDAnticipationDatabase. figshare. 2013. Publisher Full Text\n\nWagenmakers EJ, Wetzels R, Borsboom D, et al.: An agenda for purely confirmatory research. Perspect Psychol Sci. 2012; 7(6): 632–638. Publisher Full Text\n\nOpen Science Collaboration. An open, large-scale, collaborative effort to estimate the reproducibility of psychological science. Perspect Psychol Sci. 2012; 7(6): 657–660. Publisher Full Text\n\nDenburg NL, Recknor EC, Bechara A, et al.: Psychophysiological anticipation of positive outcomes promotes advantageous decision-making in normal older persons. Int J Psychophysiol. 2006; 61(1): 19–25. PubMed Abstract | Publisher Full Text\n\nHackley SA, Muñoz MÁ, Hebert K, et al.: Reciprocal modulation of eye-blink and pinna-flexion components of startle during reward anticipation. Psychophysiology. 2009; 46(6): 1154–1159. PubMed Abstract | Publisher Full Text\n\nCaes L, Uzieblo K, Crombez G, et al.: Negative emotional responses elicited by the anticipation of pain in others: psychophysiological evidence. J Pain. 2012; 13(5): 467–476. PubMed Abstract | Publisher Full Text\n\nMathys C, Daunizeau J, Friston KJ, et al.: A Bayesian foundation for individual learning under uncertainty. Front Hum Neurosci. 2011; 5: 39. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMa XS, Zotter S, Kofler J, et al.: Experimental delayed-choice entanglement swapping. Nat Phys. 2012; 8(6): 479–484. Publisher Full Text\n\nAtmanspacher H, Filk T: A proposed test of temporal nonlocality in bistable perception. J Mathematical Psychol. 2010; 54(3): 314–321. Publisher Full Text"
}
|
[
{
"id": "4300",
"date": "28 Mar 2014",
"name": "Chris Baker",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAt first glance, this appears to be a thorough demonstration that pupil diameter can be predictive of upcoming events. The study is a conceptual replication of an earlier study by the same group (Tressoldi et al, 2011) and the manuscript contains two separate experiments, each with 100 participants. Further, the second experiment was registered at http://www.openscienceframework.org before data collection (although at the time of writing this, I was unable to access any public record of the registration on the web site). Finally, the authors analyzed their results using both frequentist and Bayesian approaches, producing similar results. However, the claim being made - that somehow pupil responses can predict upcoming random events - would be incredibly remarkable (if true). Given such a bold, and some would say impossible claim, it is critical to carefully examine the experimental design and the veracity of the results. To their credit, the authors have made much of the raw data available as well as the source code for the experiments. Here, I’m going to just focus on evaluating the specific methods and analyses in this particular study, ignoring any evidence from prior work. Briefly, each experiment was composed of two phases: Preliminary Phase - The experimenters first measured individual participants’ pupil responses to two visual stimuli, a smiley face and a pointed gun (accompanied by the sound of a shot), which appeared following the presentation of a picture of a door. There were 10 trials of each condition presented in a random order (without replacement) and pupil diameter was measured during the presentation of the second stimulus (smiley or gun). Experimental Phase – In two blocks of ten trials, participants were again presented with a picture of a door followed by a smiley or a gun, but the trials were fully randomized (no replacement) and pupil diameter was now measured during the period when the door was on the screen. The authors’ claim is that in the Experimental Phase, the pupil diameter measured during the presentation of the door is predictive of the upcoming (random) stimulus. Unfortunately, the method for generating and testing predictions is not entirely clear. My understanding is that for each phase of the experiment, they separately z-scored the data across all (both smiley and gun) trials. Then for the Preliminary Phase data, they determined whether the average z-score for smileys and guns was above or below zero. This was necessary because it appears there were individual differences in the relative pupil size for the two stimuli. The prediction for each Experimental Phase trial was then generated by determining whether the z-score for that trial was above or below zero, and assigning the corresponding stimulus that had a positive or negative mean in the Preliminary Phase as the predicted stimulus. This method does not generate trial-by-trial predictions since it simply assesses whether the pupil diameter for a given stimulus tended to be in the same half of the data in both the Preliminary and Experimental Phase. Using this method the authors then generated percentage hits, comparing the predicted and actual stimuli, and determined that they were significantly above chance (for both stimuli considered separately and combined) using both frequentist and Bayesian approaches. All results were replicated in the second (pre-registered) experiment. Concerns: There are many issues that could be raised about this study, questioning whether the result should be taken to reflect unconscious prediction of random events (many, including myself, would be unwilling to accept this interpretation) or whether it is an artifact of some experimental procedure. I will admit that I don’t have a ready explanation for what sort of artifact can have produced these results (across two separate experiments), but it’s worth making a few points: Replication, in and of itself, does not prove this is a real effect. If there is a methodological or statistical artifact, this would readily replicate too. Replication is much more valuable when conducted by independent research groups. The authors could do much more to convince a skeptic as to the quality of their underlying data. As someone who has recently started to use pupil diameter as an experimental measure, I’ve become aware of how noisy this measure can be from trial-to-trial and I find it extremely hard to believe that the signals will be sufficiently robust across such a small number of trials for any kind of subtle prediction, whether it be of a physical stimulus or an upcoming random event (as is claimed here). The authors have provided no information to attest to the quality of their pupil recordings. What is particularly surprising to me is that the pupil diameter data is from completely different time periods of a trial between the Preliminary and Experimental Phases, when there are great differences in the luminance of the stimuli that are on the screen at that time. What I would like to see is much more careful presentation and analysis of the raw pupil data. For example, time course plots of individual trials for both experimental phases, assessments of trail-to-trial variance etc. Aside from the lack of clarity on the method for generating predictions, there are a couple of other details that need to be more fully specified. It’s not clear exactly what luminance measurements were taken of the stimuli and how they were “calibrated for luminance”. How were the center and periphery measurements taken? Were subjects asked to fixate, or were they free to move their eyes? Over what time period was the pupil diameter measured in each phase? Across the whole 5-second periods? What accounts for the individual differences in relative pupil diameter to the same exact stimuli with the same luminance properties? Providing this sort of information is critical if any other group is to try and replicate the current findings. Ultimately, these results do not convince me that it is possible for participants to unconsciously predict future random events. I view the results as a curiosity and suspect that there is some underlying methodological or analytical artifact that can explain the results – but this is something that may be very hard to pick up from the manuscript, even though the authors have provided much of the raw data. I remember a story a former postdoc of mine told me about a behavioral experiment he ran, in which participants had to indicate whether two sequential stimuli were the same or different. He found that some subjects were incredibly fast and accurate at the task and couldn't work out how it was possible until one of his participants told him that he was listening to the sound of the computer disk spinning – on different trials the computer would have to spin and load up a second image!",
"responses": [
{
"c_id": "792",
"date": "24 Apr 2014",
"name": "Patrizio Tressoldi",
"role": "Author Response",
"response": "Many thanks for your interest and useful comments to our paper.We completely agree that a more \"convincing\" support to our findings can derive only by independent replications. However we remind that in the cited meta-analysis by Mossbridge et al. (2012), there are independent replications, even if the basic experimental protocol is limited to the averaging of the anticipatory physiological variables.As to the differences in PD between the Preliminary and the Experimental phases, as explained on page 2 and 3, in the Preliminary phase PD was measured during the two stimuli presentation, whereas in the Experimental phase PD was measured before their presentation with participants fixating the door image. Furthermore, on page 2 we clarified that the Preliminary phase was used to familiarise the participants with the task, and not to obtain data for the prediction. In our revised article we have added \"Anticipation phase\" in Figure 2, and the sentence \"In this case pupil dilation was measured for 5 seconds during the fixation of the door and used to calculate the prediction accuracy\" to clarify that only these data were used to predict the future stimuli.The individual differences with the same exact stimuli and the same luminance properties are expected, given the individual differences in the sympathetic and parasympathetic reactivity."
}
]
},
{
"id": "4545",
"date": "23 Apr 2014",
"name": "Leo McHugh",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper presents evidence that the average person can predict random events better than chance in the context of the experimental setup described by the authors. This is a remarkable result if true. Sociologist Marcello Truzzi coined the aphorism “extraordinary claims require extraordinary proof”, from which standpoint a review of any such data must be based. It appears that the sample sizes, study design and statistical methods used are generally appropriate throughout. However I can think of a number of potential artifacts of data collection and processing in this experiment not addressed in this paper that may produce positive results inappropriately. These potential artifacts could be tested statistically within the existing dataset (i.e. without further experimentation), and the paper would be strengthened if each of these potential sources for false positive results could be eliminated. This paper is supported by a nice dataset and it would be good to see the paper refined through exposure in F1000 to accommodate data excluding other sources of bias as others read and comment on the paper to the authors. Ultimately it would be great to see a well-powered dataset (such as this one), pointing to an uncomfortable conclusion, that has addressed all the criticisms of the community and becomes an outstanding challenge for refutation. Expectation bias control: The authors report that 98% of random sequences have identical sequential elements of 5 or less, and show a constant hits percentage over lag to support this argument in Figure S1. This constant rate is an important assumption in the paper because assuming a linear trend removes the requirement to correct for gamblers fallacy, as pointed out by the authors. We would expect wider confidence intervals around larger lag values shown in S1 because these events are less frequent. It would be useful if the authors could include confidence intervals around these points else it could be argued that the ‘linear’ trend is merely point values within ever widening confidence intervals that could equally support a non-constant hits percentage. An example of the consequence of a non-constant hits percentage is that because of the smaller numbers of such events at higher lags, chance, especially if combined with a participants’ inner bias to select more often a gun or smile, may have a small effect on the overall hits accuracy. Admittedly, this would at most be only a few percentage points, but for a result a few percent above chance, this could be important. As a caveat, it would also be nice to see second order data, say looking at participant’s likelihood of selecting the same class 2 or 3 steps later. This is analogous to the gambler’s fallacy in its potential effect but is not discussed by the authors and no data is shown. Removed artifacts: The authors report 4% of trials removed due to artifacts. It would be helpful if the authors would report the number of artifacts removed for each corresponding random event. A significant imbalance would immediately be indicative of bias. False conclusions may be reached under these conditions. For example, if the smile produced a PD which was more likely to be removed as an artifact leaving an excess of guns for analysis, and the participants in general had a bias towards expecting a gun over a smile, as is supported by evidence for prospect theory (Khaneman), then the result would be a higher than ‘chance’ hits percentage merely because participants are naturally biasing towards selection of a more prevalent class. Again, this is unlikely to be more than a few percent but may still be important in this context. One way to easily show this bias as non-existent in this data would be to remove the artifacts, and randomly remove trials so that the number of guns and smiles is the same for analysis, then re-run the numbers. Minor comments (typos): “In this way a mean was always above zero and the second one below zero unless of an identical mean between the two stimuli.” Unless? Perhaps should be ‘except in the case of an identical mean…’ “This finding will be commented on further after the results of the exact replication”",
"responses": [
{
"c_id": "791",
"date": "24 Apr 2014",
"name": "Patrizio Tressoldi",
"role": "Author Response",
"response": "First of all, many thanks for your interest and useful suggestions to our paper.As suggested, in our revised article we have added confidence intervals to the expectation bias measures. As you predicted, the decreasing number of data as the lag increases makes the estimates of the average prediction more wide.As for whether the artefacts could be unbalanced between the two stimuli, they are completely random and due to anomalous or missing values, and not to participants' bias toward one specific stimulus."
}
]
}
] | 1
|
https://f1000research.com/articles/2-262
|
https://f1000research.com/articles/3-102/v1
|
08 May 14
|
{
"type": "Research Article",
"title": "Transient acid treatment cannot induce neonatal somatic cells to become pluripotent stem cells",
"authors": [
"Mei Kuen Tang",
"Lok Man Lo",
"Wen Ting Shi",
"Yao Yao",
"Henry Siu Sum Lee",
"Kenneth Ka Ho Lee",
"Mei Kuen Tang",
"Lok Man Lo",
"Wen Ting Shi",
"Yao Yao",
"Henry Siu Sum Lee"
],
"abstract": "Currently, there are genetic- and chemical-based methods for producing pluripotent stem cells from somatic cells, but all of them are extremely inefficient. However, a simple and efficient technique has recently been reported by Obokata et al (2014a, b) that creates pluripotent stem cells through acid-based treatment of somatic cells. These cells were named stimulus-triggered acquisition of pluripotency (STAP) stem cells. This would be a major game changer in regenerative medicine if the results could be independently replicated. Hence, we isolated CD45+ splenocytes from five-day-old Oct4-GFP mice and treated the cells with acidified (pH 5.7) Hank’s Balanced Salt Solution (HBSS) for 25 min, using the methods described by Obokata et al 2014c. However, we found that this method did not induce the splenocytes to express the stem cell marker Oct4-GFP when observed under a confocal microscope three to six days after acid treatment. qPCR analysis also confirmed that acid treatment did not induce the splenocytes to express the stemness markers Oct4, Sox2 and Nanog. In addition, we obtained similar results from acid-treated Oct4-GFP lung fibroblasts. In summary, we have not been able to produce STAP stem cells from neonatal splenocytes or lung fibroblasts using the acid-based treatment reported by Obokata et al (2014a, b, c).",
"keywords": [
"STAP stem cells",
"Oct4-GFP mice",
"CD45+ splenocytes",
"acid treatment."
],
"content": "Introduction\n\nTakahashi and Yamanaka (2006) reported that it was possible to induce adult fibroblasts into pluripotent stem cells using four factors: Oct3/4, Sox2, c-Myc and Klf4. The creation of these induced pluripotent stem (iPS) cells and their replication in human somatic cells has been heralded as a major breakthrough in regenerative medicine (Takahashi et al., 2007). However, the method for generating iPS cells involves complex genetic manipulation and it is beset by low efficiency of conversion and yield (Liao et al., 2008). Currently, great strides are being made to improve the efficiency of iPS cell production by incorporating small molecules, vitamin C, valporic acid, microRNAs and different combinations of transcriptional factors into the protocols (reviewed by Huo & Zambidis, 2013; Jung et al., 2014). Two recent sets of groundbreaking studies reported in Nature appear to overcome most of these problems (Obokata et al., 2014a; Obokata et al., 2014b). The authors reported that simply bathing somatic cells in a mild acid could reprogram them to become pluripotent stem cells. They harvested spleen cells from 1-week-old Oct-4-GFP transgenic mice, and isolated the CD45+ population by flow cytometry. The CD45+ splenocytes were then treated with acidified HBSS (pH 5.7) for 25 min at 37°C and maintained in DMEM/F-12 culture medium containing B27 and Leukemia Inhibitory Factor (LIF) for one to seven days. This simple procedure activated the Oct4 promoter two days post-treatment, making the splenocytes express the GFP reporter.\n\nThe authors named the cells generated by this procedure “Stimulus-triggered acquisition of pluripotency (STAP) stem cells” (Obokata et al., 2014a). STAP cells were demonstrated to be able to form all organs and tissues in chimeric embryos, when injected into host blastocysts (Obokata et al., 2014b). Furthermore, the authors reported that STAP cells could also contribute to the placenta, something that iPS and ES cells are normally incapable of doing (Rossant, 2008). In other words, STAP cells appear to be totipotent rather than pluripotent. If these surprising findings could be confirmed, it would revolutionize regenerative medicine by providing a simple, cheap, and immunocompatible source of stem cells for tissue/organ repair.\n\nThere are several fundamental questions that need to be addressed before STAP stem cells can be accepted as one of the main methods for generating stem-like iPS cells, such as: Can these findings be replicated by other researchers? And, other than mouse spleen cells, can this STAP protocol be applied to other somatic cells? Since the publication of the STAP articles, there has been a recent groundswell of comments on social media platforms, including blogs (Knoeffler Lab Stem Cell Blog), networking sites (ResearchGate) and Twitter, that have cast considerable doubt on Obokata et al.’s findings. In this context, we have attempted to create STAP cells using their most updated protocol (Obokata et al., 2014c).\n\n\nMaterials and methods\n\nOct4-GFP transgenic (CBA-Tg (Pou5f1-EGFP) 2Mnn/j) mice, obtained from The Jackson Laboratory and maintained in the CUHK Laboratory Animal Services Centre, were used for experimentation. The usage of these mice was approved by the CUHK Animal Experimentation Ethics Committee (Project No.: 11/056/GRF-5). The new born neonates were kept in a nest bedding with their mothers at 25°C room temperature, under a 12/12 dark-light cycle, and sufficient food and water. We humanely euthanized six to seven of five-day-old neonatal Oct4-GFP mice by cervical dislocation (according to the ARRIVE guidelines, Kilkenny et al., 2012) and harvested their testes, spleens and lungs. This was done on the same day as the mice left animal breeding centre.\n\nTestes were harvested from five-day-old neonates and maintained in HBSS medium. The seminiferous tubules were immediately isolated from the testes, under a Nikon SMZ745T stereo dissecting microscope. The tubules were then kept, and maintained in DMEM medium supplemented with 10% qualified Fetal bovine serum (FBS) (Gibco®, Invitrogen; Cat#10270) and penicillin (100U/mL)-streptomycin (0.1mg./mL) (Gibco®, Invitrogen; Cat#15140122). The tubules were examined under a confocal microscope to determine whether the germ cells could express Oct4-GFP. Single germ cell suspensions were also produced from the seminiferous tubules using methods described by (Garcia & Hofmann, 2012). These germ cells were analyzed using a BD LSRFortessaTM Cell Analyzer (BD Biosciences, USA) to determine whether these cells were capable of expressing Oct4-GFP.\n\nSpleens were isolated from five-day-old neonates capable of expressing Oct4-GFP when appropriately induced (e.g. using OSMK factors). The spleens were first mechanically passed through a cell strainer (grid size 70µm) to disperse the tissues and dissociate the splenocytes. The splenocytes were pelleted by centrifugation at 1200 rpm for five min and resuspended in ACK lysis buffer (65mM NH4Cl, 10mM KHCO3 and 0.1M Na2-EDTA in distilled H2O, adjusted to pH 7.3) for five min at room temperature to remove residual erythrocytes. The splenocytes were then resuspended in DMEM supplemented with 10% FBS, and 1% PS. The crude splenocytes were maintained at 37°C and 5% CO2 for one to six days.\n\nBesides splenocytes, we also isolated fibroblasts from the lungs of the Oct4-GFP neonates (Yau et al., 2011). Briefly, explants were prepared from dissected pieces of the lung (approximately 1 mm2 in size). The explants were treated with 1 mg/ml collagenase Type I (Gibco®, Invitrogen) for 30 min. The explants were then washed with PBS and plated onto collagen-coated 100 mm culture dishes (SPL; Cat#20101). DMEM/F12 plus 10% FBS were added to the explants (just enough to cover the explants). These explants were then maintained at 37°C and 5% CO2, with the culture medium changed every three days. Fibroblasts could be observed migrating out of the explants after two days. After seven days, the explants were removed and the fibroblasts on the culture dishes were trypsinized with 0.25% Trypsin-EDTA (Gibco®, Invitrogen) for two minutes and sub-cultured.\n\nThe crude splenocytes produced above were resuspended in PBS into a single-cell (1 × 107 cells/ml) suspension. The cells were incubated with Anti-mouse CD45 antibodies, which were directly conjugated with FITC (1: 100 dilutions, BD PharmingenTM FITC Rat Anti-mouse CD45) for 30 min at 4°C. The stained cells were then washed three times with PBS and resuspended in PBS supplemented with 1% FBS. The CD45+-stained splenocytes were sorted and purified using a Cell Sorter (BD LSRFortessa Cell Analyzer). The cells were sorting on 3 different occasions using the same pool of splenocytes extracted from the spleens of 6 neonates.\n\nWe treated the somatic cells with low-pH medium according to the protocol described in (Obokata et al., 2014c). Accordingly, 5×105 cells/ml of CD45+ splenocytes or lung fibroblasts were treated with low-pH HBSS (adjusted to pH 5.7 with HCl) for 25 min at 37°C. The acid-treated cells were then centrifuged at 1200 rpm for 5 min. The supernatants were removed and rechecked to confirm that the pH was still pH 5.7. All pH were measured using a calibrated pH meter (Mettler-Toledo, USA). All of acid-treated cell pellets were resuspended in DMEM/F-12 medium supplemented with 1× B27 and 1,000U LIF at a concentration of 1×105 cells/ml. The cultures were then plated onto non-adhesive culture dishes (SPL; Cat#11035) and examined for GFP expression on seven consecutive days using microscopy (LEICA SP5). The experiments were performed in triplicate.\n\nThe acid-treated and untreated cells were harvested for qPCR after six-seven days of culture. Briefly, total RNAs were isolated using an RNeasy® mini kit (Qiagen, USA) and reverse transcribed using an Omniscript RT Kit (Qiagen, USA). We used National Institutes of Health’s qPrimer Depot Database to determine the primers for Oct4 (forward: 5′GTTGGAGAAGGTGGAACCAA3′; reverse: 5′TCTTCTGCTTCAGCA GCTTG3′), Sox2 (Forward: 5′ACAAGAGAATTGGGAGGGGT3′; reverse: 5′AAAGCGTTAATTTGGATGGG3′) and Nanog (forward: 5′CCAGTGGAGT ATCCCAGCAT3′; reverse: 5′GAAGTTATGGAGCGGAG CAG3′) expressions. The master mix for each qPCR sample was prepared to a total volume of 5 µl for a 384 well-plate: 10 ng cDNA template, 0.55 µM forward primer, 0.55 µM reverse primer, SYBR green master premix (Takara Biotechnology Co Ltd, Dalian) and RNAse-free water (Ambion, Cat#AM9937). The qPCR was performed with a denaturation step at 95°C for 30 sec and thermal profiling (denaturation step: 95°C, 5 sec; annealing and extension steps: 42°C, 30 sec) for 40 cycles (ABI ViiA 7 Real Time PCR System). After the process was completed, the dissociation and amplification curves were checked to see if there was any abnormal amplification. The Ct values were further measured and acquired. The data were generated for quantitative analyses after normalization with GAPDH housekeeping gene. All samples were run in triplicate.\n\nThe data were analyzed using two-tailed, paired student’s t-test. P<0.05 was considered to be statistically significant. The statistical analysis was performed using SPSS software version 22.\n\n\nResults\n\nWe first confirmed that the cells in our Oct4-GFP transgenic mice were capable of expressing Oct4-GFP. It has been reported that all of the spermatogonia in the testes of this type of transgenic mice were capable of expressing Oct4 (Denn et al., 2008). Hence, we isolated the seminiferous tubules from the testes of our transgenic mice and directly examined the tubules under a confocal microscope. We determined that there were numerous Oct4-GFP+ spermatogonia present in the tubules (Figure 1A). This was further validated by flow cytometery of dissociated germ cells extracted from the seminiferous tubules (Figure 1B).\n\n(A) Confocal image of seminiferous tubules isolated from five-day-old neonates showing the germ cells expressing Oct4-GFP (arrows). Bar = 50µm. (B) Flow cytometry histogram confirming that the germ cells are capable of expressing the transgene. The peaks show that only Oct4-GFP germ cells but not wild type (WT) germ cells expressed GFP.\n\nTo replicate Obokata’s experiment, we isolated a pure population of CD45+ splenocytes from the spleens of five-day-old Oct4-GFP mice as shown in Figure 2A. The CD45+ splenocytes were treated with a mild acidic (pH 5.7) HBSS for 25 min at 37°C. The acid-bathed cells were then cultured in DMEM/F-12 medium supplemented with B27 and 1,000U LIF. We examined the cells for Oct4-GFP expression, every day for up to seven days, but did not observe any Oct4-GFP expression under the confocal microscope (Figures 2B, C and D). However, we did occasionally observe clusters of cells that appeared to be GFP+ but were later determined to be autofluorescence, as these cells were necrotic and stained positive with propidium iodide (Figure 2E). qPCR analysis was performed to establish whether the acid-bathed cells were capable of expressing the stemness markers. The qPCR results revealed that acid treatment did not induce the splenocytes to express Oct4, Sox2 and Nanog (Figure 2F).\n\n(A) Flow cytometry histogram showing the sorted CD45+ splenocytes used in our experiments. (B–E) Confocal images of acid-treated splenocytes after zero to six days of culture. No Oct4-GFP expression was detected in the splenocytes at any of the time-points analyzed. Bar = 50µm. (E) Confocal image of acid-treated splenocytes stained with Propidium iodide (PI) after three days of culture. The PI dye identified the necrotic splenocytes (red arrows) which occasionally also emitted a green autoflorescence (green arrows). Bar = 25µm. (F) qPCR analysis also confirmed that acid-treatment did not induce the splenocytes to express the stemness markers Oct4, Sox2 and Nanog. Error bars represent standard error of the mean (p≤0.05).\n\nWe also attempted to produce STAP cells from Oct4-GFP lung fibroblasts. Like the splenocytes, the fibroblasts were treated with acidic HBSS (pH 5.7) for 25 min at 37°C. We checked the cells for GFP expression for seven consecutive days but did not observe any GFP expression (Figure 3A and B). Likewise, our qPCR analysis also did not show induced Oct4, Sox2 and Nanog expressions in our acid-treated fibroblasts (Figure 3C).\n\n(A and B) Confocal images of acid-treated fibroblasts zero and seven days after culture. None of the acid-treated fibroblasts expressed the Oct4-GFP transgene. (C) qPCR analysis demonstrated that acid-treatment did not induce Oct4, Sox2 and Nanog expression. No expression was determined after 40 cycles of qPCR for Oct4 and Sox2, hence marked as ‘undetermined’. Error bars represent standard error of the mean (p≤0.05).\n\n\nDiscussion\n\nCurrently, there is a trend to simplify iPS cell production by minimizing genetic manipulation and incorporating the use of small chemical molecules for somatic cell reprogramming (Shi et al., 2008; Zhu et al., 2010). In this context, it has been reported that mouse iPS cells could be generated using a cocktail of seven chemical molecules without any genetic manipulation, with an efficiency of around 0.2% (Hou et al., 2013). These important developments were recently superseded by claims that hydrochloric acid treatment alone can chemically reprogram fibroblasts to become induced pluripotent stem cells (Obokata et al., 2014a; Obokata et al., 2014b). The authors claimed that chemically stressing CD45+ splenocytes (isolated from Oct4-GFP neonates) to the point of death so that approximately 25% of the cells survive will activate the Oct4-GFP transgene in over 50% of surviving cells. They called these cells “STAP cells” which, when injected into host blastocysts, could participate in the development of all tissues and organs, including the placenta. The STAP chimeric mice produced were reported to be healthy, and the STAP-derived germ cells were demonstrated to be involved in germline transmission (Obokata et al., 2014a; Obokata et al., 2014b). These are astonishing findings. Nevertheless, we have tried to replicate the first stages of Obokata’s findings using CD45+ splenocytes isolated from Oct4-GFP neonates, but could not activate the expression of the Oct4-GFP transgene. This is despite using their most updated protocol for producing STAP cells, which was reported in Protocol Exchange (Obokata et al., 2014c). We also tried using Oct4-GFP lung fibroblasts instead of splenocytes, but again we failed to detect Oct4-GFP expression after acid-treatment. Occasionally, there were cells that appeared GFP positive, but we later determined them to be autofluorescence from apoptotic cells. We made sure that the pH was exactly maintained at pH 5.7 during the experiments by measuring the pH before and after cell treatment. This is because the Protocol Exchange protocol placed a lot of emphasis on maintaining an optimal pH during the acid treatment of the cells. We found that there was a pH 0.1 increase after the acid buffer was added to treat the cells – so our starting pH was actually 5.6 to compensate. At the end of acid bath stimulation, we also measured the pH of the buffer to confirm that it was still pH 5.7. Therefore, our inability to produce STAP cells could not be attributed to changes in the pH during the cell stimulus procedures.\n\nAnother possibility why we could not replicate Obokata’s results might be the difference in the strains of Oct4-GFP transgenic mice used. We acquired our transgenic mice from The Jackson Laboratory (CBA-Tg (Pou5f1-EGFP) 2Mnn/j) while Obokata used transgenic mice generated by Ohbo et al., 2003. Their transgenic mice were developed from a C57BL.6J background, and carry the EGFP cDNA under the control of an Oct4 18-kb genomic fragment (consisting of a minimal promoter and proximal and distal enhancer). Perhaps the transgene in these mice is more easily activated than in our Jackson Laboratory mice. This could potentially explain why Obokata’s transgenic splenocytes, but not our transgenic splenocytes, expressed the EGFP reporter following acid bath treatment. Nevertheless, in the context of generating STAP stem cells, it is not the expression of the transgene that is important but rather the expression of the endogenous Oct4 gene - and related endogenous stemness genes, Sox2 and Nanog. Expression of these genes could not be demonstrated using qPCR analysis following splenocyte acid treatment and culture.\n\nIn conclusion, we have not been able to replicate Obokata et al.’s findings to produce STAP stem cells from somatic cells. It appears that the method for producing STAP stem cells is not as simple and straight forward as has been reported.\n\n\nData availability\n\nfigshare: Dataset 1 and 2. qPCR results of CD45+ splenocytes/lung fibroblasts. Doi: 10.6084/m9.figshare.1014318 (Tang et al., 2014)",
"appendix": "Author contributions\n\n\n\nMei Kuen Tang performed the confocal microscopy experiments, analysed the data and was overall in charge of the experiments performed in this study.\n\nLok Man Lo performed the experiments of the transgenic splenocytes.\n\nWen Ting Shi performed the experiments on the transgenic lung fibroblasts.\n\nYao Yao isolated the tissues from the transgenic mice and performed the flow cytometry experiments.\n\nHenry Siu Sum Lee assisted in the production of the manuscript and figures.\n\nKenneth Ka Ho Lee wrote the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research has been supported by the Hong Kong Cancer Fund (CUHK project number 6903321) assigned to Professor Kenneth Lee.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nDann CT, Alvarado AL, Molyneux LA, et al.: Spermatogonial stem cell self-renewal requires OCT4, a factor downregulated during retinoic acid-induced differentiation. Stem Cells. 2008; 26(11): 2928–2937. PubMed Abstract | Publisher Full Text\n\nGarcia T, Hofmann MC: Isolation of undifferentiated and early differentiating type A spermatogonia from Pou5f1-GFP reporter mice. Methods Mol Biol. 2012; 825: 31–44. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHou P, Li Y, Zhang X, et al.: Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds. Science. 2013; 341(6146): 651–654. PubMed Abstract | Publisher Full Text\n\nHuo JS, Zambidis ET: Pivots of pluripotency: the roles of non-coding RNA in regulating embryonic and induced pluripotent stem cells. Biochim Biophys Acta. 2013; 1830(2): 2385–94. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJung DW, Kim WH, Williams DR: Reprogram or reboot: small molecule approaches for the production of induced pluripotent stem cells and direct cell reprogramming. ACS Chem Biol. 2014; 9(1): 80–95. PubMed Abstract | Publisher Full Text\n\nKilkenny C, Browne WJ, Cuthill IC, et al.: Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. Osteoarthritis Cartilage. 2012; 20(4): 256–60. PubMed Abstract | Publisher Full Text\n\nLiao J, Wu Z, Wang Y, et al.: Enhanced efficiency of generating induced pluripotent stem (iPS) cells from human somatic cells by a combination of six transcription factors. Cell Res. 2008; 18(5): 600–603. PubMed Abstract | Publisher Full Text\n\nObokata H, Wakayama T, Sasai Y, et al.: Stimulus-triggered fate conversion of somatic cells into pluripotency. Nature. 2014a; 505(7485): 641–647. PubMed Abstract | Publisher Full Text\n\nObokata H, Sasai Y, Niwa H, et al.: Bidirectional developmental potential in reprogrammed cells with acquired pluripotency. Nature. 2014b; 505(7485): 676–680. PubMed Abstract | Publisher Full Text\n\nObokata H, Sasai Y, Niwa H: Essential technical tips for STAP cell conversion culture from somatic cells. Protocol Exchange. 2014c. Publisher Full Text\n\nOhbo K, Yoshida S, Ohmura M, et al.: Identification and characterization of stem cells in prepubertal spermatogenesis in mice. Dev Biol. 2003; 258(1): 209–25. PubMed Abstract | Publisher Full Text\n\nRossant J: Stem cells and early lineage development. Cell. 2008; 132(4): 527–531. PubMed Abstract | Publisher Full Text\n\nShi Y, Desponts C, Do JT, et al.: Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compounds. Cell Stem Cell. 2008; 3(5): 568–74. PubMed Abstract | Publisher Full Text\n\nTakahashi K, Yamanaka S: Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006; 126(4): 663–676. PubMed Abstract | Publisher Full Text\n\nTakahashi K, Tanabe K, Ohnuki M, et al.: Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007; 131(5): 861–72. PubMed Abstract | Publisher Full Text\n\nTang MK, Lo LM, Shi WT, et al.: Dataset 1 and 2. qPCR results of CD45+ splenocytes/ lung fibroblasts. figshare. 2014. Data Source\n\nYau WW, Tang MK, Chen E, et al.: Cardiogenol C can induce Mouse Hair Bulge Progenitor Cells to Transdifferentiate into Cardiomyocyte-like Cells. Proteome Sci. 2011; 9(1): 3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhu S, Li W, Zhou H, et al.: Reprogramming of human primary somatic cells by OCT4 and chemical compounds. Cell Stem Cell. 2010; 7(6): 651–655. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "4727",
"date": "09 May 2014",
"name": "Christine Mummery",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe their attempt to reproduce a study in which it was claimed that mild acid treatment was sufficient to reprogramme postnatal splenocytes from a mouse expressing GFP in the oct4 locus to pluripotent stem cells. The authors followed a protocol that has recently become available as a technical update of the original publication. They report obtaining no pluripotent stem cells expressing GFP driven over the same time period of several days described in the original publication. They describe observation of some green fluorescence that they attributed to autofluorescence rather than GFP since it coincided with PI positive dead cells. They confirmed the absence of oct4 expression by RT-PCR and also found no evidence for Nanog or Sox2, also markers of pluripotent stem cells. The paper appears to be an authentic attempt to reproduce the original study, although the study might have had additional value with more controls: “failure to reproduce” studies need to be particularly well controlled. Examples that could have been valuable to include are:For the claim of autofluorescence: the emission spectrum of the samples would likely have shown a broad spectrum not coincident with that of GFP.The reprogramming efficiency of postnatal mouse splenocytes using more conventional methods in the hands of the authors would have been useful as a comparison. Idem the lung fibroblasts.There are no positive control samples (conventional mESC or miPSC) in the qPCR experiments for pluripotency markers. This would have indicated the biological sensitivity of the assay.Although perhaps a sensitive issue, it might have been helpful if the authors had been able to obtain samples of cells (or their mRNA) from the original authors for simultaneous analysis. In summary, this is a useful study as it is citable and confirms previous blog reports, but it could have been improved by more controls.",
"responses": [
{
"c_id": "810",
"date": "12 May 2014",
"name": "Kenneth Lee",
"role": "Author Response",
"response": "Professor Mummery has provided some excellent suggestions for changes to improve the paper. We will try our best and accommodate her requests 1-3 by doing some new additional experiments.Request 4 will be next to impossible to address, I don’t think Haruko Obakata will be willing to give us her STAP stem cells- even if she does it will be problematic for us to establish they were originally derived from mouse splenocytes."
}
]
},
{
"id": "4845",
"date": "21 May 2014",
"name": "Janet Rossant",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTang et al. report attempts to replicate the findings of Obokata et al. on the derivation of pluripotent STAP cells by acid treatment of mouse splenocytes. Despite attempting to replicate the exact details of the Obokata experiments as published, they were not able to observe any activation of an Oct4-GFP transgene or of the endogenous pluripotent markers after several days of culture of the treated cells. While these results do suggest that the STAP protocol is not simple and easily replicated, negative findings are always hard to interpret and need to be very carefully controlled. For this reason there are some points that need to be considered: It is not clear how much cell death occurred after the acid treatment - was this at the same level as reported by Obokata et al.? What was the sensitivity of the PCR analysis of the expression of the pluripotent genes? A control analysis of different levels of expression in existing pluripotent lines would help. Is it possible to repeat the analysis with a different Oct4-reporter to avoid the potential confusion with green autofluorescence?There is merit in this replication study being published in the scientific literature, however when trying to reproduce a null finding the controls used need to be well defined to prevent any ambiguity.",
"responses": [
{
"c_id": "835",
"date": "24 May 2014",
"name": "Kenneth Lee",
"role": "Author Response",
"response": "Professor Rossant has made some excellent suggestions for improving the paper. Points 2 and 3 will be address by adding positive and negative controls into a revised version of the paper. This will demonstrate the sensitively of our Oct4-GFP transgene. qPCR data will also accompany these results which will address the sensitivity of the PCR analysis question. To address point 1, how “much cell death occurred after the acid treatment”, we are currently checking over all our stored confocal images that have been stained with PI dye to derive an estimate. We will try and integrate this data into the revised version of the paper. To date, we have already generated a set of negative control results but the positive control will take some time to generate. We will do it by chemical induction of our Oct4-GFP lung fibroblasts using a cocktail of small molecules. We will try and have a revised version of the paper uploaded as soon as possible."
}
]
}
] | 1
|
https://f1000research.com/articles/3-102
|
https://f1000research.com/articles/3-99/v1
|
01 May 14
|
{
"type": "Case Report",
"title": "Congenital duplication of the urethra with urethral diverticulum: a case report",
"authors": [
"Darshan H Shah",
"Arvind P Ganpule",
"Ravindra B Sabnis",
"Mahesh R Desai",
"Darshan H Shah",
"Ravindra B Sabnis",
"Mahesh R Desai"
],
"abstract": "Duplication of the urethra is a rare congenital anomaly. Urethral duplication with the presence of diverticulum is a rare combination and to the best of our knowledge has not been previously reported. We report a case of a 16 month old male child with duplication of the urethra and diverticulum arising from the ventral urethra. We also cover the intricacies and challenges in the management of such a case.The opening of the narrowed accessory dorsal urethra at the verumontanum was cauterized and gradually the dorsal urethra became atrophied. The ventral urethral diverticulum was excised. This case is unique due to:The unusual presentation of swelling over the dorsum of the penis, together with duplication of the urethra with diverticulum.The use of cauterization as a treatment modality. Cauterization of the ventral urethra with a Bugbee electrode and diverticulectomy was performed. A glidewire helped in identifying the small opening of the dorsal urethra at the level of the verumontanum. The case also highlights the importance of endoscopic management of this clinical entity.",
"keywords": [
"Duplication of the urethra is a rare congenital anomaly. Most cases involve incomplete duplication of the urethra. This anomaly is more common in males1. The etiology of urethral duplication is unclear",
"no hypothesis explains the basis for all cases2",
"3. Urethral duplication with the presence of a diverticulum is a rare combination and to the best of our knowledge has not been previously reported."
],
"content": "Introduction\n\nDuplication of the urethra is a rare congenital anomaly. Most cases involve incomplete duplication of the urethra. This anomaly is more common in males1. The etiology of urethral duplication is unclear, no hypothesis explains the basis for all cases2,3. Urethral duplication with the presence of a diverticulum is a rare combination and to the best of our knowledge has not been previously reported.\n\nIn this report we describe a rare case of urethral duplication presenting as a urethral diverticulum in the ventral urethral passage, whilst the dorsal opening was abnormal. We also allude to the intricacies and challenges in the management of such a case.\n\n\nCase report\n\nA 16 month old male child from India with duplication of the urethra and a diverticulum arising from ventral urethra was presented at our clinic in 2013. He presented with swelling of the penis, which increased in size with urination. General examination revealed an otherwise healthy child. Local examination revealed an approximately 4×2cm sized soft tissue swelling on the ventral aspect of the distal part of penis, cystic in nature (Figure 1A). The meatus at the tip of the glans (dorsal urethral opening) of the penis was tiny and admitted a no. 22 G Intracath tip. On compression, drops of urine egressed from the meatal opening. Another meatal opening (ventral urethral opening) was seen 10mm proximal and ventral to first opening, which was wide and admitted a 10fr (3.33mm) infant feeding tube. A micturating cystourethrogram (MCUG) showed complete duplication of the urethra with a diverticulum arising from ventral urethra near its terminal part with a small para ureteral bladder diverticulum (Figure 1B).\n\n1A) Clinical photograph showing diverticulum at the distal part of the penis. 1B) Micturating cystourethrogram showing complete duplication of the urethra in the sagittal plane. Large diverticulum arising from distal part of ventral urethra with small bladder diverticulum.\n\nCytsoscopy was done with a 9.5Fr cystoscope (KarlStorz, Germany). A Bugbee electrode, 3fr (KarlStorz, Germany) that could pass through a 3fr working channel of the cystoscope was used. Cystoscopy revealed a normal ventral urethra with a diverticulum. The dorsal urethra was abnormal and was narrowed in the proximal part. Its opening into the normal ventral urethra was localized with difficulty after passing a 0.025”/0.64mm glidewire (Terumo Corporation, Tokyo-Japan) through it (Figure 2A). The opening was just proximal to the verumontanum at 10 o’clock. The dorsal urethra was cauterized at its opening into the ventral urethra using a Bugbee electrode and open diverticulectomy of the ventral urethral diverticulum was performed.\n\n2A) Pictorial diagram showing the glidewire passed from the dorsal urethra to identify its opening into the normal ventral urethra. The cystoscope was passed from the ventral urethra up to the opening of dorsal urethra with a Bugbee electrode for fulguration of this opening. 2B) Post-operative micturating cystourethrogram showing normal urethra, no urethral diverticulum and complete obliteration of the dorsal urethra is noted.\n\nThe patient was doing well at 6 months follow up with a good urinary stream (Figure 2B).\n\n\nDiscussion\n\nDuplication of the urethra can occur with complete duplication of the penis or urinary bladder in the most extreme cases4. Urethral duplication may be sagittal or collateral. In our case it was sagittal. Sagittal duplication takes the form of two channels running one above the other in the sagittal plane, whereas in the collateral form, the duplicate urethras run side by side. Most urethral duplications occur in the sagittal plane within a single penis and most are incomplete. Usually in such cases the ventral urethra is the dominant one5. The most common sagittal variety is an orthotopic principal urethral channel and an epispadiac accessory urethra lying dorsal to it.\n\nThere are several different classifications describing urethral duplications. The classification by Effman et al. is the most widely used6. According to this classification; the present case was type II A-2 (complete urethral duplication with the second urethra arising from first one and coursing independently into separate meatus).\n\nClinical presentation varies from type to type. Double urinary stream is one of the presentations of urethra duplication and may be bothersome when the ventral meatus is too proximal over the penis. Presentation may also include repeated urinary tract infections, incontinence or it may be asymptomatic and the only concern being a double meatus7. This happens particularly when both meatus are very nearby.\n\nClinical examination and retrograde urethrogram (RGU) with MCUG should be sufficient for diagnosis in most cases. However, sonourethrograms and magnetic resonance imaging (MRI) are now also being used as adjunct procedures. Both will give excellent soft tissue details such as plaque or calcification which are associated with chordee in such cases8.\n\nDetailed knowledge of urethral duplication is important when planning for any surgical procedure for its correction. Many patients are asymptomatic and do not require any surgery. Indications for surgery are bothersome symptoms and cosmetic or functional deformity. Surgical reconstruction varies from case to case. It may range from simple meatoplasty to complex staged urethroplasty, depending on the severity of case. Most procedures involve excision of the accessory urethra with reconstruction of the dominant urethra9. A favorable outcome is achieved in most of cases after reconstructive surgery. Dilatation of the orthotopic urethra is s more controversial option10. Holst et al. have described fulguration of an atypical urethra as another treatment option11.\n\nOur treatment technique in this case was unique (minimally invasive) and successful. The opening of narrowed accessory urethra at the verumontanum was cauterized and the dorsal narrow accessory urethra gradually atrophied and had disappeared at a 6 month follow up MCUG. The ventral urethral diverticulum was excised at same time. This approach was chosen, given the concerns of infertility and incontinence associated with excision of such a long abnormal urethral tract5.\n\nIn summary, the uniqueness of our case lies in the following facts:\n\n– Unusual presentation of swelling over the dorsum of the penis, duplication of urethra with diverticulum.\n\n– Cauterization was used as a treatment. Cauterization of the ventral urethra with a Bugbee electrode and diverticulectomy was offered as a treatment modality. The glidewire helped in identifying the small opening.\n\nThe case also highlights the importance of endoscopic management of this clinical entity.\n\n\nConsent\n\nBefore surgical procedure written informed consent obtained from patient’s parents. Written informed consent for publication of clinical details and clinical images was also obtained.",
"appendix": "Author contributions\n\n\n\nDHS and APG prepared the initial case report and literature review. RBS and MRD were involved in critical interpretation and revision of the manuscript. All authors have agreed to the final content of the case report.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nPippi Salle JL, Sibai H, Jacobson AI, et al.: Bladder exstrophy associated with complete urethral duplication: a rare malformation with excellent prognosis. J Urol. 2001; 165(6 Pt 2): 2434–37. PubMed Abstract | Publisher Full Text\n\nDas S, Brosman SA: Duplication of the male urethra. J Urol. 1977; 117(4): 452–54. PubMed Abstract\n\nCasselman J, Williams DI: Duplication of the urethra. Acta Urol Belg. 1996; 34(4): 535–41. PubMed Abstract\n\nUrakami S, Igawa S, Shiina H, et al.: Congenital collateral urethral duplication in the frontal plane. J Urol. 1999; 162(6): 2097–98. PubMed Abstract | Publisher Full Text\n\nSalle JL, Sibai H, Rosenstein D, et al.: Urethral duplication in male: review of 16 cases. J Urol. 2000; 163(6): 1936–40. PubMed Abstract | Publisher Full Text\n\nEffmann EL, Lebowitz RL, Colodny AH: Duplication of the urethra. Radiology. 1976; 119(1): 179–85. PubMed Abstract | Publisher Full Text\n\nErdil H, Mavi A, Erdil S, et al.: Urethral duplication. Acta Med Okayama. 2003; 57(2): 91–3. PubMed Abstract\n\nBhadury S, Parashari UC, Singh R, et al.: MRI in congenital duplication of urethra. Indian J Radiol Imaging. 2009; 19(3): 232–34. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPodesta ML, Medel R, Castera R, et al.: Urethral duplication in children: surgical treatment and results. J Urol. 1998; 160(5): 1830–33. PubMed Abstract | Publisher Full Text\n\nOrtolano V, Nasrallah PF: Urethral duplication. J Urol. 1986; 136(4): 909–12. PubMed Abstract\n\nHolst S, Peterson NE: Fulguration-ablation of atypical accessory urethra. J Urol. 1988; 140(2): 347–48. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "5280",
"date": "01 Aug 2014",
"name": "Ali Tourchi",
"expertise": [],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe case reported in this paper is an extremely rare presentation of urethral duplication which led me to read it three times. Urethral duplication is a rare congenital finding in pediatric urology. Most patients with this anomaly are asymptomatic. When they are symptomatic, they present with a double urinary stream (the most common presentation), urinary tract infection, incontinence, epididymitis, and bladder outflow obstruction. The authors have not described the type of duplication very well and the reader will be confused whether it is a complete, Y-type or some other type of duplication. Most pediatric urologists and surgeons agree upon identifying the functional urethra prior to any surgical correction of this anomaly. The functional urethra can be determined by urodynamic studies of both urethrae but it was not done in this study. I humbly appreciate the authors performance of cauterization of the ventral urethra; however, the remnant of the ventral urethra is observable on post-operative MCUG. This remnant is concerning for its potential to cause urinary tract infection or incontinence in the future.",
"responses": []
},
{
"id": "5659",
"date": "08 Aug 2014",
"name": "Sanjay B. Kulkarni",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a good case report.It highlights the fact that urethral duplication has varied presentations.Ventral divertculum actually helped in diagnosing the patient early as it was a symptomatic cosmetic presentation.The authors are highly skilled in Endourology and skillfully performed the cauterization of the dorsal urethra.This suggests that minimally invasive endourological technique can be used for treatment of duplication.The follow-up MCU shows excellent result.",
"responses": []
},
{
"id": "5660",
"date": "20 Aug 2014",
"name": "Tamsin Jillian Greenwell",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a nicely written case report of a novel treatment of an unusual problems and deserves indexing.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-99
|
https://f1000research.com/articles/2-182/v1
|
11 Sep 13
|
{
"type": "Research Article",
"title": "Short- and long-term habituation of auditory event-related potentials in the rat",
"authors": [
"Kestutis Gurevicius",
"Arto Lipponen",
"Rimante Minkeviciene",
"Heikki Tanila",
"Rimante Minkeviciene",
"Heikki Tanila"
],
"abstract": "An auditory oddball paradigm in humans generates a long-duration cortical negative potential, often referred to as mismatch negativity. Similar negativity has been documented in monkeys and cats, but it is controversial whether mismatch negativity also exists in awake rodents. To this end, we recorded cortical and hippocampal evoked responses in rats during alert immobility under a typical passive oddball paradigm that yields mismatch negativity in humans. The standard stimulus was a 9 kHz tone and the deviant either 7 or 11 kHz tone in the first condition. We found no evidence of a sustained potential shift when comparing evoked responses to standard and deviant stimuli. Instead, we found repetition-induced attenuation of the P60 component of the combined evoked response in the cortex, but not in the hippocampus. The attenuation extended over three days of recording and disappeared after 20 intervening days of rest. Reversal of the standard and deviant tones resulted is a robust enhancement of the N40 component not only in the cortex but also in the hippocampus. Responses to standard and deviant stimuli were affected similarly. Finally, we tested the effect of scopolamine in this paradigm. Scopolamine attenuated cortical N40 and P60 as well as hippocampal P60 components, but had no specific effect on the deviant response. We conclude that in an oddball paradigm the rat demonstrates repetition-induced attenuation of mid-latency responses, which resembles attenuation of the N1-component of human auditory evoked potential, but no mismatch negativity.",
"keywords": [
"The auditory oddball paradigm",
"in which a series of repeated standard stimuli are interrupted by occasional deviant stimuli",
"has been used extensively in cognitive psychology to study early stages of auditory processing in humans1. Typically a sufficiently rare deviant stimulus evokes a long-duration negative potential shift beginning 100–200 ms after the stimulus onset",
"referred to as mismatch negativity (MMN). MMN has been considered an electrophysiological correlate of a mismatch between the incoming stimulus and a sensory memory trace2. MMN is not dependent on a subject’s attention and can be evoked even during sleep or anesthesia1. Electrical and magnetic recordings in human subjects have localized the MMN generator to the auditory cortex3",
"although a frontal component has also been observed4. In addition",
"there are some speculations on subcortical generators",
"especially the hippocampus",
"but those cannot be indisputably verified in noninvasive recordings5",
"6."
],
"content": "Introduction\n\nThe auditory oddball paradigm, in which a series of repeated standard stimuli are interrupted by occasional deviant stimuli, has been used extensively in cognitive psychology to study early stages of auditory processing in humans1. Typically a sufficiently rare deviant stimulus evokes a long-duration negative potential shift beginning 100–200 ms after the stimulus onset, referred to as mismatch negativity (MMN). MMN has been considered an electrophysiological correlate of a mismatch between the incoming stimulus and a sensory memory trace2. MMN is not dependent on a subject’s attention and can be evoked even during sleep or anesthesia1. Electrical and magnetic recordings in human subjects have localized the MMN generator to the auditory cortex3, although a frontal component has also been observed4. In addition, there are some speculations on subcortical generators, especially the hippocampus, but those cannot be indisputably verified in noninvasive recordings5,6.\n\nWhereas the psychophysical details of conditions evoking MMN have been thoroughly analyzed, relative little is known about the neuropharmacology and molecular mechanisms of MMN. This is largely due to the paucity of experimental studies of MMN in standard laboratory animals. MMN-like responses have been reported in various animal species such as cats7, guinea pigs8, rabbits9, monkeys10 and rats11,12. However, there are also reports of negative findings in rats implying that the evoked potentials are modified by the preceding stimuli or adapted in a stimulus-specific manner not resembling MMN13,14.\n\nTherefore, the aim of the present study was to address a number of unresolved issues related to MMN in the rat. First, anesthesia was reported to attenuate MMN in the cat7. Therefore, we wanted to test whether MMN can be evoked in freely moving rats14,15 rather than in the anesthetized preparation11,12. Second, we compared the event-related potentials (ERPs) recorded using cortical and hippocampal electrodes to reveal a possible hippocampal generator. Third, to distinguish between MMN and long-term adaptation to standard auditory stimuli as suggested by Lazar and Metherate13, we repeated the oddball stimulus set on two daily sessions and on consecutive days. Fourth, to shed light on the neuropharmacology of MMN, we manipulated the cholinergic input to the cortex and hippocampus by systemic administration of scopolamine. We report evidence for repetition-induced attenuation of the mid-latency auditory ERPs but no correspondence to the sustained negativity around 100–200 ms in response to the deviant sound that is referred to as MMN in humans.\n\n\nMethods\n\nMale Wistar rats (National Laboratory Animal Center, University of Eastern Finland, Finland, n=12, weight 412 ± 9 g) were reared in groups of 2–4 until 5 months of age and individually thereafter in a controlled environment (temperature +21°C, lights on from 7:00 h to 19:00 h, water and food available ad libitum) Animals were housed in stainless steel metal cages, floor 31 cm x 45 cm, height 18 cm as according to the guidelines of the Council of Europe ETS123. At the age of 5–6 months, the rats were chronically implanted with two recording electrodes made of 50 μm insulated stainless steel wire (California Fine Wire Company Co, Grover Beach, CA, USA) in the hippocampus at the following stereotactic coordinates: AP (from Bregma) - 3.8, L (from Bregma) +3.1, V (from brain surface) - 3.1 with a vertical separation of the tips of 0.6 mm. In addition, two cortical screw electrodes (Wurth Electronics, Finland) were fixed on the (left and right) parietal bones (L ± 2.0 mm and A -7.5 mm from Bregma). A frontal screw served as the ground and a common reference electrode. The hippocampal electrode closest to the pyramidal cell layer and the right parietal cortical electrode were selected for the final analysis of evoked potentials. The rats were anesthetized with a mixture of pentobarbital and chloral hydrate (40 mg/kg i.p. each), and, for post-operative analgesia, they received 5 mg/kg of carprofen (Rimadyl®, Vericore, Dundee, UK) intraperitoneally. The rats were housed in individual cages after the surgery. Recordings started after at least 2 weeks of recovery period. The rats were involved in an EEG study for three weeks before the current study on evoked potentials. All animal procedures were carried out in accordance with the guidelines of the European Community Council Directives 86/609/EEC and approved by the Animal Experiment Board of Finland.\n\nIn total 10 rats were recorded for the study but due to poor signal in some channels, the number of records in the analysis varies from 6 to 9. During the recordings the rat was able to freely move in a brown paste-board cylinder (70 cm diameter, 50 cm height) that was highly familiar to the rat due to previous EEG recordings. Two conventional speakers were placed on the opposite sides outside the cylinder. Auditory stimuli were created through a computer sound card (Sound Blaster 16, Creative Technology Ltd, Singapore, Singapore) and included pure sinusoidal tones of 7, 9 or 11 kHz pitch (tone duration 150 ms, 70 dB, rise/fall time 5 ms). The signal was analog filtered for the 1–1000 Hz band, amplified (× 1000–5000), and digitized at 2 kHz per channel for further processing using a commercial software (Experimenter’s Workbench, DataWave Technologies, Longmont, CO, USA).\n\nAt the end of the experiment, the rats were euthanized by an overdose of anesthetic pentobarbital and chloral hydrate each 80 mg/kg i.p. and the sites of the electrode tips were marked by passing a 30 μA anodal current for 5 s through each hippocampal electrode. Subsequently, the brains were immersion-fixed overnight with 4% formalin (formalin was diluted from 37% formaldehyde solution (Sigma-Aldrich)) and sectioned at 50 μm with a vibratome (Leica VT1000s). The sites of the electrolytic lesions were verified in sections stained with cresyl violet Sigma-Aldrich) by using a light Olympus CX microscope Figure 1.\n\nThe arrows point to the lesion marks corresponding to the two electrode tips, the upper one in the alveus/oriens and the deeper one in the fissure. Scale bar = 2000 μm.\n\nThe basic study protocol was a conventional mismatch (or oddball) paradigm consisting of one standard tone and one or two deviant tones. Under most conditions, the standard was 9 kHz and the deviants were 7 and 11 kHz tones. Both a low and a high deviant were used to exclude the contribution of tonotopy to auditory evoked potential (AEP) amplitudes. Every run consisted of 400 repetitions with a 1-s inter-stimulus interval. The three tones (7, 9 and 11 kHz) were presented in a pseudo-random order, so that the proportions of the standard, deviant 1 and deviant 2 tones were 85%, 7.5% and 7.5%, respectively.\n\nExperiment 1 consisted of three consecutive days with the 9 kHz tone as the standard, and 7 and 11 kHz tones as the deviants. Similar recordings were performed during Experiment 2 (three weeks after Experiment 1) that also consisted of three consecutive runs. Day 1 replicated Day 1 of the Experiment 1, and was followed by a similar run on Day 2. In addition, Day 2 included a second run with the mismatch contingency reversed, so that 7 kHz became the standard and 9 kHz the deviant. Experiment 3 (one week later) included pharmacological manipulations and consisted only of two runs, one on Day 1 and the second on Day 4. In the first run the standard tone was 9 kHz and the deviants 7 and 11 kHz. In the second run the standard tone was 7 kHz and the deviant 9 kHz. Four rats received scopolamine (0.2 mg/kg, s.c.; Sigma-Aldrich) 20 min before the first run, and five rats before the second run. Saline was used as control treatment.\n\nFirst, all signals were corrected for amplification. Waveform averaging and AEP peak detection were conducted by custom made routines in Visual Basic under Microsoft Excel® (version 2002).\n\nThe AEP in a typical rat had three middle-latency components, N40, P60 and P110 (N40 means a negative deflection at 40 ms). In addition, these components were followed by a broad negativity from 150 ms to 250 ms after the stimulus onset (Figure 2A, B). The amplitude of these components was calculated as a maximum deviation from the baseline. The baseline was calculated for each rat from the averaged response between 0 and 100 ms before stimulus onset. When calculating mismatch effect between standard and deviant AEP, we focused on the middle-latency components only (N40, P60 and P110).\n\nCortical (A) and hippocampal (B) AEPs. The thin line denotes the response to the deviant tone and the thick line the response to the standard tone. The triangle marks the tone onset. The horizontal bar corresponds to 100 ms, the vertical bar to 0.04 mV (scale for the cortex is five times smaller than that for the hippocampus). Negativity is downward.\n\nThe statistical analysis was conducted by using SPSS for Windows 11.5 software. The standard and deviant responses were compared within-subjects using ANOVA with repeated measures with the run (1–3) or drug (placebo or scopolamine) as additional within-subject factors. The threshold for significance was set to p < 0.05.\n\n\nResults\n\nHistology verified the location of the hippocampal electrodes in the intended layers: the top electrode in the stratum pyramidale – stratum radiatum and the deeper one in the hippocampal fissure – outer molecular layer of the dentate gyrus. The typical location of the hippocampal electrodes is illustrated in Figure 1.\n\nRepresentative examples of an averaged cortical and hippocampal AEPs obtained in the auditory mismatch paradigm are shown in Figure 2. The components N40, P60 and P110 were identified for each rat and pooled for standards and deviants for all drug-free days. The exact latencies of these components are summarized in Table 1 and their mutual correlations in Table 2. The mutual Pearson correlation coefficients were high and significant for all components of the hippocampal response (if the absolute value of one component grows there is a high probability that other components will also grow). This suggests that physiological source(s) of AEP’s components is not completely independent. On the other hand, only the mutual correlations of the P60–P110 components in the cortical response reached a comparable significance level. Furthermore, neither cortical P60 nor P110 correlated with any hippocampal component, which suggests that the cortical and hippocampal responses are largely independent, with the exception of the early N40 component.\n\n* Correlation is significant at the 0.05 level (2-tailed).\n\n** Correlation is significant at the 0.01 level (2-tailed).\n\nThe overall analysis of all three days of Experiment 1 revealed larger cortical responses to the deviant tone compared to the standard tone (Figure 2A, and Figure 3). The difference was significant for N40 [F(1,7) = 7.7, p = 0.03] and P60 (p = 0.04) components and approached significance for P110 (p = 0.06). However, the shape of the average evoked response remained the same, and there was no evidence for the typical mismatch negativity as reported in human studies2. In contrast, the hippocampal response did not differentiate between the standard and the deviant tones (p ≥ 0.10 for all components). Together with the correlation table (Table 2) this finding speaks against the notion that the cortical response is a simple volume conducted signal from the hippocampus.\n\nMean amplitudes of AEP components (N40, P60, P110) ± SEMs are given. In each chart the x-axis represent different runs of the test. Note the break between Run 3 of Experiment 1 and Run 1 of Experiment 2 to indicate the intervening days.\n\nR under Run 3 of Experiment 2 indicates reversal of the mismatch contingency.\n\n* significant difference between consecutive Runs (including between Run 3 of Experiment 1 and Run 1 of Experiment 2);\n\n# significant difference between standard and deviant responses;\n\n& significant repetition effect on component attenuation.\n\nThe amplitude of cortical N40 response was relatively stable in Experiment 1, but the P60 component attenuated significantly between days [F(2,6) = 5.9, p = 0.04], and the P110 showed a similar, but non-significant trend [F(2,6) = 1.9, p = 0.24]. This trend could be observed for both standard and deviant tones (Figure 3). In contrast, none of the hippocampal components attenuated between days (all p values > 0.40).\n\nThe time dependency of AEP attenuation was further investigated in Experiment 2. First, we replicated the standard mismatch condition after 20 intervening days of rest. The cortical response to the standard tones reached the original (or higher) amplitude of Day 1 in Experiment 1 (Figure 3). The ANOVA for repeated measures revealed significant enhancement of cortical P60 [F(1,6) = 12.9, p = 0.01] and P110 (p = 0.03) components between Day 3 of Experiment 1 and Day 1 of Experiment 2. Interestingly, these were the same components that were also attenuated over three daily sessions in Experiment 1. Although a similar trend was observed in the N40 component in some animals, the difference did not reach significance at the group level (p > 0.15). The response enhancement after 20 intervening days could be observed to some extent for both standard and deviant stimulus (Figure 3). In contrast, hippocampal responses, which did not change significantly over the three days of Experiment 1, did not increase after the 20 intervening days of rest, either (all p > 0.35).\n\nNext, we repeated the same mismatch condition on Day 2 of Experiment 2 to see whether this habituation of responses between days could be replicated. This time we saw an attenuation of cortical N40 [F(1,6) = 8.6, p = 0.03] and P60 [F(1,6) = 20.0, p = 0.004] components; and a similar, but not significant trend of P110 component [F(1,6) = 1.7, p = 0.24] (Figure 3). In addition, habituation of hippocampal N40 reached significance [F(1,5) = 12.9, p = 0.02]. Again habituation was similar for the standard and deviant responses. Furthermore, the difference between AEPs to the standard and deviant tones could be replicated. However, this time the most robust oddball effect was observed for cortical P110 [F(1,6) = 29.3, p = 0.002], while P60 showed only a trend (p = 0.07), and N40 no effect (p > 0.30). Unlike in Experiment 1, the hippocampal P60 component showed a clear oddball effect [F(1,5) = 15.2, p = 0.01].\n\nFinally, we reversed the mismatch contingency on the second run of Day 2. The reversal resulted in a robust enhancement of both cortical [F(1,6) = 12.2, p = 0.01] and hippocampal N40 [F(1,5) = 28.7, p = 0.003] components, which increased even above the Day 1 (of Experiment 2) level (Figure 3). This change was observed for both the standard and deviant tones. No other cortical or hippocampal components were enhanced after the reversal (all p > 0.14), but the reversal removed the oddball effect for hippocampal P60 and cortical P110 components (Figure 3).\n\nMuscarinic receptors in the central nervous system (CNS) play an important role in the regulation of arousal, attention and synaptic plasticity16,17. To test the contribution of muscarinic receptors on the mismatch effect, we used the subtype nonspecific muscarinic antagonist, scopolamine18, in Experiment 3.\n\nScopolamine resulted in general attenuation of the cortical response, with significant effects in the N40 and P60 components (Figure 4; p = 0.03 and p = 0.04, respectively). In the hippocampal response, only the P60 component decreased significantly (p = 0.002). In Experiment 3, differences were no longer detected between the responses to the standard and deviant sounds for any of the cortical or hippocampal components. Furthermore, the effect of scopolamine did not differ for the standard vs. deviant response (for all sound × drug interactions p > 0.45).\n\nRepresentative example obtained from one rat in the auditory oddball paradigm. The thin line indicates the response to the deviant tone and the thick line the response to the standard tone. The triangle marks the tone onset. Horizontal bar corresponds to 100 ms, vertical bar to 0.04 mV (cortical scale is 5.7 times smaller than that for the hippocampus). Negativity is downward.\n\n* significant difference between scopolamine and saline runs.\n\n\nDiscussion\n\nThe mismatch negativity (MMN) is well established phenomena in humans and widely studied within the field of cognitive neuroscience and psychology. However, MMN studies in laboratory animals are sparser and somehow controversial. We found evidence for repetition-induced attenuation of the mid-latency auditory ERPs but no correspondence to the sustained negativity around 100–200 ms in response to the deviant sound that is referred to as MMN in humans.\n\nThe rat auditory sensitivity as a function of stimulus frequency is very different from that of humans. The human auditory system is sensitive to frequencies from about 20 Hz to a maximum of around 20,000 Hz, although it is most sensitive between 2 and 5 kHz. In rats the auditory evoked potential increases in amplitude from 2 to 8 kHz reaching a plateau until 20 kHz19. Therefore having the deviant sounds higher than the standards can yield a false impression of MMN. This possibility was excluded in the present study by using a balanced number of higher and lower deviants and averaging their responses when comparing them to the standard. Nevertheless, the cortical ERPs in Experiment 1 had higher amplitudes in response to the deviant than the standard tones. Notably, the overall shape of the ERP did not change, and we found no evidence for a sustained shift – whether negative of positive – that would resemble the human MMN. Interestingly, no augmentation of the ERP to the deviant tone was observed in the hippocampus.\n\nWhereas the number of high vs. low deviants was balanced in the present study, the standard and deviant responses differed in an important parameter, the repetition rate. The standard was presented at the proportion of 85%, while each deviant was presented only at 7.5%. One of the studies in anesthetized rats13 reported augmented responses to deviant sounds, which the authors interpreted in terms of repetition rate. In the present study, the cortical ERPs gradually decreased over three daily sessions (Experiment 1) and returned to the original levels after a three-week break between Experiments 1 and 2. The decrement of ERP from session to session was again replicated in Experiment 2. Notably, this decrement in ERP amplitude was roughly the same for the standard- and deviant-evoked responses. The most parsimonious way to interpret these findings is that both the response enhancement to deviant stimuli and general ERP decrement over time reflects gradual attenuation of auditory ERPs to stimulus repetition. This interpretation is also consistent with the disappearance of all differences between standard- vs. deviant-evoked responses after the standard and deviant stimuli were reversed. Namely, after the reversal the cumulative number of the former deviant stimuli soon approached that of the standard for that session. Thus our findings largely support the conclusion of Lazar and Metherate13 that the enhanced response to the deviant sound in an oddball paradigm can be attributed to differences in repetition rate.\n\nSome of the present findings, however, cannot be explained by differences in repetition rate. First, after reversal of the task contingency, the N40 responses (for both the standard and the deviant tone) increased markedly in amplitude. A change in repetition rate could explain why the responses increased to the 9 kHz stimulus, the former standard that now became the deviant (proportion change from 85% to 15%, as only one deviant was used in this part of the experiment). However, this enhancement was also found for the 7 kHz stimulus that became much more frequent (7.5% vs. 85%). Moreover, the enhancement could be observed not only in the cortical channel that was sensitive to the repetition rate, but also in the hippocampus. A similar response to the reversal in the cortex and hippocampus may reflect general arousal or response enhancement in the thalamus or brainstem. A second finding that is at odds with the repetition rate hypothesis was the enhanced deviant-evoked hippocampal P60 and cortical P110 responses. It is possible that these changes after a three-week break in the experiment reflect a ‘declarative’ kind of memory recall as opposed to gradual response attenuation as a function of stimulus presentation. This finding warrants further studies.\n\nOur conclusion that no auditory MMN exists in non-anesthetized rats contrasts with another studies conducted in anesthetized rats11,12,20. These studies found a sustained positive response over the auditory cortex to the deviating sound between 63 and 253 ms after the stimulus onset by electrocorticogram recording. In addition, the comparison between the responses to deviant alone vs. oddball deviant (i.e. deviant after the standard stimulus) revealed also a robust late positive response to the deviant alone between 220 and 350 ms after the stimulus onset. The shape of the latter response (Figure 2 in Ruusuvirta et al.20) is remarkably similar to a sinusoidal oscillation at 4 Hz, which is the theta frequency under urethane anesthesia. One interpretation of the study is that the deviant alone, as the most infrequent stimulus, induced a so-called theta reset and phase-locking of the theta rhythm to the auditory stimulus21, whereas the same deviant stimulus in the oddball context was less arousing and induced theta reset only occasionally, resulting in a flat positive response in the average. The sensitivity to theta reset may be much higher under urethane anesthesia than in urethane/xylazine combination anesthesia13 or in awake animals (present study), thus explaining the lack of these late components in these later studies. However, as for the P60 component all three studies seem to agree in that it is slightly larger for the deviant stimulus and dependent of the repetition rate (deviant alone is larger than the standard alone irrespective of stimulus pitch).\n\nIn light of previous studies and the present study it is highly unlikely that the awake rat has a similar auditory MMN response as reported for humans. Instead, stimulus repetition results in gradual attenuation of the mid-latency responses, which resembles attenuation of the N1-component of human auditory ERP22. One possible neuropharmacological mechanism underlying this repetition-related attenuation of auditory ERP is reduced cholinergic tone, as administration of scopolamine in the present study reduced the amplitudes of standard- and deviant evoked mid-latency responses. Thus the rat provides a model to study neuropharmacological regulation of the human N1-component, but other animal models need to be employed for the modeling of human MMN.",
"appendix": "Author contributions\n\n\n\nK.G. and H.T. designed the experiments. K.G. analyzed the data. K.G., H.T. and A.L. wrote the paper. A.L. carried out experiments. R.M. manufactured the electrodes, implanted them and conducted the brain histology.\n\n\nCompeting interests\n\n\n\nThe authors have no competing interests to declare.\n\n\nGrant information\n\nThe authors declare that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe thank Dr. M. Penttonen for constructive comments on the manuscript.\n\n\nReferences\n\nNaatanen R, Alho K: Mismatch negativity--a unique measure of sensory processing in audition. Int J Neurosci. 1995; 80(1–4): 317–337. PubMed Abstract | Publisher Full Text\n\nNaatanen M, Tervaniemi E, Sussman P, et al.: \"Primitive intelligence\" in the auditory cortex. Trends Neurosci. 2001; 24(5): 283–288. PubMed Abstract | Publisher Full Text\n\nAlho K: Cerebral generators of mismatch negativity (MMN) and its magnetic counterpart (MMNm) elicited by sound changes. Ear Hear. 1995; 16(1): 38–51. PubMed Abstract | Publisher Full Text\n\nGiard MH, Perrin F, Pernier J, et al.: Brain generators implicated in the processing of auditory stimulus deviance: A topographic event-related potential study. Psychophysiology. 1990; 27(6): 627–640. PubMed Abstract | Publisher Full Text\n\nKumaran D, Maguire EA: Match mismatch processes underlie human hippocampal responses to associative novelty. J Neurosci. 2007; 27(32): 8517–24. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDuncan K, Ketz N, Inati SJ, et al.: Evidence for area CA1 as a match/mismatch detector: a high-resolution fMRI study of the human hippocampus. Hippocampus. 2012; 22(3): 389–98. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCsepe V, Karmos G, Molnar M: Evoked potential correlates of stimulus deviance during wakefulness and sleep in cat--animal model of mismatch negativity. Electroencephalogr Clin Neurophysiol. 1987; 66(6): 571–578. PubMed Abstract | Publisher Full Text\n\nKraus N, McGee T, Littman T, et al.: Nonprimary auditory thalamic representation of acoustic change. J Neurophysiol. 1994; 72(3): 1270–1277. PubMed Abstract\n\nRuusuvirta T, Korhonen T, Penttonen M, et al.: Hippocampal evoked potentials to pitch deviances in an auditory oddball situation in the rabbit: No human mismatch-like dependence on standard stimuli. Neurosci Lett. 1995; 185(2): 123–126. PubMed Abstract | Publisher Full Text\n\nJavitt DC, Schroeder CE, Steinschneider M, et al.: Demonstration of mismatch negativity in the monkey. Electroencephalogr Clin Neurophysiol. 1992; 83(1): 87–90. PubMed Abstract | Publisher Full Text\n\nAstikainen P, Stefanics G, Nokia M, et al.: Memory-based mismatch response to frequency changes in rats. PLoS One. 2011; 6(9): e24208. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRuusuvirta T, Lipponen A, Pellinen E, et al.: Auditory cortical and hippocampal-system mismatch responses to duration deviants in urethane-anesthetized rats. PLoS One. 2013; 8(1): e54624. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLazar R, Metherate R: Spectral interactions, but no mismatch negativity, in auditory cortex of anesthetized rat. Hear Res. 2003; 181(1–2): 51–56. PubMed Abstract | Publisher Full Text\n\nvon der Behrens W, Bäuerle P, Kössl M, et al.: Correlating stimulus-specific adaptation of cortical neurons and local field potentials in the awake rat. J Neurosci. 2009; 29(44): 13837–13849. PubMed Abstract | Publisher Full Text\n\nUmbricht D, Vyssotki D, Latanov A, et al.: Deviance-related electrophysiological activity in mice: Is there mismatch negativity in mice? Clin Neurophysiol. 2005; 116(2): 353–363. PubMed Abstract | Publisher Full Text\n\nSarter M, Bruno JP:Cognitive functions of cortical acetylcholine: toward a unifying hypothesis. Brain Res Brain Res Rev. 1997; 23(1–2): 28–46. PubMed Abstract | Publisher Full Text\n\nPerry E, Walker M, Grace J, et al.: Acetylcholine in mind: a neurotransmitter correlate of consciousness? Trends Neurosci. 1999; 22(6): 273–80. PubMed Abstract | Publisher Full Text\n\nKatzung BG, Masters SB, Trevor AJ, et al.: Basic & clinical pharmacology (McGraw-Hill Medical; McGraw-Hill distributor. New York; London, ed. 11th,. 2009). Reference Source\n\nKnight RT, Brailowsky S, Scabini D, et al.: Surface auditory evoked potentials in the unrestrained rat: Component definition. Electroencephalogr Clin Neurophysiol. 1985; 61(5): 430–439. PubMed Abstract | Publisher Full Text\n\nRuusuvirta T, Penttonen M, Korhonen T: Auditory cortical event-related potentials to pitch deviances in rats. Neurosci Lett. 1998; 248(1): 45–48. PubMed Abstract | Publisher Full Text\n\nVinogradova OS: Expression, control, and probable functional significance of the neuronal theta-rhythm. Prog Neurobiol. 1995; 45(6): 523–583. PubMed Abstract | Publisher Full Text\n\nLu ZL, Williamson SJ, Kaufman L: Behavioral lifetime of human auditory sensory memory predicted by physiological measures. Science. 1992; 258(5088): 1668–1670. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "1785",
"date": "23 Sep 2013",
"name": "James Knierim",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper provides data on auditory evoked potentials in rats using a mismatch (oddball) paradigm and also tests the role of scopolamine on the evoked potentials. The work appears to have been conducted well and analyzed appropriately.I have a few minor suggestions for improvement.Fig 2A. 'P6' should be 'P60'How much of the AEP was due to the reference screw v.s. the recording electrode? This is an issue that the authors may wish to address, as the reference screw presumably contributed to the evoked potentials measured in the differential recordings.Table 1. How were the n's determined in this table? Is this the total number of recording sites? Are any recording sites counted multiple times?Need to provide F values and d.f. for all comparisons, not just some, in the paragraph under the heading \"Increased cortical response to the deviant tone\" as well as in other places in the manuscript. The authors should make sure that the appropriate test statistics and degrees of freedom are provided for all of the measurements not just the resulting p values.",
"responses": []
},
{
"id": "2641",
"date": "20 Dec 2013",
"name": "Timm Rosburg",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe mismatch negativity (MMN) represents the cortical response to sound deviance in an otherwise uniform stimulation. This component of auditory evoked potentials (AEPs) has gained large interest in clinical neurophysiology. In order to understand underlying cortical mechanisms of the MMN, it is of great importance to further establish animal models that allow, for example, investigation into the effects of pharmacological interventions on MMN generation.Given this, the study of Gurevicius and co-workers addresses an important issue of clinical neurophysiology. In their study, AEPs in response to standard tones and deviants were recorded from the hippocampus and parietal cortex of awake, freely moving rats. However, as a main finding, a MMN-like signal could not be observed. Overall, I think the study would benefit from a clearer description of the relation between MMN findings in humans and animals, a clearer outline of the study purpose, the clarification of some methodological details, re-calculation of the amplitude values, and some stronger focus on significant results (rather than on null-findings). I have the following additional comments:The relation between human and animal MMN data is not sufficiently described: Invasive AEP recordings in humans did not reveal any evidence that the MMN is generated in the hippocampus (Halgren et al., 1995; Kropotov et al.,1995; 2000; McCarthy et al.,1989; Rosburg et al., 2007). In contrast, MMN-like signals in humans have been recorded from the temporal cortex (Kropotov et al., 1995; 2000) and in some few instances also from the frontal cortex (Rosburg et al., 2005). Consequently, I find it a little surprising that the authors did not observe a MMN-like hippocampal signal in rats. Moreover, it is not fully clear to me whether the authors doubt the value of other, already established animal models on MMN generation, like the mice model of Umbricht et al. (2005) or the cat model of Csepe et al. (1987). The study is entitled as “Short- and long-term habituation…”, and indeed much space is dedicated to the description on how the recorded AEPs varied from one recording day to the next or from one experiment to the next. However, it is not evident what the authors actually mean by ‘short-term’ and ‘long-term’. In human recordings, ‘short-term decrement’ is usually conceptualized as response decrease from one stimulus to the next. For this kind of decrement, it has been argued that it reflects a consequence of refractoriness (rather than a process of habituation) (Barry et al., 1992; Budd et al., 1998; Rosburg et al., 2004; 2006; 2010; 2013). However, Gurevicius and co-workers did not assess this kind of response decrements, concentrating exclusively on different forms of long-term decrement. Such long-term decrements might reflect a process of habituation, albeit further studies are warranted to support this notion (Rosburg et al., 2002). In consequence, the title of the study should be modified. Furthermore, I propose that the term ‘long-term decrement’ should be used, rather than ‘long-term habituation’. The authors present only exemplary AEP data, but no grand average data. Since the statistics are based on group data, the grand average AEP data need to be depicted (at least across experiment 1). Full evaluation of the study is not possible on the basis of exemplary data. Moreover, there are a couple of technical details that require clarification:(a) The morphology of the depicted AEPs in the hippocampal and cortical recordings look rather similar. Based on the study of Ruusuvirta et al. (2013), I would have expected to find clear phase differences between the two recording sites.(b) Related to this issue, the choice of the active and reference electrode sites needs to be justified. Is the parietal electrode assumed to record activity from the auditory cortex? Is the frontal reference electrode assumed to be electrically silent for auditory stimulation?(c) Only analog filtering of the recordings (1-1000 Hz) is mentioned. Were data additionally offline-filtered?(d) The authors mention that some recordings were excluded due to “poor signal” (p.2). What were the exact criteria for excluding data?(e) The authors do not describe how the data were screened for artefacts and how artefacts were handled.(f) According to the text, Table 1 refers to correlations between peak latency values. Is this really the case? Moreover, it is not clear on what data Tables 1 and 2 are based. Finally, the peak labels in Table 1 do not correspond to the labels used in the rest of the study.(g) The authors should describe how the stimulus intensity was measured. The N40, P60, and P110 peaks were defined as maximum deviations from the baseline. This kind of quantification introduces a bias for obtaining higher (absolute) amplitude values for AEPs to deviants than to standards because the noise levels affect peak amplitude measures (the maximum peak amplitude is increased by the overlying noise signal, and the noise level decreases with the number of trials, used for calculating the AEP). Thus, the peaks should be quantified as mean amplitudes instead. Moreover, the authors should not imply the existence of differences when the statistical analysis revealed non-significant results (e.g. “the P110 showed a similar, but non-significant trend [F(2,6) = 1.9, p = 0.24]”, p. 4). Scopolamine had no differential effect on the AEPs to standards and deviants, but scopolamine resulted in general reductions of AEP components (N40, P60). However, the current study cannot differentiate whether this effect is due to peripheral or central effects of the drug. In human recordings, scopolamine administration often leads to delayed AEP responses, in particular of the N100 (e.g. Pekkonen et al.,2005), but not to an amplitude reduction. One study even described an enhancement of middle-latency auditory evoked neuromagnetic fields (Jääskelainen et al., 1999). This divergence between human and animal data should be discussed. There were some AEP differences between standards and deviants in the 2nd experiment. For evaluation, these differences should be depicted as waveform.",
"responses": []
},
{
"id": "2955",
"date": "10 Mar 2014",
"name": "Colin Lever",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI have read the previous two referee reports, which were made several months ago, so I will try to confine myself to new points.Overview:This paper is acceptable but needs improvements, including a better narrative. In its current state, it will not be easy for a reader to know exactly how the paper adds to the current state of knowledge. The presentation is a little confusing, sometimes focused on the negative result that there is no hippocampal MMN, sometimes on the claim that there is no awake-rat MMN at all, sometimes on habituation of auditory ERP components that can be observed. The strands of all these stories should run throughout the report, including the Introduction, Results, and Discussion.The authors seem to claim that they show that there is no MMN in the awake rat (“Our conclusion that no auditory MMN exists in non-anesthetized Rats…”). This claim needs further justification and clarification.Introduction:The introduction should better set out what has been established about the electrophysiological correlates of the hippocampus in novelty, and why it might be reasonable to test if there is a hippocampal MMN. For instance in the rat, the species examined here, various investigators find increased theta power and/or reduced theta frequency and/or theta reset in novelty. (Theta reset is important to their arguments on the bottom of page 7.) In humans, Knight (1996) produced evidence that a P3 component of the novelty reaction was hippocampus dependent. These and other such evidences would link the hippocampus to novelty, and where available to ‘mismatch novelty’, and perhaps suggest some larger novelty-related function of the hippocampus. This kind of intellectual context would improve the paper. The introduction should then go on to set out a better rationale for why specifically the authors thought the MMN should be studied in a rat, and why they should look in the hippocampus for this. To my knowledge, it is not a standard current view to suggest that the hippocampus might be a generator of the MMN, but presumably some kind of case can be made? Much more reference to the literature is required. If the aim was simply to detect a rodent MMN, why not look at the auditory and frontal cortex?Electrical and magnetic recordings in human subjects have localized the MMN generator to the auditory cortex, although a frontal component has also been observed. In addition, there are some speculations on subcortical generators, especially the hippocampus, but those cannot be indisputably verified in noninvasive recordings5,6.\"This is misleading. The last two references are not attempts to detect hippocampal origins of the MMN. Therefore, the aim of the present study was to address a number of unresolved issues related to MMN in the rat. First, anesthesia was reported to attenuate MMN in the cat7. Therefore, we wanted to test whether MMN can be evoked in freely moving rats14,15 rather than in the anesthetized preparation11,12. Second, we compared the event-related potentials (ERPs) recorded using cortical and hippocampal electrodes to reveal a possible hippocampal generator. Third, to distinguish between MMN and long-term adaptation to standard auditory stimuli as suggested by Lazar and Metherate13, we repeated the oddball stimulus set on two daily sessions and on consecutive days. Fourth, to shed light on the neuropharmacology of MMN, we manipulated the cholinergic input to the cortex and hippocampus by systemic administration of scopolamine.\" Add test/rewrite to incorporate the idea that the paper does not have ANY results about the MMN, and to study the auditory-evoked components that are seen.Methods:\"The hippocampal electrode closest to the pyramidal cell layer and the right parietal cortical electrode were selected for the final analysis of evoked potentials\"How was it determined which electrode was closest to the layer - purely by histology? Did this turn out to matter much? \"The rats were involved in an EEG study for three weeks before the current study on evoked potentials.\"The procedure of the previous study should be briefly described in a supplementary note together with a comment that they think the other study made no difference to this (assuming the authors think that). \"In total 10 rats were recorded for the study but due to poor signaling some channels, the number of records in the analysis varies from 6 to 9.\"What were the minimum threshold criteria used to determine acceptability of signal? Results: I would suggest splitting up the results by cortical and hippocampal regions even more to avoid ambiguity and perhaps using results headings which summarise the results. That might improve the readability of this paper. The figures and indeed main text should state more clearly what values are being entered into averages and so on. E.g. Figure 2, it says ‘averaged AEPs’. How many trials per rat, how many rats, an equivalent number of trials per rat? Figure 3 and Results text 2nd paragraph in Repetition effect on the responses: \"The cortical response to the standard tones reached the original (or higher) amplitude of Day 1 ,in Experiment 1 (Figure 3). The ANOVA for repeated measures revealed significant enhancement of cortical P60 [F(1,6) = 12.9, p = 0.01] and P110 (p = 0.03) components between Day 3 of Experiment 1 and Day 1 of Experiment 2.\"I suggest they use a different statistical symbol than a star to make this point in Figure 3, emphasising the between-experiment changes. The star is already used. Table 2 legend. What is the measure being correlated (one amplitude value, mean amplitude over a set period?), and from which datasets? How does the n= 94 and 76 break down? Discussion: \"Our conclusion that no auditory MMN exists in non-anesthetized rats contrasts with another studies conducted in anesthetized rats11,12,20.\"Overclaiming? On what basis can the authors say that “no auditory MMN exists in non-anesthetized rats”, when they have not recorded from the auditory cortex? Or are they claiming they did sample the auditory cortex in some way? See the queries in Reviewer Rosburg Point 3b.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-182
|
https://f1000research.com/articles/3-63/v1
|
27 Feb 14
|
{
"type": "Short Research Article",
"title": "Modulation of gene expression in guinea pig paraflocculus after induction of hearing loss",
"authors": [
"Wilhelmina H. A. M. Mulders",
"Jennifer Rodger",
"Clarissa G. Yates",
"Donald Robertson",
"Jennifer Rodger",
"Clarissa G. Yates",
"Donald Robertson"
],
"abstract": "Hearing loss often results in plastic changes in the central auditory pathways, which may be involved in the generation of tinnitus, a phantom auditory sensation. However, although animal studies have consistently shown increased neural activity in auditory structures after hearing loss, tinnitus does not always develop. It has therefore been suggested that non-auditory structures perform a gating or regulatory role that determines whether the increased activity in auditory structures leads to conscious perception. Recent evidence points to the paraflocculus of the cerebellum as having such a role. Therefore, we investigated the early effects of hearing loss on gene expression in guinea pig paraflocculus. Gene expression was investigated after two weeks recovery from either acoustic or mechanical cochlear trauma. The genes investigated in our study were associated with inhibitory neurotransmission (GABA-A receptor subunit alpha 1; glutamate decarboxylase 1), excitatory neurotransmission (glutamate receptor NMDA subunit 1), and regulation of transmitter release (member of RAB family of small GTPase). Our results show increased mRNA levels of glutamate decarboxylase 1 in ipsilateral paraflocculus with no difference between the different methods of cochlear trauma. Early modulation of gene expression in the paraflocculus suggests that an early effect of hearing loss may affect the influence of this structure on auditory processing.",
"keywords": [
"It is well known that trauma to the cochlea results not only in a reduced sensitivity to sound1",
"2",
"but also leads to a variety of physiological changes in the central nervous system. Central neural changes following hearing loss have been described using many different animal models and include changes in tonotopic maps3",
"4",
"increased synchronous firing patterns5–7 and increased spontaneous firing rates6–11."
],
"content": "Introduction\n\nIt is well known that trauma to the cochlea results not only in a reduced sensitivity to sound1,2, but also leads to a variety of physiological changes in the central nervous system. Central neural changes following hearing loss have been described using many different animal models and include changes in tonotopic maps3,4, increased synchronous firing patterns5–7 and increased spontaneous firing rates6–11.\n\nThis abnormal neural activity observed in the central auditory pathways following cochlear trauma has been suggested to play a role in the development of tinnitus, a phantom auditory sensation that is often associated with the presence of hearing loss12–14. However, although animal studies have shown that central changes are consistently present after trauma to the cochlea, tinnitus is not always present15–17. This is in accordance with human data showing that not all subjects with hearing loss develop tinnitus18.\n\nTherefore, although a trauma to the peripheral auditory receptor could serve as a trigger for the abnormal changes seen in the central auditory pathways, other brain regions are likely to be involved in giving rise to the eventual phantom auditory perception. Some brain regions that have been suggested are limbic structures19–21, which may be involved in gating mechanisms for the suppression of unwanted noise. Another structure that may be involved in this process is the paraflocculus of the cerebellum, which is not considered part of the classical auditory pathway22. Brozoski and co-workers reported that neural activity increases in the paraflocculus of rats displaying behavioural evidence of tinnitus23. Furthermore, ablation of the paraflocculus in rats before the induction of tinnitus results in the prevention or reduction of subsequent tinnitus17.\n\nInterestingly, anatomical data suggest that the paraflocculus receives direct sensory inputs from the cochlea24. This is in line with the observation that the paraflocculus neurons of rats25 and bats26 respond to auditory stimuli. In addition, Azizi et al. showed evidence of connectivity between central auditory structures and the paraflocculus. In particular, they showed that rat paraflocculus neurons can respond to electrical stimulation of the auditory cortex and inferior colliculus. Using anterograde and retrograde tracer techniques, the authors also showed evidence for a corticopontocerebellar connection from the auditory cortex to the paraflocculus27.\n\nIn central auditory structures, hearing loss might result in transcriptional modulation of genes regulating inhibitory and excitatory neurotransmission, regulation of pre-synaptic transmitter release and intrinsic neuronal membrane excitability9,28–33. In view of the direct cochlear input to the paraflocculus and the indirect innervation arising from the auditory cortex, gene expression changes might also be found in paraflocculus soon after hearing loss. We therefore investigated peripheral hearing loss and used quantitative real-time PCR (qRT-PCR) to measure the expression of four genes involved in inhibitory and excitatory neurotransmission and regulation of pre-synaptic transmitter release in paraflocculus of guinea pigs following acoustic and mechanical cochlear trauma. Both of these methods of trauma are known to result in hearing loss, hyperactivity and associated changes in gene expression in the central auditory pathway9,10,33. We studied the expression of GABA-A receptor subunit alpha 1 (GABRA1), glutamate decarboxylase 1 (GAD1), glutamate receptor NMDA subunit 1 (GRIN1) and a member of Rab family of small GTPase (RAB3A) using guinea pig-specific primers previously designed in our laboratory9.\n\n\nMethods\n\nTwelve (8 males and 4 females) adult pigmented guinea pigs (tricolor strain obtained from a breeding colony at the University of Western Australia) weighing between 245 and 385 g at the time of final recovery surgery were used in the experiment. Animals were housed in cages with clear plastic walls (51 cm × 41 cm base, 28 cm high) based on sex (2 per cage) in a SPF facility at 22°C with a 12 hour light/dark cycle. Guinea pigs had ad libitum access to food and water. Animals were supplied with Perspex hutch and hay for environmental enrichment. We investigated the effects of three treatments (sham, acoustic trauma and mechanical trauma) on peripheral thresholds and measured gene expression in the paraflocculus by RT-PCR. Animals were randomly allocated to an experimental group (n=4 for each). All experimental protocols were in line with the Code of Practice of the National Health and Medical Research Council of Australia, and were approved by the Animal Ethics Committee of The University of Western Australia, approval ID 03/100/1007.\n\nAnaesthesia and surgical procedures have been described in detail previously9,34. For recovery experiments, all animals were injected with 0.1 ml atropine sulphate (0.6 mg/ml; APEX Laboratories, Australia) subcutaneously, followed by an intraperitoneal injection of diazepam (5 mg/kg; Pamlin, Ceva, Australia), and an intramuscular injection of Hypnorm 20 minutes later (0.315 mg/ml fentanyl citrate and 10 mg/ml fluanisone; 1 ml/kg; VetaPharma, UK). Absence of the foot withdrawal reflex was used to ascertain deep anaesthesia, after which animals were placed on a heating blanket in a soundproof room and the head mounted in hollow ear bars. A small opening was made in the bulla on the left side and an insulated silver wire was placed on the round window in order to record a compound action potential (CAP) audiogram (frequency range 4–24 kHz in 2 kHz steps)35 using a closed sound system. All sound stimuli were presented through a ½″ condenser microphone driven in reverse as a speaker (Bruel and Kjaer, type 4134). Pure tone stimuli were synthesized by a computer equipped with DIGI 96 soundcard connected to an analog/digital interface (ADI-9 DS, RME Intelligent Audio Solution). Sample rate was 96 kHz. The interface was driven by a custom-made computer program (Neurosound, MI Lloyd), which was also used to collect single neuron data during the non-recovery experiment. CAP signals were amplified, filtered (100 Hz-3 kHz bandpass) and recorded with a second data acquisition system (Powerlab 4SP, AD Instruments). If cochlear thresholds were within the normal range, the animal was randomly assigned to either the sham group, acoustic trauma or mechanical lesion group.\n\nSham animals received no further treatment after the measurement of the CAP audiogram, but this group was treated identically to the acoustic trauma and mechanical trauma group in every other aspect; they were maintained under anaesthesia for two hours and underwent identical recovery treatment.\n\nFor acoustic trauma groups, the contralateral ear was blocked with plasticine and the animal was subjected to a continuous loud tone for 1 hour (10 kHz, 124 dB SPL). After the acoustic trauma, CAP thresholds were again recorded to determine the magnitude of the immediate hearing loss.\n\nFor mechanical lesions groups, a small hole was hand drilled in the wall of the cochlea at the level of the basal turn. A glass micropipette electrode (tip diameter ~20 μm) filled with 150 mM KCI was inserted through the hole passing through the scala tympani and the organ of Corti into the scala media (signalled by the sudden appearance of a large positive potential between 80 and 100 mV). The pipette was then further advanced until it penetrated Reissner's membrane (signalled by a drop in the positive voltage). The pipette was then removed and a CAP audiogram was determined to establish loss of neural sensitivity. This procedure was repeated up to 3 times to ensure a substantial change in CAP thresholds, after which the hole in the cochlear wall was covered by a small piece of gelfilm.\n\nAll animals remained under full surgical anaesthesia throughout the acoustic and mechanical trauma procedures. Finally, in all animals the incision was sutured and buprenorphine (0.05 mg/kg subcutaneously; Temgesic, Reckitt Benckiser, Australia) was given post-operatively as analgesic. Survival time from the recovery experiment till the final non-recovery experiment was 2 weeks.\n\nFor the final non-recovery experiments anaesthesia consisted of a subcutaneous injection with 0.1 ml atropine followed by an intraperitoneal injection of pentobarbitone sodium (30 mg/kg; Ilium, Australia) and an intramuscular injection of Hypnorm (0.15 ml). Animals were placed on a heating blanket in a sound proof room and artificially ventilated on carbogen (95% O2 and 5% CO2). After the animals were mounted in hollow ear bars, the left and right cochleae were exposed and CAP audiograms were recorded on both sides with a silver wire placed on the round window as described above for the recovery experiments.\n\nFollowing the measurements of the CAP audiograms during the non-recovery experiment, for tissue collection, animals were terminally anaesthetised with Pentobarbitone (Lethabarb, Virbac, Australia) and decapitated using an animal guillotine (World precision Instruments, USA) and their brains rapidly removed in ice-cold phosphate-buffered saline (0.1 M). The paraflocculus, on both sides of the brain, was removed quickly using either a sharp scalpel or fine scissors, and then transferred into 1.5 ml RNase-free tubes. The samples were immediately stored at −80°C until RNA extraction.\n\nThe qRT-PCR procedures have been described in detail previously28. The total RNA was isolated using a tissue homogenizer (Invitrogen, Mount Waverley, VIC, Australia) and a PureLink RNA Mini Kit Total RNA Purification System (Invitrogen), according to the manufacturer’s protocol for purifying RNA from frozen animal tissue followed by a standard ethanol precipitation. Nucleic acid concentration was measured by a NanoDrop 1000 Spectrophotometer (Thermo Scientific, Waltham, MA, USA). Subsequently, genomic DNA contamination was removed by RQ1 RNase-free DNase (Promega, Alexandria, NSW, Australia) treatment (1 U/µg nucleic acid). The RNA integrity was confirmed by agarose gel electrophoresis before storage at −80°C.\n\nThe following four genes were selected for qRT-PCR: the GABA-A receptor subunit alpha 1 (GABRA1, GAD1, GRIN1 and RAB3A). The guinea pig-specific primers for these genes were designed previously in our laboratory9. Synthesis of first-strand cDNA from RNA was carried out using GoScript Reverse Transcription System (Promega) and 500 ng of RNA was reverse transcribed in a 20 µl reaction with random primers according to the manufacturer’s instructions. The resultant cDNA was purified in a PCR clean-up kit (Promega Wizard PCR clean-up system). The purified cDNA was quantified again on a Nanodrop 1000 Spectrophotometer and diluted 40 times with nuclease-free water before being stored at −80°C.\n\nqRT-PCR was performed in a Rotor-Gene Q real-time thermocycler (Corbett Life Science, Sydney, NSW, Australia). Amplification was carried out in a total volume of 20 µl reaction mixture containing 10 µl of 2× QuantiTect SYBR Green PCR Master Mix (catalogue number: 204141; Qiagen, Doncaster, VIC, Australia), 0.5 µM of each specific gene primer and 9 µl (10 ng) of diluted cDNA prepared as described above. Real-time PCR reactions were cycled 40 times after initial denaturation (50°C for 2 min, 95°C for 15 min) under the following parameters: 94°C for 15 s (denaturation), 54°C for 30 s (annealing) and 72°C for 30 s (extension and fluorescence data collection). Samples were run in duplicate and accompanied by negative controls (‘no reverse transcription’ and ‘no template’). The specificity of all amplicons was further assessed by using the melting curve protocol on the Rotor-Gene Q (Corbett Life Science). In order to avoid problems created by any inter-run variability, qRT-PCR for tissue samples (controls, acoustic and mechanical trauma) from the same side of the brain was conducted in the same runs. All analyses were replicated for each gene and the mean of the two reactions was used to calculate the expression level of that gene in each animal. Using the housekeeping genes ribosomal protein S16 (RPS16) and beta-2-microglobulin (B2M) for normalization29, relative quantification of target gene expression for all groups was performed following the comparative CT method36. In order to reflect clearly the different expression levels of different genes, the data are reported only as the ratio of target to housekeeping gene without converting to fold change. To calculate the ratio of target to housekeeping genes, for each target gene the mean of the replicates was calculated as well as the mean of the replicates of both the housekeeping genes.\n\nStatistical analysis of CAP threshold changes following each treatment and at each frequency was performed using a Kruskall-Wallis test and a Dunn’s multiple comparison post-test. Statistical significance (estimated at p<0.05) for qRT-PCR data was evaluated using ANOVA with Bonferroni’s multiple comparison post-tests (GraphPad Prism software).\n\n\nResults\n\nThe effects of different treatments on CAP peripheral thresholds are illustrated in Figure 1. Sham surgery had no significant effect on peripheral thresholds at 2 weeks recovery (Figure 1A). Acoustic and mechanical trauma both resulted in a frequency restricted hearing loss after recovery, though there was large variation in the patterns of hearing loss between the individual animals (Figure 1B,C, thin black lines for individual animals). Statistical comparisons of the groups showed that there were significant threshold losses at 12 kHz and 14 kHz in both groups (acoustic trauma p<0.05 at 12 kHz and p<0.01 at 14 kHz; mechanical trauma p<0.01 at 12 and 14 kHz). There were no statistically significant differences in threshold loss between the acoustic and mechanical trauma group. Thresholds measured in the right ear were not significantly different from the left ear before trauma (Data Set 1).\n\nChanges in cochlear sensitivity measured as CAP threshold loss recorded in the left cochlea after recovery from sham surgery (A), acoustic trauma (B) or mechanical trauma (C). Thick black lines indicate the mean ± SEM (n=4 for all), thin black lines represent individual animals. * p<0.05, ** p<0.01 compared to before treatment data.\n\nFigure 2 shows the pattern of gene expression in the paraflocculus ipsilateral and contralateral to the cochlea exposed to sham surgery, acoustic trauma or mechanical trauma. No statistically significant changes were observed between the ipsi- and contralateral side in sham animals for any of the genes investigated. Two genes (GRIN1, involved in excitatory neurotransmission and RAB3A, involved in regulation of pre-synaptic neurotransmitter release Figure 2E–H) did not show any change compared to sham animals after either acoustic or mechanical trauma to the cochlea.\n\nHowever, for the genes involved in inhibitory neurotransmission (GABRA1 and GAD1) there was an upward trend compared to sham animals following both acoustic and mechanical trauma (Figure 2A–D). For GABRA1 the increase after acoustic and mechanical trauma compared to sham animals varied from 64% to 88% and for GAD1 increases varied from 27% to 49%. The percentage of increase was calculated as: (trauma value-sham value)/(sham value/100). ANOVA performed on the ratios of the target to housekeeping genes showed a significant effect of treatment only for GAD1 expression in the ipsilateral paraflocculus F(2, 9) = 10.19, p=0.0049 (ANOVA test). Bonferroni’s post-hoc analysis showed significant increases of 49% (p<0.01) and 45% (p<0.05) compared to the sham group after acoustic and mechanical trauma, respectively (Figure 2C,D).\n\n\n\nChanges in mRNA expression levels of 4 genes in the left (ipsilateral; A, C, E, G) and right (contralateral; B, D, F, H) paraflocculus in sham (white bars), acoustic trauma (black bars) and mechanical trauma animals (grey bars), after 2 weeks recovery, as shown by qRT-PCR. Gene abbreviations: GABR1: GABA-A receptor subunit alpha 1; GAD1: glutamate decarboxylase 1; GRIN1: glutamate receptor NMDA subunit 1; RAB3A: a member of RAB family of small GTPase. Values are mean ± SEM. Statistical analysis: * p<0.05, ** p<0.01. Asterisks indicate comparison with sham data.\n\n\nDiscussion\n\nThe present study shows the transcriptional modulation of genes regulating neurotransmission in the paraflocculus of the cerebellum of guinea pigs, following peripheral hearing loss due to damage of the cochlea. Changes in genetic expression are caused by either acoustic or mechanical trauma to the sense organ. These early changes in paraflocculus observed after two weeks may be evoked by an alteration in the direct input from the cochlea or in the indirect pathway described from the cortex as outlined below.\n\nThe hearing loss after acoustic trauma observed in our experiment showed a notch–like loss that was largest just above the exposure frequency, as reported in previous studies10,28,37–39. This pattern of hearing loss can be attributed to the nonlinear properties of the basilar membrane at high sound levels40,41. Mechanical trauma in this study also resulted in notch-like losses with the largest loss observed at the same frequency as after acoustic trauma.\n\nOur RT-PCR analysis performed in the paraflocculus following mechanical or acoustic trauma of the cochlea revealed no significant changes in the expression of the glutamate receptor NMDA subunit GRIN1, associated with excitatory neurotransmission, or RAB3A, involved in regulation of neurotransmitter release42. However, both types of trauma induced a slight increase in mRNA levels of genes associated with inhibitory neurotransmission (GABRA1 and GAD1), which was statistically significant for GAD1 in the ipsilateral paraflocculus. Changes in the ipsilateral paraflocculus may be a direct effect of an altered cochlear input to this cerebellar structure after trauma, since it has been demonstrated in chinchilla that following an injection with biotinylated dextran amine in the cochlea, labelled axons were found in the ipsilateral paraflocculus24. In addition, paraflocculus neurons in rats25 and bats26 have been reported to respond to free field auditory stimuli. The apparent increase in gene expression in the contralateral paraflocculus, although not statistically significant, may be an indirect effect due to changes in neuronal activity in the inferior colliculus or cortex. Azizi and co-workers demonstrated that paraflocculus neurons respond to electrical stimulation of the auditory cortex and inferior colliculus and showed evidence for a corticopontocerebellar connection25,27.\n\nInterestingly, changes to the ipsilateral rather than the contralateral paraflocculus are in line with the changes in the neural activity in paraflocculus of rats with tinnitus induced by acoustic trauma reported by Brozoski and co-workers23. However, our finding of an increase in gene expression levels associated with inhibitory actions seems counter-intuitive in view of the increase in activity reported23. However, Brozoski et al. tested their rats approximately 12 months after acoustic trauma, whereas the present study was performed two weeks after acoustic trauma. Other studies have shown that gene expression after acoustic trauma can vary significantly at different time-points28 and the increase in inhibition observed in the present study may represent only an early transient change. In addition, depending on the circuitry affected by the altered gene expression, the downstream effect on the activity in the core auditory pathway may well be disinhibition and therefore it may cause an increase in physiological activity. It is still unclear whether there are direct efferent projections from the paraflocculus to the auditory system and whether these are excitatory or inhibitory.\n\nOur data demonstrate early changes in the paraflocculus following two different types of cochlear trauma that both result in hearing loss and hyperactivity in the central auditory nuclei9,37. At present, the functional consequences of these changes in gene expression, in particular for tinnitus related activity in the auditory pathway, remain to be determined.\n\n\nData availability\n\nfigshare: Data of paraflocculus cochlear trauma and modulation of gene expression. http://dx.doi.org/10.6084/m9.figshare.93819343.",
"appendix": "Author contributions\n\n\n\nWHAMM, JR and DR conceived the study and designed the experiments. WHAMM, CGY and DR carried out the research. WHAMM prepared the first draft of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research was supported by grants from the Royal National Institute for Deaf People (UK) G55, the Neurotrauma Research Program Western Australia, the Medical Health and Research Infrastructure Fund (WA) and The University of Western Australia.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nLiberman MC: Single-neuron labeling and chronic cochlear pathology. I. Threshold shift and characteristic-frequency shift. Hear Res. 1984; 16(1): 33–41. PubMed Abstract | Publisher Full Text\n\nLiberman MC, Kiang NY: Single-neuron labeling and chronic cochlear pathology. IV. Stereocilia damage and alterations in rate- and phase-level functions. Hear Res. 1984; 16(1): 75–90. PubMed Abstract | Publisher Full Text\n\nNorena AJ, Eggermont JJ: Enriched acoustic environment after noise trauma reduces hearing loss and prevents cortical map reorganization. J Neurosci. 2005; 25(3): 699–705. PubMed Abstract | Publisher Full Text\n\nRobertson D, Irvine DR: Plasticity of frequency organization in auditory cortex of guinea pigs with partial unilateral deafness. J Comp Neurol. 1989; 282(3): 456–71. PubMed Abstract | Publisher Full Text\n\nFinlayson PG, Kaltenbach JA: Alterations in the spontaneous discharge patterns of single units in the dorsal cochlear nucleus following intense sound exposure. Hear Res. 2009; 256(1–2): 104–17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSeki S, Eggermont JJ: Changes in spontaneous firing rate and neural synchrony in cat primary auditory cortex after localized tone-induced hearing loss. Hear Res. 2003; 180(1–2): 28–38. PubMed Abstract | Publisher Full Text\n\nBauer CA, Turner JG, Caspary DM, et al.: Tinnitus and inferior colliculus activity in chinchillas related to three distinct patterns of cochlear trauma. J Neurosci Res. 2008; 86(11): 2564–78. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKaltenbach JA, Zhang J, Afman CE: Plasticity of spontaneous neural activity in the dorsal cochlear nucleus after intense sound exposure. Hear Res. 2000; 147(1–2): 282–92. PubMed Abstract | Publisher Full Text\n\nDong S, Mulders WH, Rodger J, et al.: Changes in neuronal activity and gene expression in guinea-pig auditory brainstem after unilateral partial hearing loss. Neuroscience. 2009; 159(3): 1164–74. PubMed Abstract | Publisher Full Text\n\nMulders WH, Ding D, Salvi R, et al.: Relationship between auditory thresholds, central spontaneous activity, and hair cell loss after acoustic trauma. J Comp Neurol. 2011; 519(13): 2637–47. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKomiya H, Eggermont JJ: Spontaneous firing activity of cortical neurons in adult cats with reorganized tonotopic map following pure-tone trauma. Acta Otolaryngol. 2000; 120(6): 750–6. PubMed Abstract | Publisher Full Text\n\nEggermont JJ, Roberts LE: The neuroscience of tinnitus. Trends Neurosci. 2004; 27(11): 676–682. PubMed Abstract | Publisher Full Text\n\nAxelsson A, Ringdahl A: Tinnitus--a study of its prevalence and characteristics. Br J Audiol. 1989; 23(1): 53–62. PubMed Abstract | Publisher Full Text\n\nSalvi R, Lobarinas E, Sun W: Pharmacological treatments for tinnitus: new and old. Drugs Future. 2009; 34(5): 381–400. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDehmel S, Eisinger D, Shore SE: Gap prepulse inhibition and auditory brainstem-evoked potentials as objective measures for tinnitus in guinea pigs. Front Syst Neurosci. 2012; 6: 42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang H, Brozoski TJ, Turner JG, et al.: Plasticity at glycinergic synapses in dorsal cochlear nucleus of rats with behavioral evidence of tinnitus. Neuroscience. 2009; 164(2): 747–59. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBauer CA, Kurt W, Sybert LT, et al.: The cerebellum as a novel tinnitus generator. Hear Res. 2013; 295: 130–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMazurek B, Olze H, Haupt H, et al.: The more the worse: the grade of noise-induced hearing loss associates with the severity of tinnitus. Int J Environ Res Public Health. 2010; 7(8): 3071–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRauschecker JP, Leaver AM, Muhlau M: Tuning out the noise: limbic-auditory interactions in tinnitus. Neuron. 2010; 66(6): 819–26. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDe Ridder D, Elgoyhen AB, Romo R, et al.: Phantom percepts: tinnitus and pain as persisting aversive memory networks. Proc Natl Acad Sci U S A. 2011; 108(20): 8075–80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKraus KS, Canlon B: Neuronal connectivity and interactions between the auditory and limbic systems. Effects of noise and tinnitus. Hear Res. 2012; 288(1–2): 34–46. PubMed Abstract | Publisher Full Text\n\nShulman A, Strashun A: Descending auditory system/cerebellum/tinnitus. Int Tinnitus J. 1999; 5(2): 92–106. PubMed Abstract\n\nBrozoski TJ, Ciobanu L, Bauer CA: Central neural activity in rats with tinnitus evaluated with manganese-enhanced magnetic resonance imaging (MEMRI). Hear Res. 2007; 228(1–2): 168–79. PubMed Abstract | Publisher Full Text\n\nMorest DK, Kim J, Bohne BA: Neuronal and transneuronal degeneration of auditory axons in the brainstem after cochlear lesions in the chinchilla: cochleotopic and non-cochleotopic patterns. Hear Res. 1997; 103(1–2): 151–68. PubMed Abstract | Publisher Full Text\n\nAzizi SA, Woodward DJ: Interactions of visual and auditory mossy fiber inputs in the paraflocculus of the rat: a gating action of multimodal inputs. Brain Res. 1990; 533(2): 255–62. PubMed Abstract | Publisher Full Text\n\nSun DX, Sun XD, Jen PH: The influence of the auditory cortex on acoustically evoked cerebellar responses in the CF-FM bat, Rhinolophus pearsonic chinesis. J Comp Physiol A. 1990; 166(4): 477–87. PubMed Abstract | Publisher Full Text\n\nAzizi SA, Burne RA, Woodward DJ: The auditory corticopontocerebellar projection in the rat: inputs to the paraflocculus and midvermis. An anatomical and physiological study. Exp Brain Res. 1985; 59(1): 36–49. PubMed Abstract | Publisher Full Text\n\nDong S, Mulders WH, Rodger J, et al.: Acoustic trauma evokes hyperactivity and changes in gene expression in guinea-pig auditory brainstem. Eur J Neurosci. 2010; 31(9): 1616–28. PubMed Abstract | Publisher Full Text\n\nCui YL, Holt AG, Lomax CA, et al.: Deafness associated changes in two-pore domain potassium channels in the rat inferior colliculus. Neuroscience. 2007; 149(2): 421–33. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHolt AG, Asako M, Duncan RK, et al.: Deafness associated changes in expression of two-pore domain potassium channels in the rat cochlear nucleus. Hear Res. 2006; 216–217: 146–53. PubMed Abstract | Publisher Full Text\n\nHolt AG, Asako M, Lomax CA, et al.: Deafness-related plasticity in the inferior colliculus: gene expression profiling following removal of peripheral activity. J Neurochem. 2005; 93(5): 1069–86. PubMed Abstract | Publisher Full Text\n\nMossop JE, Wilson MJ, Caspary DM, et al.: Down-regulation of inhibition following unilateral deafening. Hear Res. 2000; 147(1–2): 183–7. PubMed Abstract | Publisher Full Text\n\nSun W, Zhang L, Lu J, et al.: Noise exposure-induced enhancement of auditory cortex response and changes in gene expression. Neuroscience. 2008; 156(2): 374–80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMulders WH, Robertson D: Progressive centralization of midbrain hyperactivity after acoustic trauma. Neuroscience. 2011; 192: 753–60. PubMed Abstract | Publisher Full Text\n\nJohnstone JR, Alder VA, Johnstone BM, et al.: Cochlear action potential threshold and single unit thresholds. J Acoust Soc Am. 1979; 65(1): 254–7. PubMed Abstract | Publisher Full Text\n\nLivak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001; 25(4): 402–8. PubMed Abstract | Publisher Full Text\n\nMulders WH, Robertson D: Hyperactivity in the auditory midbrain after acoustic trauma: dependence on cochlear activity. Neuroscience. 2009; 164(2): 733–46. PubMed Abstract | Publisher Full Text\n\nSalvi RJ, Hamernik RP, Henderson D: Auditory nerve activity and cochlear morphology after noise exposure. Arch Otorhinolaryngol. 1979; 224(1–2): 111–6. PubMed Abstract | Publisher Full Text\n\nCody AR, Robertson D: Variability of noise-induced damage in the guinea pig cochlea: electrophysiological and morphological correlates after strictly controlled exposures. Hear Res. 1983; 9(1): 55–70. PubMed Abstract | Publisher Full Text\n\nCody AR, Johnstone BM: Acoustic trauma: single neuron basis for the \"half-octave shift\". J Acoust Soc Am. 1981; 70(3): 707–11. PubMed Abstract | Publisher Full Text\n\nSellick PM, Patuzzi R, Johnstone BM: Measurement of basilar membrane motion in the guinea pig using the Mossbauer technique. J Acoust Soc Am. 1982; 72(1): 131–41. PubMed Abstract | Publisher Full Text\n\nSchluter OM, Schmitz F, Jahn R, et al.: A complete genetic analysis of neuronal Rab3 function. J Neurosci. 2004; 24(29): 6629–37. PubMed Abstract | Publisher Full Text\n\nMulders WH, Yates C, Robertson D: Data of paraflocculus cochlear trauma and modulation of gene expression. Figshare. 2014. Data Source"
}
|
[
{
"id": "3990",
"date": "06 Mar 2014",
"name": "Thomas Brozoski",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe title and abstract are appropriate. Overall, the methods, results, and conclusions appear to be sound. The study contributes to our evolving understanding of the central nervous system consequences of hearing loss. The results also temper the general hypothesis that loss of peripheral hearing sensitivity leads to a loss of inhibition. Unfortunately, the actual story may be more complex than that.Specific Comments:There is a typographical error in the methods paragraph describing gene expression analysis (missing parenthesis). Hearing threshold data are displayed as dB loss. This is okay, but I prefer to see hearing threshold levels simply plotted as dB SPL. Elevations (loss) can then be interpreted by the reader. It is surprising that GABA indictors were elevated in the trauma animals, while glutamatergic indicators were not. But that is why research is done. It would have been informative to look at longer-term consequences of the trauma as well, and perhaps those experiments are in progress.",
"responses": [
{
"c_id": "778",
"date": "23 Apr 2014",
"name": "wilhelmina Mulders",
"role": "Author Response",
"response": "We will add the missing parenthesis. Our spreadsheet with the raw data shows the hearing thresholds for each animal. We agree that the lack of change in glutamatergic indicators is surprising, especially in view of the reviewer's own result using NMDA antagonist. However, as we point out in our Discussion, our data was derived at an early time-point after trauma. In addition, it is possible that a glutamatergic pathway is involved but not necessarily upregulated in this circuitry."
}
]
},
{
"id": "3906",
"date": "22 Apr 2014",
"name": "Ramesh Rajan",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting area of study and a timely one. I have only a couple of comments to make the article clearer:“Central neural changes following hearing loss have been described using many different animal models and include changes in tonotopic maps, increased synchronous firing patterns and increased spontaneous firing rates\" This statement focuses on the “positive” side effects of cochlear hearing loss, but there are many “negative” changes as well such as reduced sensitivity, loss of inhibition (positive and negative used in the same sense as in schizophrenia, not to imply good or bad). The first paragraph of the Introduction is couched in terms of tinnitus, but this article has nothing to do with tinnitus since the authors have examined changes after loud sound but not measured tinnitus, and the link in the Introduction to tinnitus is reported in one brief line. This is not a compelling argument. The demonstration that neural activity increases in the paraflocculus in cases of some behavioural evidence of tinnitus is not an argument that the cerebellum is involved in the generation of tinnitus, as is implied in the first sentence of the paragraph “other brain regions are likely to be involved in giving rise to the eventual phantom auditory perception.” The evidence that the authors cite in the rest of the paragraph shows that the paraflocculus gets auditory input, not that it modulates auditory input or percepts as needed for the argument that paraflocculus activity is linked to tinnitus. More compelling is the more recent Brozoski et al. (2013) article which reports that blockade of NMDA receptor activity in the paraflocculus reduces tinnitus, but the authors don’t cite this. Finally, it is unclear to me how many animals contributed the various data. The authors report n=4 per group in the Methods, but the Table presents data for 4 shams and 2 mechanical trauma animals, and Figure 2 does not cite the n for each group.",
"responses": [
{
"c_id": "777",
"date": "23 Apr 2014",
"name": "wilhelmina Mulders",
"role": "Author Response",
"response": "The reviewer is quite correct to point out that our list of changes is not comprehensive. The reviewer is correct that we cannot make a direct link between our findings and tinnitus. However, in the Introduction we cite two articles by Brozowski and co-workers (paraflocculus ablation and MEMRI) that do directly implicate the paraflocculus in the modulation of tinnitus. We are grateful for the reviewer pointing out the 2013 article and have included this in the paper. The spreadsheet files with our raw data need to be opened and they show the information the reviewer is requesting. We had 4 animals per group as described in the Methods."
}
]
}
] | 1
|
https://f1000research.com/articles/3-63
|
https://f1000research.com/articles/2-282/v1
|
20 Dec 13
|
{
"type": "Research Article",
"title": "Quality parameters for a multimodal EEG/EMG/kinematic brain-computer interface (BCI) aiming to suppress neurological tremor in upper limbs",
"authors": [
"Giuliana Grimaldi",
"Mario Manto",
"Yassin Jdaoudi",
"Giuliana Grimaldi",
"Yassin Jdaoudi"
],
"abstract": "Tremor is the most common movement disorder encountered during daily neurological practice. Tremor in the upper limbs causes functional disability and social inconvenience, impairing daily life activities. The response of tremor to pharmacotherapy is variable. Therefore, a combination of drugs is often required. Surgery is considered when the response to medications is not sufficient. However, about one third of patients are refractory to current treatments. New bioengineering therapies are emerging as possible alternatives. Our study was carried out in the framework of the European project “Tremor” (ICT-2007-224051). The main purpose of this challenging project was to develop and validate a new treatment for upper limb tremor based on the combination of functional electrical stimulation (FES; which has been shown to reduce upper limb tremor) with a brain-computer interface (BCI). A BCI-driven detection of voluntary movement is used to trigger FES in a closed-loop approach. Neurological tremor is detected using a matrix of EMG electrodes and inertial sensors embedded in a wearable textile. The identification of the intentionality of movement is a critical aspect to optimize this complex system. We propose a multimodal detection of the intentionality of movement by fusing signals from EEG, EMG and kinematic sensors (gyroscopes and accelerometry). Parameters of prediction of movement are extracted in order to provide global prediction plots and trigger FES properly. In particular, quality parameters (QPs) for the EEG signals, corticomuscular coherence and event-related desynchronization/synchronization (ERD/ERS) parameters are combined in an original algorithm which takes into account the refractoriness/responsiveness of tremor. A simulation study of the relationship between the threshold of ERD/ERS of artificial EEG traces and the QPs is also provided. Very interestingly, values of QPs were much greater than those obtained for the corticomuscular module alone.",
"keywords": [
"EEG",
"EMG",
"gyroscopes",
"multimodal",
"brain-computer interface",
"tremor"
],
"content": "Introduction\n\nTremor is the most common movement disorder encountered during daily practice1. It causes functional disability and social inconvenience, disturbing daily life activities. Its incidence and prevalence increase with ageing1. The response of tremor to pharmacotherapy is variable and a combination of drugs is often required after a few years of therapy. Neurosurgical procedures are considered when the clinical response is not sufficient or when the patient becomes refractory. However, a number of patients do not respond to current therapies. Therefore novel strategies are being developed. New bioengineering therapies are now emerging as viable solutions2. In particular, recent studies aim to develop and validate a new treatment for upper limb tremor based on the combination of functional electrical stimulation (FES) with a brain-computer interface (BCI)2–3. The main goal is to set up a semi-automatic procedure to reduce/stop upper limb tremor, with a selective cancellation of tremor oscillations while preserving natural voluntary movement. The intentionality of movement is tracked by the BCI, in order to trigger FES in the upper limbs. Such concepts open new doors for the treatment of numerous neurological disorders affecting the upper limbs.\n\nWe describe a multimodal detection of the intentionality of movement by fusing signals from EEG, EMG and kinematic sensors (in particular gyroscopes and accelerometers). A kinematic module is applied purely for analyzing tremor, but also finds a specific application for the early detection of movement in patients presenting with a rest tremor - a tremor occurring while in a rest position - on the basis of a decrement of the rest tremor before movement onset4. Why use a multimodal detection of the intentionality of movement? Although the potential for BCIs in neurological disorders is huge, the applicability of current BCI systems has been limited by several factors5. One of them is the poor performance of BCIs based on conventional EEG analysis. Therefore, this multimodal processing is assumed to add accuracy in the prediction of movements, thus improving the effectiveness of the system.\n\n\nMaterials and methods\n\nFigure 1 gives a schematic glance at the multimodal approach. From each module (described in detail in sections B–F) quality parameters (QPs) for the detection of the intentionality of movement or the early detection of movement are extracted (we extract QPs and probabilities of stimulation for the EEG and Kinematic modules, and probability of stimulation for the corticomuscular module). Patients and the experimental procedure were as detailed in the next sections.\n\nDifferent modules are used to predict a voluntary movement: EEG, EMG, Corticomuscular (CM) and a Kinematic module.\n\nAcquisition of data was carried out on 4 neurological patients exhibiting a bilateral upper limb tremor (combinations of rest, postural and/or kinetic tremor), following approval of the Ethical Committee of ULB – Hôpital Erasme. All the patients were followed at the Erasme Hospital and gave their written informed consent to participate in the study. Patients were affected by: Parkinsonism of vascular origin (n=1), Parkinson’s disease (n=1), essential tremor (n=1) and post-traumatic brain injury (n=1). Male/female ratio was 3:1. Mean age of the patients was 62 ± 20 years. The patients were all right-handed and presented with upper limb tremor of grade 1 to 3/4. The ADL-T24 score range was 4–17/246. The Schwab and England ADL score ranged from 50 to 100%7.\n\nThe patients were comfortably seated and performed sequences of \"finger-to-nose\" movements cued by acoustic signals. The patients kept their eyes open. The finger-to-nose task consists of touching the nose with the index finger, keeping the index finger on the nose for about one second and then putting it back onto the thigh (starting position). After (1) hearing an acoustic signal, the patient (2) prepared themselves mentally for the execution of movement and (3) performed the task. The dominant arm was studied. During a single run, the task was repeated about 10 times. Patients were first trained in order to perform the task correctly. Each patient executed a maximum of 6 runs. The nomenclature used for the recorded files is “pppFNnn\" standing for patient’s code, task executed (\"Finger-to-nose\") and run number, respectively.\n\nPatients were equipped with:\n\n(i) IMU sensors (inertial measurement units; tri-axial gyroscopes, accelerometers, magnetometers). Two IMUs were located on the anterior face of the upper limb at about 4 cm above and below the elbow, respectively. Sensors were attached with tape.\n\n(ii) a conventional EEG cap with the following location of EEG electrodes (international 10–20 system): FC3, FCz, FC4, C5, C3, C1, CZ, C2, C4, C6, CP3, CPZ, and CP4 (POz: ground; linked ear-lobes: reference). Artifacts were minimized by restraining head movements, keeping the jaw and face relaxed and by avoiding swallowing or blinking during the recordings. Artifact rejection was applied by visual inspection of traces. EEG signals were sampled at 256 Hz (re-sampling at 1000 Hz for synchronization purposes) and band-pass filtered at 0.5–60 Hz.\n\n(iii) EMG multi-array electrodes (arrays of 16 electrodes) located on the flexor carpi radialis (FCR), extensor carpi radialis (ECR), biceps and triceps muscles. EMG data were sampled at 1 KHz.\n\nThe main goal of this module is to identify the beginning and the end of a movement in the time domain. In order to build a “movement window”, the signal from the magnetometer (which provides a very clean signal), is processed first. The delay generated with magnetometers is then corrected with accelerometer and gyroscope signals. This results in an “extended movement window” within a time frame of 500 ms before the “basic movement window” generated by the magnetometer signal alone (Figure 2). Each variation (in accelerometer and gyroscope channels) larger than the standard deviation channel will extend the basic ‘movement window’ until the detected variation. This module, in the multimodal strategy, will be used by all other modules to determine whether a context is well predicting a movement or a false positive is occurring.\n\nThe dotted line corresponds to a voluntary movement.\n\nCortical activation occurring during the preparation of movement is detected by the EEG module thanks to a method based on the event-related desynchronization/synchronization (ERD/ERS) phenomenon6. By considering: (i) the changes in the β²/α and β/α ratio (representing bursts of β-γ frequencies) during the pre-movement period; (ii) an appropriate threshold indicating which peaks of ratios are actually followed by a movement (and therefore may be considered as a predictor of movement); (iii) the number of movements executed, we extracted a QP for the detection of intentionality of movement8.\n\nUpsampled EEG data were processed with a Hamming window of 256 samples, an overlap of 250 in the time domain. Spectrograms were computed at the frequencies from 1 Hz to 40 Hz with the Goertzel algorithm using a short time Fourier Transform (STFT)9. A one-sided power spectral density (PSD) matrix was then obtained with the following formula:\n\n\n\nWhere P contains the PSD of each segment for the frequency range 1–40 Hz, w(n) denotes the Hamming window function and Fs is the sampling frequency (1000 Hz).\n\nThree time intervals were studied: pre-movement period, movement period, post-movement period. The pre-movement period (lasting 2 seconds) was defined according to the acoustic order given to the patients and the detection of the beginning of movement via the gyroscopes.\n\nThe α, β and γ frequency bands were compared by calculating β/α and β²/α ratios. PSD in a β-γ frequency band was divided by the PSD in the α frequency band:\n\n\n\nWhere n = {1,2} depends on the ratio considered (squared or not), f is the interval of β-γ frequencies (e.g.: from 26 to 33 Hz), and f’ is the interval of α frequency (e.g.: from 8 to 10 Hz).\n\nTo extract these sub-bands, the following intervals in the α, β and γ frequency bands were first studied: 8–12 Hz (8 Hz, 9 Hz, 10 Hz, 11 Hz, 12 Hz), 8–10 Hz, 10–12 Hz, 13–40 Hz, 13–26 Hz, 26–40 Hz, 13–20 Hz, 20–26 Hz, 26–33 Hz, 33–40 Hz, 13–16 Hz, 16–20 Hz, 20–23 Hz, 23–26 Hz, 26–30 Hz, 30–33 Hz, 33–40 Hz. Therefore, each β-γ interval was compared with the α intervals. A total amount of 105 pairs of intervals were thus analyzed. By applying (2) to the EEG power spectra from all the EEG channels and the successive runs, we obtained ratiograms which are spectrogram-like representations of EEG activities on the skull. The peaks (β/α and β²/α ratios) higher than a defined threshold were considered as indicators of a potential voluntary movement8, given that they represent the detection of the cortical motor preparation of the movement. To determine the occurrence of false positive results, the number of movements detected was added. QP is the geometric mean of the probability of movement (true positive stimulations) and the percentage of movements predicted8.\n\nA low-pass filter at 30 Hz was applied to EMG data. Corticomuscular coherence is a function of frequency (with values between 0 and 1) and indicates the degree of correlation between the two signals10. The Welch's averaged modified periodogram method was used to compute the magnitude squared coherence of an EEG channel and an EMG electrode along the frequency band6,9,10. More than 800 possible EEG/EMG combinations (n=832) were tested for the cortico-muscular coherence analysis:\n\n\n\nWhere Pxx and Pyy are the PSDs and Pxy is the cross-spectral density, f is the frequency and Cxy is the magnitude squared coherence. The signal was first segmented in 200 ms squared windows. Each window was then processed with an FFT (length 512, window of 8 samples, and overlap of 6).\n\nAs mentioned above, the kinematic module was applied both to characterize tremor and for the early detection of movement in patients presenting with a rest tremor. Up-sampled gyroscope signals (from 50 Hz to 1 KHz) were processed with a Hamming window of 256 samples, an overlap of 250 in the time domain. The spectrogram was computed at the frequencies from 1 Hz to 20 Hz with the Goertzel algorithm using a STFT. A one-sided PSD matrix was then obtained with the following equation:\n\n\n\nWhere P contains the PSD of each segment for the frequency range 1–20 Hz, w(n) denotes the Hamming window function and Fs is the sampling frequency (1000 Hz).\n\nThe prediction of the movement was based on two major features of the pre-movement period:\n\n- a 200 ms gap in the spectrum corresponding to a temporary dramatic decrease of the tremor\n\n- a rise of low frequencies.\n\nThe rise of low frequencies was used for a mathematical modeling which considered:\n\n- the ratio of high frequencies (7–20 Hz) divided by low frequencies (0–7 Hz)\n\n- the max PSD in the low frequency band over time.\n\nA threshold was then applied and the prediction was based upon the following algorithm:\n\nIf (ratio(t) > T and maxPSD(t) > T) then\n\nPotential movement predicted\n\nElse\n\nNo movement detected\n\nEnd\n\nWhere t is the time and T stands for threshold and is defined as the standard deviation of the ratio and the maxPSD.\n\nThe kinematic QP extracted is the following:\n\n\n\nWhere p is the probability of movement and n the number of movements predicted. A QP can be derived for one axis (X or Y or Z) or from several axes combined. Probability of movement p represents a key signal for the BCI-triggered delivery of FES to launch the muscle stimulation. Thus, this parameter is also named “probability of stimulation”. QP is an index of prediction of movement, while the “probability of stimulation/of movement” is the accuracy of this index, corresponding to the true positives.\n\n\nResults\n\nThe EEG QP allowed the prediction of the voluntary movement with a probability between 70% and 90%. The mean QP was 82±12% (median = 83.5%) for the β/α ratio and 79.5±10.4% (median = 80%) for the β²/α ratio. We found no significant difference between the QP calculated from β/α ratio and β²/α ratio (p = 0.502)8. The highest QPs were found when the selected sub-band of frequency included the 30–35 Hz (Figure 3). A sub-band of interest was more difficult to identify for the α band. However, the entire α band and its sub-bands never provided low values of QP. In terms of QP distribution on the scalp, the central areas of the brain showed the highest values of QPs8. The highest probability to predict efficiently the intention of the upper limb movement corresponded to the contralateral central area of the brain.\n\nTwo peaks can be identified for a beta frequency of 20 Hz and 35 Hz. Polynomial fitting (order 3) for an alpha frequency of 11 Hz (blue; R² = 0.9555) and 9 Hz (red; R² = 0.8632).\n\nBy applying the process described in the Corticomuscular module section of the Materials and methods for each EEG channel compared to an EMG electrode, we obtained a graphical representation of the corticomuscular coherence (coherogram, Figure 4). A coherogram can be designed in different ways: either combining all EEG channels with one EMG electrode or associating all electrodes of an EMG device with one EEG channel. Here the first option was chosen because the possibility of a practical implementation of this approach in clinical applications is greater. Statistics for one coherogram channel were obtained by applying a threshold equal to the standard deviation. The same process was then applied on all channels. Data from two patients (3 trials for each patient) were analyzed in-depth (see additional Data Set). Probabilities of stimulation were extracted. For example, the coherence probability reached a maximum of 35.97% for patient 009FN03 (ECR muscle). Figure 5 shows the maximum values from all the possible EEG/EMG channels combinations.\n\nThe black vertical dotted lines correspond to the detected movements.\n\nResults of corticomuscular coherence from patient 001 (presenting a Parkinsonism of vascular origin) and from patient 009 (presenting essential tremor). Three trials were analyzed for each patient.\n\nThe selection of the axis of tremor is extremely important in this module. Indeed, if a patient has a pure mono-axial tremor on x-axis then better results are expected for this axis (as compared to the y- or z-axis). In our group of patients, the y-axis provided better results. Figure 6 shows the results of predictions of movements with the kinematic module. Figure 7 shows a comparison of kinematic QPs for the x-axis, the y-axis, and a combination. Values above 70% were reached for the y-axis. Figure 8 illustrates the probability of stimulation (see the Kinematic module section in Materials and methods). Clear differences between the x-axis and the y-axis were observed.\n\nRed and green lines represent the predictions from the x-axis and y-axis, respectively. The black vertical dotted line corresponds to the voluntary movement. In this case, the movement is predicted approximately 250 ms in advance with the y-axis of the gyroscope (green).\n\nResults for axis x, y and xy combined. Note the better results from channel y.\n\nTo select the appropriate parameters for the BCI, a probability tree was built in order to identify the best associations of parameters. As an example, probability trees from 2 patients are shown in Figure 9. The probability can be extended with several combinations (probabilities for each EEG channel, each EMG electrode or combinations). From the statistical point of view, it is important to note that in some cases the association of several parameters can worsen the prediction of the intentionality of movement, as compared to a single parameter. For example, the association of the channel x and y in the kinematic module yielded lower statistics than the y-axis alone (Figure 9).\n\nEEG QPs and the probabilities of stimulation for the kinematic and the cortico-muscular modules are visualized in a tree form. For each leaf of the tree, a parameter of prediction can be computed. Values are given in %. Once the tree is filled, it is possible to identify the best parameters and associate them.\n\nOne main issue and challenge for the use of a BCI-based on ERD/ERS in neurological patients is to predict whether a given patient would exhibit a sufficient ERD/ERS to be enrolled in therapies based on BCIs. To this aim, we simulated an EEG signal and specifically looked for the relationship between ERD/ERS and QPs. EEG signal was simulated according to a method reported earlier11. The signal generated was a sum of four sinusoids with frequencies chosen randomly from specified ranges of frequencies (delta, theta, alpha, beta, gamma), with a random initial phase. The phase of the oscillations was reset at a specified timing for the simulation. The following parameters were used for generation of EEG signals: sampling frequency of 250 Hz, range of delta band: 0.5–4 Hz, range of theta band: 4–8 Hz, range of alpha band: 8–13 Hz, range of beta band: 13–40 Hz, range of gamma band: 36–44 Hz. Segments of 8 seconds were generated for each of these bands and were superimposed to obtain an artificial EEG trace (Figure 10 and Figure 11). We repeated this procedure to obtain an EEG signal of 32 seconds. The spectrogram was computed with the Goertzel algorithm between 0.5 and 44 Hz (window of 256 samples, overlap of 250). A number of four events of desynchronization (with a duration of the desynchronization period of 2–4 sec for each of them) were introduced. Ratios beta/alpha and beta²/alpha were extracted. Figure 12 illustrates an example of the true positives (in green) for the QP for a threshold of desynchronization set as -4.8 and 9.5 for the simple and squared ratio, respectively. The threshold varied from mean – 10*SD to mean + 10*SD, using steps of 0.1. We performed a simulation for 2875 trials similar to the trial shown in Figure 12. Figure 13 illustrates how QP evolved as a function of the threshold values used. The traces of averaged QPs were characterized by values around 70% (Figure 14). Very interestingly, these values are much greater than the values obtained for the corticomuscular module alone.\n\nExample of spectral analysis of an artificial EEG containing alpha (A), beta (B) and delta theta gamma sub-bands (C). A color-code is used for the representation of spectral densities (bottom panels). Note the red bands corresponding to the highest spectral densities.\n\nBlue trace: ratio beta/alpha; red trace: ratio beta²/alpha.\n\nThreshold varying from mean – 10*SD to mean + 10*SD (steps of 0.1). QP is expressed in %.\n\nContinuous trace: mean values. Dotted lines: mean ± SD.\n\n\n\n\nDiscussion\n\nWe present a novel method to predict the intentionality of movement in neurological patients presenting tremor in the upper limbs. We use a multimodal approach based on the combination of several parameters, in order to decrease the rate of false positive and false negative detections. Starting from the EEG and the kinematic signals, we have extracted a QP, defined as the geometric mean of the probability of movement prediction and the number of movements detected. For the EEG module, the extraction of QP is based on the changes in ratios of sub-bands according to the ERD/ERS phenomenon. We suggest that values equal or higher than 70% correspond to a good QP, as compared to values in the literature12,13. QP values greater than 90% were observed in some of the runs performed by our patients. However, an inter-patient and intra-patient variability was found and further evaluations with a larger number of patients and more runs per patient are required. The complexity of EEG recordings in patients with tremor performing upper limb movements should not be underestimated, especially when tremor genesis involves deep nuclei in the brain. The QP parameter has been defined as a geometric mean in order to force both the true positive stimulation rate (in case of FES application) and the percentage of detected movements to be high enough to obtain a good QP value. Adaptive algorithms could be implemented to take into account variations of the standard deviation and, thus, to adapt to different kinds of activities that have different ratio profiles. We suggest that the choice of the thresholding method and the convenient sub-band ratio for the application of QP in the framework of a BCI-driven system should be made for each patient, depending on the neurological disorder considered. Neuroscience and engineering research support the hypothesis that the inclusion of non-invasive EEG data in the pre-movement period (which corresponds to motor preparation and planning) is useful to reach more effective rehabilitation procedures and to decrease the response time of BCIs14. It is very likely that the design of more advanced neuroprostheses and robot-assisted neurorehabilitation will benefit from EEG-based BCIs15.\n\nThe study of the kinematic data has revealed interesting features in terms of detection of voluntary movements in patients with rest tremor. This tremor occurs mainly in extra-pyramidal disorders such as Parkinson’s disease, which is a very common neurological disorder in the elderly. Assessments of kinematic data per se are particularly interesting because of their simplicity and their direct access without intrusion in the body. Three main neuronal mechanisms have been hypothesized for rest tremor: a cortico-subthalamo-pallido-thalamic loop generating tremor, a pacemaker consisting of the external pallidum and the subthalamic nucleus, and an abnormal synchronization within the whole striato-pallido-thalamic pathway leading to a loss of segregation16. The findings of a decrement of rest tremor before voluntary movement in Parkinson’s disease patients suggest an involvement of a neuronal input from the cerebellum to the thalamus, which may occur sufficiently early to suppress the resting tremor before the voluntary movement8. However, it remains unclear how the understanding of these oscillators in the brain will impact directly on the design of BCIs.\n\nProbability trees show a global visualization of the parameters proposed for the prediction of movement and allow the identification of the best ones (or the best association of them). When all the possible combinations of EEG/EMG/kinematic are tested, the probability tree could yield an optimal efficiency. An exhaustive list of the probabilities for the entire amount of data recorded is not provided, because of the huge amount of time that this analysis requires. The global multimodal plot improves the effectiveness of the system by providing redundant parameters for the prediction of movements: Moreover, it could be particularly helpful during the training phases of the BCI implementation in a given patient. These training phases are known to be time-consuming in some patients.\n\nIn patients responding to FES, we propose a novel closed-loop approach (Figure 15). FES is applied to the upper limbs following the detection of the intentionality to move by the multimodal platform reported here and taking into account the analysis of the QPs, in order to prevent the emergence of tremor just before the start of action. FES is triggered to reduce or cancel tremor. In case of detection of rest tremor by the kinematic sensors, FES is applied accordingly to the muscles in the upper limbs. The parameters of FES (intensity of stimulation, duration of stimuli, modes selected) are adapted according to the severity of tremor and the tolerance. Refractory rest tremor may occur in patients in whom FES is not effective to suppress tremor. In these patients, the on-line multimodal prediction is used to trigger FES once the intentionality to move is detected. Indeed, the various forms of upper limb tremor may react differently to FES due to their distinct pathogenesis.\n\nThe detection of the intentionality to move is based on the quality parameters (QPs) reported here.\n\n\nConclusion\n\nWe suggest a multimodal approach to identify the intentionality of movement. The QP is a promising index in the field of the ERD/ERS-based methods to detect the intention of movement for future BCI applications. This parameter could be also used to process EEG recordings from wearable dry electrodes. Novel wearable devices developed for the treatment of motor disturbances outside the field of neurological tremor might benefit from this approach. We propose that the QP can be complemented by the cortico-muscular coherence and the analysis of the changes in the kinematic signals which occur prior to the voluntary movements. We suggest a fusion of the parameters. Global multimodal plots may become attractive with the development of wearable technologies. They will have to take into account the various pathologies of the central nervous system, especially the localization of the lesions and their course with time. It is very likely that in progressive neurological disorders, the parameters selected in global multimodal plots will have to be modified or adapted accordingly. This is in agreement with adaptive methods which are being developed currently with the goal of improving the classifiers5. Also, our approach will have to be tested in a large sample of patients in the future in order to demonstrate its real clinical usefulness in daily practice.",
"appendix": "Author contributions\n\n\n\nOverall study design and protocol development: GG, MM. Data analysis: GG, MM, JY. Writing of manuscript: GG, MM, JY. Final version reviewed and approved by all the authors.\n\n\nCompeting interests\n\n\n\nGG received fundings (covering the salary) from the European Commission. The authors did not apply for any patent and are not preparing a patent application. No financial return is expected from the present article. The authors declare that they have no non-financial competing interests (political, personal, religious, ideological, academic, intellectual, commercial).\n\n\nGrant information\n\nThe multi-disciplinary research project “Tremor” (ICT-2007-224051) was supported by the European Commission (FP7). The grant was assigned to MM for the contribution of ULB to the research project. GG received a salary from the grant.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nPreliminary results were presented at the annual conference of the Engineering in Medicine and Biology Society, EMBC, 2011, Boston, USA.\n\n\nReferences\n\nLouis ED, Marder K, Cote L, et al.: Differences in the prevalence of essential tremor among elderly African Americans, whites, and Hispanics in northern Manhattan, NY. Arch Neurol. 1995; 52(12): 1201–5. PubMed Abstract | Publisher Full Text\n\nGrimaldi G, Manto M: \"Old\" and emerging therapies of human tremor. Clin Med Insights: Ther. 2010; 2: 169–178. Publisher Full Text\n\nProchazka A, Elek J, Javidan M: Attenuation of pathological tremors by functional electrical stimulation. I: Method. Ann Biomed Eng. 1992; 20(2): 205–24. PubMed Abstract | Publisher Full Text\n\nKinoshita M, Hitomi T, Matsuhashi M, et al.: How does voluntary movement stop resting tremor? Clin Neurophysiol. 2010; 121(6): 983–5. PubMed Abstract | Publisher Full Text\n\nLlera A, van Gerven MA, Gomez V, et al.: On the use of interaction error potentials for adaptive brain computer interfaces. Neural Netw. 2011; 24(10): 1120–7. PubMed Abstract | Publisher Full Text\n\nGrimaldi G, Manto M: Neurological tremor: sensors, signal processing and emerging applications. Sensors. 2010; 10(2): 1399–1422. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGillingham FJ, Donaldson MC: Third Symp. of Parkinson's Disease, Edinburgh, Scotland, E&S Livingstone, 1969; pp.152–157.\n\nGiuliana G, Mario M, Yassin J: A quality parameter for the detection of the intentionality of movement in patients with neurological tremor performing a finger-to-nose test. Conf Proc IEEE Eng Med Biol Soc. 2011; 2011: 7707–10. PubMed Abstract | Publisher Full Text\n\nProakis JG, Manolakis DG: Digital Signal Processing: Principles, Algorithms, and Applications, Upper Saddle River, NJ: Prentice Hall, 1996; pp. 480–481.\n\nTimmer J, Lauk M, Köster B, et al.: Cross-spectral analysis of tremor time series. Int J Bifurc Chaos. 2000; 10(11): 2595–2610. Reference Source\n\nMäkinen V, Tiitinen H, May P: Auditory event-related responses are generated independently of ongoing brain activity. Neuroimage. 2005; 24(4): 961–8. PubMed Abstract | Publisher Full Text\n\nPfurtscheller G, Flotzinger D, Neuper C: Differentiation between finger, toe and tongue movement in man based on 40 Hz EEG. Electroencephalogr Clin Neurophysiol. 1994; 90(6): 456–460. PubMed Abstract | Publisher Full Text\n\nShishkin SL, Ganin IP, Kaplan AY: Event-related potentials in a moving matrix modification of the P300 brain-computer interface paradigm. Neurosci Lett. 2011; 496(2): 95–9. PubMed Abstract | Publisher Full Text\n\nAhmadian P, Cagnoni S, Ascari L: How capable is non-invasive EEG data of predicting the next movement? A mini review. Front Hum Neurosci. 2013; 7: 124. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLew E, Chavarriaga R, Silvoni S, et al.: Detection of self-paced reaching movement intention from EEG signals. Front Neuroeng. 2012; 5: 13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDeuschl G, Raethjen J, Baron R, et al.: The pathophysiology of parkinsonian tremor: a review. J Neurol. 2000; 247(Suppl 5): V33–48. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "2881",
"date": "03 Jan 2014",
"name": "Christoph M Michel",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript evaluates the use of a combination of neurophysiological signals to detect movement intention in tremor patients. Multichannel scalp EEG, multi-array MEG and kinematic sensors were jointly analyzed in a trigger-initiated finger-to-nose movement task. The aim was to optimize detection of movement initiation. Four patients with upper limb tremor were studied.The paper is technically sound and the analysis methods are clear. However, some of the analyzed parameters are irrelevant, redundant or highly correlated, making it difficult to understand why they were all performed separately. I have the following major comments: EEG analysis of Beta-Alpha ratio was analyzed in 105 different combinations of frequency bands that were highly overlapping. The rational for this fine distinction of different frequency bands is not very clear given the existing (but not cited) literature on event-related synchronization/desynchronization (work by Pfurtscheller, for example). While not explicitly stated, I guess that these 105 combinations were done for each electrode. Studying the existing literature would have allowed the authors to restrict to the known electrodes and frequency-bands of interest. EEG-MEG coherence analysis: each EEG channel is correlated with one MEG channel. Figure 4 shows very similar results for each electrode. This is not surprising given that each EEG electrode measures part of the activity of the same generators in the brain. Volume conductance makes the different EEG signals highly correlated when referred to a common reference and thus they are all correlated with the MEG waveform. It is not clear whether the results are specific to tremor patients - a control group is missing. The results are not clear. One would have expected a conclusion that informs the reader which of the multiple parameters seem to be most promising. The presented probability trees are very abstract and do not allow one to make a conclusion on specific parameters. The Table at the end of the manuscript is not clear. What do the different numbers represent? The abstract should not be a verbatim copy of the Introduction. A better description of the literature on ERD in BCI is needed in the Introduction. Important work is missing.",
"responses": [
{
"c_id": "790",
"date": "24 Apr 2014",
"name": "Mario Manto",
"role": "Author Response F1000Research Advisory Board Member",
"response": "We thank the referee for their criticisms.1. “EEG analysis of Beta-Alpha ratio was analyzed in 105 different combinations of frequency bands that were highly overlapping. The rational for this fine distinction of different frequency bands is not very clear given the existing (but not cited) literature on event-related synchronization/desynchronization (work by Pfurtscheller, for example). While not explicitly stated, I guess that these 105 combinations were done for each electrode. Studying the existing literature would have allowed the authors to restrict to the known electrodes and frequency-bands of interest.”We have added the following sentences in the Discussion:“Our protocol in neurological patients with tremor differs from those in the literature, hence our study on the multiple combinations of frequency bands. When a neurological patient with tremor is seated and assessed, he/she may exhibit a tremor of the head and trunk. This tremor may be pretty stable or rather intermittent. There may even be an overlap with the main frequencies of the EEG signal, for instance in the alpha band (a rapid head tremor may be found). Therefore, we decided to have a close look to each of these bands. For instance, we have seen patients with cerebellar disorders and orthostatic tremor in whom the sub-band 8-10 Hz was much less informative as compared with the sub-band 10-12 Hz. We would like to point out that in the study of Pfurtscheller et al. on single-trial classification of EEG and imagination (Neuroimage. 2006;31(1):153-9), the frequency of the most reactive components was 11.7 +/- 0.4 Hz (mean +/- SD). The SD was thus small. Although the desynchronized components were centered at 10.9 Hz +/- 0.9 Hz, the synchronized components were narrow-banded, with higher frequencies at 12.0 Hz +/- 1.0 Hz. We agree with the authors that the classification of single EEG trials improves when ERD and ERS patterns are combined for multiple tasks. We aim to pursue the use of narrow bands of frequency in multiple tasks.”2. “EEG-MEG coherence analysis: each EEG channel is correlated with one MEG channel. Figure 4 shows very similar results for each electrode. This is not surprising given that each EEG electrode measures part of the activity of the same generators in the brain. Volume conductance makes the different EEG signals highly correlated when referred to a common reference and thus they are all correlated with the MEG waveform.”The combinations of EEG/EMG were done for each electrode. We agree that in theory the volume conductance effect and the common generators could impact on the coherence analysis in healthy subjects. The rationale is the following: EEG signals taken from nearby areas may be very different in patients with brain lesions (the density of MEG channels is also much higher as compared to our set-up). This is due to structural lesions in the brain. These MRI images (T2-weighted axial sections) illustrate the subcortical lesions in a patient with Parkinsonism of vascular origin. The lesions disrupt the subcortical tracts of the periventricular white matter. The attempt to translate data obtained in healthy subjects cannot be efficient. In these patients, a case-by-case BCI scenario is required. We want also to point out that fMRI studies show an activation of selected areas of the homunculus during limb movements. Hence also the selection of the central area for the EEG channels (see the reply to Yvonne Hoeller). Several groups working on BCIs are now attempting to focus on the EEG signals located nearby the pre-central sulcus. We would like to stress that our patients do exhibit tremor (including of the head-trunk), unlike control subjects. The algorithm suggested for tremor suppression requires tremor. It might be interesting to investigate patients showing tremor likely induced by a disruption of the peripheral nervous system, and to compare the results with those obtained in patients showing brain lesions.This figure illustrates an example of the results of ERD/ERS in a patient with post-traumatic tremor (multiple brain lesions) pressing on a force transducer during a pinch task of the right hand. The patient is seated in front of a computer: (a) baseline measurement (rest) during 2 seconds, followed by (b) the patient prepares himself by mental imagery for 4 seconds, and (c) the patient presses a force transducer during 2 seconds. The channels C3-Cz and FC3-Cz are compared. The relative results for the main sub-bands (delta, theta, alpha, beta) are shown. Note the clear difference between the ratio beta/alpha. The ERD/ERS is much stronger for C3-Cz as compared to FC3-Fz.3. “It is not clear whether the results are specific to tremor patients - a control group is missing.”Our project focused on tremor patients and not on healthy subjects. This has been addressed by other partners of the project. We would like to point out that the attempt to translate findings in healthy subjects to neurological patients with brain lesions is very likely to be poorly productive. See also the reply to the previous query.“The results are not clear. One would have expected a conclusion that informs the reader which of the multiple parameters seem to be most promising. The presented probability trees are very abstract and do not allow one to make a conclusion on specific parameters.”We have modified the Discussion as follows:“Our data provide a ground for the concept of multimodal approach developed for the early detection of the intentionality of movement. The presented probability trees are general schemes. A case-by-case analysis is required. In order to provide the most possible accurate BCI-driven FES system, each subject needs to be studied in order to define the best combination of QPs. For instance the kinematic QPs may be more efficient than the EEG QPs in a given patient (as it may happen when ERD/ERS is not stronger enough to be detected). The system would take into account these features. By analysing a larger group of patients, we might identify subgroups of patients on the basis of the results of the probability trees. In other words, the probability trees would be used as an eligibility procedure to multimodal BCI-driven treatments in neurological patients with tremor”.- “The Table at the end of the manuscript is not clear. What do the different numbers represent?”Data from experiments have been included, so that the referee or the reader can compare them with his/her own software.- “The abstract should not be a verbatim copy of the Introduction.”The introduction has been re-written.4. “A better description of the literature on ERD in BCI is needed in the Introduction. Important work is missing.”The following references have been added: · Pfurtscheller and Lopes da Silva (1999)· Pfurtscheller et al. (2006)· Birbaumer et al. (2006)"
}
]
},
{
"id": "2883",
"date": "13 Jan 2014",
"name": "Yvonne Hoeller",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article presents a highly promising idea of using a multimodal control unit for FES to treat tremor during movement. I think this is a valuable contribution to the literature since the possible application (closed-loop FES) is an auspicious field of treatment research.The manuscript is well-written and technically sound. However, due to lack of clarity I have several questions regarding the methods and the results. Moreover, given the exhaustive analysis of EEG frequency ratios and EEG-MEG coherence, I would suggest applying some feature selection methods in order to overcome the problem of multidimensional data, which is hard to present and to interpret.AbstractThe sentence \"A BCI-driven detection of voluntary movement is used to trigger FES in a closed-loop approach.\" sounds to me like you have used FES in this study. Perhaps it would be less misleading if you wrote this in the subjunctive.IntroductionIn the introduction you emphasize the limitations of BCIs, which are based on conventional EEG analysis. Although I fully agree with you, this is a rather unspecific statement. Does this mean that \"conventional\" EEG analysis is less reliable for BCI-performance than \"high-tech\" EEG-analysis? Although we both know that this is not the case (BCIs are fed with highly sophisticated markers of the EEG and still do not perform at 100%), the sentence could be understood in this way. Therefore, I would suggest that you specify that you mean BCIs which ware based on EEG analysis only.I would like to make a further point on this: BCI users differ between each other in the way they can voluntarily modify their brain activity, and there is even an interindividual difference in the detectability of movement-related EEG-activity. Moreover, the fact that movement disorders often show up with brain atrophy or neuroplastic changes makes it difficult to generalize BCI markers that have been tested in a healthy population to patients with varying pathologies. I think this variance is a major reason why your multimodal processing could do so much better than a BCI-system that is solely based on EEG analysis.Methods(A) I don't think that the sentence \"Patients and the experimental procedure were as detailed in the next sections.\" in the first paragraph of the Methods section is necessary (the reader sees that there is a section for patients etc.) (A) Maybe in this first paragraph it would be nice to read that the different modules act during different time-windows. (B) Why did you prefer acoustically triggered movement over self-paced movement? How did you ensure that the acoustic signal did not influence the EEG signal which was used for detection of movement planning? (B) Why did you restrict your EEG-setup to centrally positioned electrodes? Movement planning involves frontal regions (e.g. electrodes F3, F4, F7, F8) and it is highly likely that you get a better prediction of movement if you include signals from these positions. (B) How did you avoid blinking during the recordings? If you tell the participants that they should not blink, they might concentrate on this instruction instead on the task, making EEG-data less valid. In addition, participants who are told not to blink usually blink more frequently than if you would not mention blinks. (Fig. 2) I did not understand the reason why you only used three colors for the lines in figure 2. The reader can distinguish the three cases but not which sensor is represented by what line. Moreover, the first blue line, entitled \"Mouvement...\" should be entitled Movement... and should be a dashed line as indicated below the figure. Are the three cases three different sensors, three patients or three movements? (D) You write \"upsampled EEG data...\" and indicate that there were 256 samples - is this one second? Does an overlap of 250 in the time domain refer to the original sampling rate or to the upsampled data, resulting in 250ms? Did I get this right; the Hamming window was 1 sec but overlapped with 250ms? (D) I do not exactly understand how you realized that the pre-movement period lasted for 2 sec, but participants performed the task following the acoustic signal. Were participants instructed to wait 2 sec? Or is this the average time the participants took until they performed the task? (D) I did not understand what you intended by the (8 Hz, 9 Hz, ... 12 Hz) after the first interval. (E) Possibly because this is the first time I have read about this very interesting method of correlating brain and muscle signals - I did not fully understand the segmentation/processing of the data. You segmented data into epochs of 200ms and calculated the FFT on this window - what then does the \"window of 8 samples\" mean? Moreover, how did you determine the optimal window length? I see that the epochs vary between the modules.Results(A) Why do you refer to reference no. 8 when you report your results? Aren't these your present results? (A) The central areas showed the highest values of QPs - indeed, you used only a central montage? (A) You talk about means - obviously, you may indicate averages over patients, but did you also average over electrodes? (Fig. 3) What electrode positions are used to build this figure? (Fig. 4) The vertical dashed lines seem to occur periodically (each 20ms) with a certain time-distance but not necessarily on the highest peaks - do you have an interpretation/explanation for this phenomenon? (Fig. 5) With what rationale were the 2 patients and 3 trials chosen? (Fig. 6) It would be helpful to rescale the x-axis, so that the beginning of movement is time 0 and then indicate steps of +-250ms. (Fig. 9) What is the EEG/CorticoMuscular probability based on - averages across channels/channel combinations or one single channel/combination (the best one?)? (Fig 14) There are not only continuous and dotted lines but also external lines with \"big dots\" - is this the range (min-max)?Supplementary dataWhat do the rows in e.g. 001FN03.mat_EE...csv mean? Are these samples?DiscussionYou state that QP values greater than 90% were observed in some of the runs...That is, QP was calculated for each run? This is not clear from the methods section. Why are EEG recordings more complex when tremor is generated in nuclei deep in the brain? In the discussion of kinematic data it seems to me that you discuss the background generally without referring to your own results. You should rather discuss why the y axis alone is superior to x axis or a combination of the two. Do probability trees really show the best association of parameters? In a subsequent sentence you write that all possible combinations need to be tested. To me probability trees as used in this paper seem to give the impression of how complex the problem is, but they do not answer the question: Which feature/combination of features leads to highest detection rates? This problem is solved by feature selection algorithms (see general remark 2). I am not an FES-expert, but wonder if there is any literature about the instance of time when FES has to be applied in order to make a movement free of tremor. Should it be applied at movement onset, or before the movement is carried out, i.e., in the planning phase? It probably depends on each individual patient, since tremor can occur pre- movement or during movement. Thus, the kind of FES should be different for each patient and similarly, depending on the type of application, the EEG-part may or may not be useful. Also if planning the action induced the tremor, detection of movement intention based on EEG could be too late? There is no discussion about the simulation results.ConclusionIn the conclusion it seems that the QP is an EEG-specific value which can be complemented by values from other modules (kinematic...) - instead, the QP is a value for estimating the performance in detecting movement and can be applied to each modality. But is this really a main conclusion? I think the conclusion should be that the EEG can be supplemented by other modalities. However, the extent to which the detection rate of the EEG can be supplemented, i.e., how much the QP can be increased by combining several modalities, has not been evaluated. The last minus one sentence: The term classifiers comes out of the blue. This technique deserves some place in the discussion (as suggested in general remark 2). Last sentence: The approach has firstly to be implemented - the present manuscript is a pre-study. The next step is to integrate all of the modalities. Then you could evaluate the approach on a large sample.General remarksYou use BCI in terms of a control unit. BCI is an interface between brain and computer. As such, your system would be a multimodal control unit, including also an EEG-module like often used for BCIs, but also body modules to control a stimulation unit. I think the term BCI is not the best choice here, since it does not take into consideration the EMG module etc. The way such a problem would be solved by the current BCI-community is a classification by use of machine learning techniques i.e., you apply some feature selection algorithm and train your machine in discriminating movements based on the multimodal input. These two steps could further be included in one step e.g. by use of random forests. By doing so you evaluate the performance of your modules and find out which ones contribute most to a high detection rate - separately for each patient (thus, you take into account interindividual variability). I would at least include this option in the discussion. I think the data presented here could help to determine what detection rate is possible by combining several modalities. This is what the reader could expect when they read the abstract and the introduction. Then, the conclusion could be that combining several modalities increases the detection rate or it does not change it or it decreases the detection rate (I expect that it increases it). Instead, the authors presented the detection rate by calculating a detection-rate affine measure (QP) separately for each module, and in the conclusion the authors just suggest to fusion the parameters. I think a deeper analysis (e.g. by using random forests...) could answer the very important question of how to increase EEG-based detection of movement intention by including information from other modules. I see that the authors want to leave this important question for future publications, but the abstract and the introduction should be clear in determining the aim of this study. The authors separately evaluated the value of multimodal parameters, in order to determine if it could be interesting to integrate them in one system. The presentation of the results is rather descriptive, the authors report QP, SDs etc. but no statistical test is applied. It would have been interesting if the QPs differ between modalities, within modalities with respect to electrode locations etc. I think some statistical evaluation could allow more concrete conclusions to be drawn. Furthermore, a feature-selection algorithm can be based on some statistics. I would suggest having a look at the recent BCI-research which provides many ideas on how to reduce multidimensional data. The cited references in the introduction and discussion are reduced to a minimum. I would suggest doing some extensive research on movement-related EEG studies etc.",
"responses": [
{
"c_id": "786",
"date": "24 Apr 2014",
"name": "Mario Manto",
"role": "Author Response F1000Research Advisory Board Member",
"response": "We thank the referee for their criticisms. “Moreover, given the exhaustive analysis of EEG frequency ratios and EEG-MEG coherence, I would suggest applying some feature selection methods in order to overcome the problem of multidimensional data, which is hard to present and to interpret.”We have attempted to simplify the analysis, but unfortunately simplification would result in poorly informative findings. The field of BCI is highly complex and data often require an exhaustive approach in order to make BCIs really applicable to neurological patients. These patients are often heterogeneous in terms of mental imagery (see also the reply to Christoph M Michel) and a simple scheme could not be applicable to more than a single patient. This would mean that very few patients would benefit from BCI-based programs.Abstract“The sentence \"A BCI-driven detection of voluntary movement is used to trigger FES in a closed-loop approach.\" sounds to me like you have used FES in this study. Perhaps it would be less misleading if you wrote this in the subjunctive.”The relevant paragraph of the abstract has been changed to the following:“The main purpose of this challenging project was to develop and validate a new treatment for upper limb tremor based on the combination of functional electrical stimulation (FES; which has been shown to reduce upper limb tremor) with a brain-computer interface (BCI); thus providing a BCI-driven detection of voluntary movement which can be used to trigger FES in a closed-loop approach.”Since this article’s publication, our global concept has now been validated by the MENRVA Group who used an affordable, BCI-controlled, wearable robot and electrical stimulation to assist subjects in drinking a glass of water. In this very recent study, the set-up required an EEG headset on the head of subjects along with FES electrodes on their arm. An embedded potentiometer was used to measure wrist rotation angle whereas an encoder in the elbow motor was used to quantify elbow angle. A gyroscope was used for the shoulder (Looned et al., 2014). The general approach of the project has been presented earlier (Rocon et al., 2010).Introduction“In the introduction you emphasize the limitations of BCIs, which are based on conventional EEG analysis. Although I fully agree with you, this is a rather unspecific statement. Does this mean that \"conventional\" EEG analysis is less reliable for BCI-performance than \"high-tech\" EEG-analysis? Although we both know that this is not the case (BCIs are fed with highly sophisticated markers of the EEG and still do not perform at 100%), the sentence could be understood in this way. Therefore, I would suggest that you specify that you mean BCIs which ware based on EEG analysis only.”The word “only” has been added. As stated previously, recent studies point out that for some specific tasks a conventional affordable EEG can be used (Looned et al., 2014). We do not want to reduce the importance of EEG. “I would like to make a further point on this: BCI users differ between each other in the way they can voluntarily modify their brain activity, and there is even an interindividual difference in the detectability of movement-related EEG-activity. Moreover, the fact that movement disorders often show up with brain atrophy or neuroplastic changes makes it difficult to generalize BCI markers that have been tested in a healthy population to patients with varying pathologies. I think this variance is a major reason why your multimodal processing could do so much better than a BCI-system that is solely based on EEG analysis.”We have added these sentences to the article.Methods“I don't think that the sentence \"Patients and the experimental procedure were as detailed in the next sections.\" in the first paragraph of the Methods section is necessary (the reader sees that there is a section for patients etc.)”The sentence has been removed. “Maybe in this first paragraph it would be nice to read that the different modules act during different time-windows”The paragraph has been changed to the following:“From each module, acting during different time-windows, (EEG, Kinematic and corticomuscular - described in detail in sections C-F) quality parameters (QPs) for the detection of the intentionality of movement or for the early detection of movement are extracted.” “Why did you prefer acoustically triggered movement over self-paced movement? How did you ensure that the acoustic signal did not influence the EEG signal which was used for detection of movement planning”This protocol did not generate artefacts as a consequence of the acoustic signal. Indeed, after the acoustic signal the subject first relaxes, then prepares the movement and subsequently executes it. This protocol provides a defined time window during which influences on EEG are minimized. Several groups in the world working with patients have observed that patients tend to execute automatic movements (probably with a smaller contribution of the cortical potential) when the task is self-paced. This is in contrast with experimental findings in control subjects (Niazi et al., 2012). Providing an acoustic cue is particularly interesting in Parkinson’s disease (Lohnes and Earhart, 2011). It is plausible that a visual or somatosensory cue could also be used (Rochester et al., 2007). “Why did you restrict your EEG-setup to centrally positioned electrodes? Movement planning involves frontal regions (e.g. electrodes F3, F4, F7, F8) and it is highly likely that you get a better prediction of movement if you include signals from these positions”.Our ultimate aim is to propose a wearable EEG cap with a few channels, in order to reach the goal of a BCI which could be used in daily life (outside the laboratory and the clinic) by neurological patients. Using 14 electrodes, we have detected the ERD/ERS phenomenon. The conventional caps which include the prefrontal electrodes require an important signal processing step, because of the artefacts generated - in particular by the frontalis muscle. This muscle often contaminates the EEG traces in neurological patients. Note McFarland et al. (2000)’s study which focused on the central areas of the brain for the principal component analysis related to motor imagery and movement. Schröder et al. (2003) have shown that choosing physiologically motivated channels improves classification accuracy when compared to all-channels. The choice of a subset of EEG channels is one of the selection features for the classification of EEG signals in BCI systems in order to avoid dealing with high dimensional and noisy data. “How did you avoid blinking during the recordings? If you tell the participants that they should not blink, they might concentrate on this instruction instead on the task, making EEG-data less valid. In addition, participants who are told not to blink usually blink more frequently than if you would not mention blinks”Patients were told not to blink only in the period after the acoustic signal until the end of the movement. They were free to blink or swallow after the end of the movement until the successive acoustic signal. Therefore, we did not encounter difficulties related to blinking prevention.“I did not understand the reason why you only used three colours for the lines in figure 2. The reader can distinguish the three cases but not which sensor is represented by what line. Moreover, the first blue line, entitled \"Mouvement...\" should be entitled Movement... and should be a dashed line as indicated below the figure. Are the three cases three different sensors, three patients or three movements?”We have modified Figure 2. “You write \"upsampled EEG data...\" and indicate that there were 256 samples - is this one second? Does an overlap of 250 in the time domain refer to the original sampling rate or to the upsampled data, resulting in 250ms? Did I get this right; the Hamming window was 1 sec but overlapped with 250ms?”Data were upsampled from 256 samples to 1000/sec. Subsequently, signal processing is applied to these upsampled data. “I do not exactly understand how you realized that the pre-movement period lasted for 2 sec, but participants performed the task following the acoustic signal. Were participants instructed to wait 2 sec? Or is this the average time the participants took until they performed the task?”The acoustic signals indicate the beginning of the test. After hearing the signal, the patients relaxed their face (no more blinking, swallowing....), prepared by mental imagery, and then executed the movement. In the study of Defebvre et al. (1999) desynchronization of EEG was recorded 2 s before to 0.5 s after voluntary wrist flexions from 11 leads covering the primary sensorimotor cortex (central), supplementary motor area (frontocentral) and parietal cortex (parietocentral). This has been added to the Methods. “I did not understand what you intended by the (8 Hz, 9 Hz, ... 12 Hz) after the first interval.”These are frequencies of interest that are used for the analysis. “Possibly because this is the first time I have read about this very interesting method of correlating brain and muscle signals - I did not fully understand the segmentation/processing of the data. You segmented data into epochs of 200ms and calculated the FFT on this window - what then does the \"window of 8 samples\" mean? Moreover, how did you determine the optimal window length? I see that the epochs vary between the modules.”This is a conventional method to calculate the FFT on epochs of data. According to the module used, the window may vary because the window is adapted to obtain the more relevant spectral information (see the details in McNames (2013)). The values used are based on our experience with signal processing in movement disorders.Results “Why do you refer to reference no. 8 when you report your results? Aren't these your present results?”The present study provides a detailed analysis and novel data as compared to the previous reference, which corresponded to a conference presentation. In addition, the present article includes the simulation study which is novel and was not published earlier. “The central areas showed the highest values of QPs - indeed, you used only a central montage?”We represent here the QPs that reached a good value of accuracy. “You talk about means - obviously, you may indicate averages over patients, but did you also average over electrodes?”No, we did not average the traces over the electrodes. This results in a lower spatial resolution, especially in neurological patients with focal brain lesions or disorders. We have observed that individual channels provide more meaningful information as compared to averages for the task of finger-to-nose. Other studies have been performed in the framework of this project and have not shown a superiority of averages (they rather showed a lower performance when all the channels are averaged). “(Fig. 3) What electrode positions are used to build this figure?”These data correspond to the central area of the brain. This has been added to the revised article. “(Fig. 4) The vertical dashed lines seem to occur periodically (each 20ms) with a certain time-distance but not necessarily on the highest peaks - do you have an interpretation/explanation for this phenomenon?”This is well known for the cortico-muscular coherence. This is why some groups use a flexible window in their software to extract the best cortico-muscular coherence. There are several explanations: the variability in the preparation of movement, the variability in the corticospinal tract command from trial-to-trial, the variability in the muscle contraction from trial-to-trial and the electro-mechanical delay in muscle contraction (see Jenkins, Palmer and Cramer (2013) and Howatson et al. (2009)). “(Fig. 5) With what rationale were the 2 patients and 3 trials chosen?”These patients exhibited reproducible low values for the cortico-muscular coherence, by contrast to reproducible high values for the QP. This highlights the importance of our approach. This has been added to the revised article. “(Fig. 6) It would be helpful to rescale the x-axis, so that the beginning of movement is time 0 and then indicate steps of +-250ms.”It might be interesting to keep the same presentation in order to highlight the prediction phase from the post-movement period. “(Fig. 9) What is the EEG/CorticoMuscular probability based on - averages across channels/channel combinations or one single channel/combination (the best one?)?”The combinations of EEG/EMG were done for each electrode. Averages across channels decrease the values obtained, and therefore reduce the strength of the study. This is in agreement with the current wearable EEG caps, which include a few selected channels only (and this is also a better approach in terms of ergonomics and aesthetics). “(Fig 14) There are not only continuous and dotted lines but also external lines with \"big dots\" - is this the range (min-max)?”External lines with larger dots indicate the 95% confidence interval. This has been added in the legend of the figure in the revised article.Supplementary data\"What do the rows in e.g. 001FN03.mat_EE...csv mean? Are these samples?\"Yes, these are samples. The referee and the reader can use the data to extract QPs and compare with other methods they might develop in the future.Discussion“You state that QP values greater than 90% were observed in some of the runs...That is, QP was calculated for each run? This is not clear from the methods section.”QPs were calculated for each movement executed by the patients. One run contains several movements. QPs extracted showed good accuracy (fixed for values higher that 70%), with some QPs which were extremely good (accuracy greater than 90%). The methods section has been modified accordingly in the revised version of the article. “Why are EEG recordings more complex when tremor is generated in nuclei deep in the brain?”Because EEG activity is mainly driven by the cortical brain electrical activity, which is detectable through the scalp. Activities generated in depth, for instance in basal ganglia (putamen, caudate nucleus…), are very weakly recorded with conventional EEG electrodes. They require the use of invasive deep brain electrodes. The same observation can be put forward for tremor related to lacunar stroke. “In the discussion of kinematic data it seems to me that you discuss the background generally without referring to your own results. You should rather discuss why the y axis alone is superior to x axis or a combination of the two.”We have added one sentence explaining that there is very often a so-called dominant axis which provides the most meaningful information. For instance, in cases of cerebellar kinetic tremor, the vertical axis perpendicular to the direction of motion is the most meaningful (relevant for kinetic tremor). “Do probability trees really show the best association of parameters? In a subsequent sentence you write that all possible combinations need to be tested. To me probability trees as used in this paper seem to give the impression of how complex the problem is, but they do not answer the question: Which feature/combination of features leads to highest detection rates? This problem is solved by feature selection algorithms (see general remark 2).”The best association of QPs is a case by case decision. We confirm that the probability trees can be used to extract the best combination. However, these probability trees have been built for a given task and it is very likely that the ultimate choice will rely on the selection of the best probability trees related to a given number of selected tasks representative for daily life. A specific study should address this very interesting but very complex question. “I am not an FES-expert, but wonder if there is any literature about the instance of time when FES has to be applied in order to make a movement free of tremor. Should it be applied at movement onset, or before the movement is carried out, i.e., in the planning phase? It probably depends on each individual patient, since tremor can occurr pre- movement or during movement. Thus, the kind of FES should be different for each patient and similarly, depending on the type of application, the EEG-part may or may not be useful. Also if planning the action induced the tremor, detection of movement intention based on EEG could be too late?”Muscular FES induces mainly a peripheral suppression of tremor, since electrical stimulation exerts its effect on the activities of the agonist and antagonist muscles of the trembling limb, rather than on the central sources of tremor. Our aim is to trigger the electrical stimulator on the basis of an early detection of the intention of movement. FES appears as a viable option to suppress the different forms of tremor. Several articles have been published on this topic. For instance, our group has now demonstrated that FES is effective to block physiological tremor - Grimaldi, Fernandez and Manto (2013). The intensity of stimulation and the pattern of stimulation can be adapted as a function of the percentage of tremor reduction. “There is no discussion about the simulation results.”The following sentences have been added to the revised version of the article:“Results obtained with the simulation study provide useful information about EEG QP in order to select patients more effectively for a BCI-based treatment, including rehabilitation. The simulation demonstrates the relationship between the threshold and the QP. Future studies could take advantage of these findings to select the best neurological candidates on the basis of the ERD/ERS for BCI-based management.\"Conclusion“In the conclusion it seems that the QP is an EEG-specific value which can be complemented by values from other modules (kinematic...) - instead, the QP is a value for estimating the performance in detecting movement and can be applied to each modality. But is this really a main conclusion? I think the conclusion should be that the EEG can be supplemented by other modalities. However, the extent to which the detection rate of the EEG can be supplemented, i.e., how much the QP can be increased by combining several modalities, has not been evaluated.”The paragraph has been changed to the following:“We propose that the EEG QP can be complemented by the QPs extracted from the cortico-muscular coherence and the QPs obtained by the analysis of the changes in the kinematic signals, which occur prior to the voluntary movements. We suggest a fusion of the QP parameters in order to increase the likelihood to detect the intentionality of movement. The analysis of the corticomuscular coherence shows that this parameter alone cannot be used to predict voluntary motion and be implemented in a BCI”. Some studies focusing only on corticomuscular coherence have failed. “The last minus one sentence: The term classifiers comes out of the blue. This technique deserves some place in the discussion (as suggested in general remark 2). ”The word “classifier” has been changed to:“classification algorithms for BCI system in order to extract EEG patterns related to a cognitive or motor status”.The following reference has been added: Pfurtscheller et al. (1996). “Last sentence: The approach has firstly to be implemented - the present manuscript is a pre-study. The next step is to integrate all of the modalities. Then you could evaluate the approach on a large sample.”The sentence has been changed to:“Our approach will have to be tested in a large sample of patients in the future, in order to demonstrate its real clinical usefulness in daily practice. We propose to select a larger group of neurological patients to confirm the strength of the multimodal prediction.” in the revised version of the article.General remarks“You use BCI in terms of a control unit. BCI is an interface between brain and computer. As such, your system would be a multimodal control unit, including also an EEG-module like often used for BCIs, but also body modules to control a stimulation unit. I think the term BCI is not the best choice here, since it does not take into consideration the EMG module etc.”The terminology is used in the literature, but we understand the point made by the referee. In the revised version of the Discussion, we have introduced the concept of a “multimodal control unit”:“In theory, BCI is an interface between brain and computer. As such, our system would be a multimodal control unit, including also an EEG-module like often used for BCIs, but also body modules to control a stimulation unit.” “The way such a problem would be solved by the current BCI-community is a classification by use of machine learning techniques i.e., you apply some feature selection algorithm and train your machine in discriminating movements based on the multimodal input. These two steps could further be included in one step e.g. by use of random forests. By doing so you evaluate the performance of your modules and find out which ones contribute most to a high detection rate - separately for each patient (thus, you take into account interindividual variability). I would at least include this option in the discussion.”This is a great idea. We have added the following sentence to the Discussion:“Future works could apply some feature selection algorithm and train the multimodal control unit in discriminating movements based on the multimodal input. These two steps could further be included in one step e.g. by use of random forests. By doing so, the performance of the modules would be evaluated to find out which ones contribute most to a high detection rate. This would be done separately for each patient, thus taking into account the inter-individual variability.” “The way such a problem would be solved by the current BCI-community is a classification by use of machine learning techniques i.e., you apply some feature selection algorithm and train your machine in discriminating movements based on the multimodal input. These two steps could further be included in one step e.g. by use of random forests. By doing so you evaluate the performance of your modules and find out which ones contribute most to a high detection rate - separately for each patient (thus, you take into account interindividual variability). I would at least include this option in the discussion.”We have added the following sentence to the Conclusion:“The present study opens the door for future studies in terms of how to increase EEG-based detection of movement intention by incorporating information from multiple modules.”We feel that we respond partially to this critical question, for instance by showing that a cortico-muscular module cannot provide a good evaluation of the intentionality of movement (some groups still try to rely on this parameter to detect the preparation of movement and to manipulate an effector). “The presentation of the results is rather descriptive, the authors report QP, SDs etc. but no statistical test is applied. It would have been interesting if the QPs differ between modalities, within modalities with respect to electrode locations etc. I think some statistical evaluation could allow more concrete conclusions to be drawn. Furthermore, a feature-selection algorithm can be based on some statistics. I would suggest having a look at the recent BCI-research which provides many ideas on how to reduce multidimensional data.”We have added the references below and have included the following sentence in the Discussion:“Techniques of multichannel EEG compression, phase congruency and graphical representations aiming at a reduction of multidimensional data have been proposed [Gasser and Möcks, 1983; Logesparan and Rodriguez-Villegas, 2010; Dauwels et al. 2013]. However, no technique has been widely accepted so far.”Gasser and Möcks (1983)Logesparan and Rodriguez-Villegas (2010)Dauwels et al. (2013)The following references have been added:Rektor, Sochůrková and Bocková (2006)Shibasaki and Hallett (2006)"
}
]
},
{
"id": "3358",
"date": "27 Jan 2014",
"name": "Carlos M. Gomez",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis report tries to identify EEG, EMG and kinematics features that permit prediction of movement production, in order to use functional electrical stimulation (FES) to compensate for tremor in neurological patients. The rational and the methodology is sound, but several points must be clarified in order to improve the validity of the result and to increase readability of the results of these important and worthy results, not only for academics but also in the clinical settings. The queries can be answered, clarified or considered for more advanced reports in the future.Introduction:In Figure 1 the dashed line seemed to indicate the movement, but how it is possible that the movement itself is predicted by the movement kinematic - that should be zero before movement. Except if some thresholds are defined, pre-thresholds movements are the predictors. Please clarify the sentence \"decrement of the rest tremor before movement onset\" - does it means that the reduction in tremor is not the initial voluntary movement but a different neural command which is being inhibited by the voluntary control? Then define physiological characteristics, frequency amplitude, of tremor. Otherwise it would be difficult to distinguish between the reduction of tremor and the initial phases of movement. Methods: \"Mean age of the patients was 62 ± 20 years\" . It would be more precise to describe the age of each individual subjects. Means are useful for high number of subjects. Same for other parameters. \"After (1) hearing an acoustic signal, the patient (2) prepared themselves mentally for the execution of movement and (3) performed the task.\" It is not clear how long it takes this preparation. Was it induced by the experimenter instructions or it was an spontaneous strategy? How long does each of the phases take? Is it neccesary to indicate the files code? Otherwise please suppress it. \"EEG signals were sampled at 256 Hz (re-sampling at 1000 Hz for synchronization purposes). \" It seems that the data were re-sampled to a higher frequency. Could it be considered an interpolation rather than a re-sampling? Anyway it would be better to sample at 1000 Hz, if needed. Figure 2 is very difficult to follow, maybe doing a composite figure with accelerometer, gyroscopes and magnetometers separated would be better. One example of tremor suppression would be appreciated. \"The peaks (β/α and β²/α ratios) higher than a defined threshold were considered as indicators of a potential voluntary movement. given that they represent the detection of the cortical motor preparation of the movement\" The authors are very confident with this option, but some information should be given to the non-specialist of BCI. \"Three time intervals were studied: pre-movement period, movement period, post-movement period. The pre-movement period (lasting 2 seconds) was defined according to the acoustic order given to the patients and the detection of the beginning of movement via the gyroscopes.\" Please give a more precise description of the time window analyzed - 2000 ms before the movement? 2000 ms after the auditory signal? In the middle? Was there always 2000 ms between auditory signal and movement? \"The peaks (β/α and β²/α ratios) higher than a defined threshold were considered as indicators of a potential voluntary movement\" Was the threshold pre-defined a priori (which value?), or adjusted following the predictive value. \"More than 800 possible EEG/EMG combinations (n=832)\", please clarify the origin of these 832 combinations. \"Where p is the probability of movement and n the number of movements predicted\", please define more precisely the probability of movement. In general, it would be desirable to have an experimental protocol in which the subjects have the opportunity to decide if he/she wants to move or not. Or still better to go to a more ecological situation in which the subject is instructed to do the finger-to-nose movement at its own pace. And compare false positive and false negative predictions. Results: \"The mean QP was 82±12% (median = 83.5%) for the β/α ratio and 79.5±10.4% (median = 80%) for the β²/α ratio.\" Please report individual subjects' values. Figure 3 reports values of QP much lower than the mean values reported in the text. Please clarify. Which are the coherence values for Figure 4? The Y axis is missing. Figure 6: Is not the predicted predicting? Or, is not the movement pre-threshold predicting the post-threshold movement? Is that the reason why the Y channel is so good predictor of movement? Please clarify. If it is reduction of tremor, please show some examples. The motivation of the simulation is not clear, because only the EEG can be simulated, not the movement. Please clarify the interest of the simulations study or remove it. Discussion: The discussion seems to clarify that the excellent predictor of reduction of tremor is produced by a different command than the voluntary control. But tremor should have a definite frequency, and this frequency property of the tremor seems not be taken in account in present report.",
"responses": [
{
"c_id": "788",
"date": "24 Apr 2014",
"name": "Mario Manto",
"role": "Author Response F1000Research Advisory Board Member",
"response": "We thank the referee for their criticisms.Introduction:“In Figure 1 the dashed line seemed to indicate the movement, but how it is possible that the movement itself is predicted by the movement kinematic - that should be zero before movement. Except if some thresholds are defined, pre-thresholds movements are the predictors. Please clarify the sentence \"decrement of the rest tremor before movement onset\" - does it means that the reduction in tremor is not the initial voluntary movement but a different neural command which is being inhibited by the voluntary control? Then define physiological characteristics, frequency amplitude, of tremor. Otherwise it would be difficult to distinguish between the reduction of tremor and the initial phases of movement.”The initiation phase of movement in a patient with rest tremor has been characterized in details in the literature (Papengut et al. 2013). In patients exhibiting a rest tremor, tremor suppression at movement onset (reduction in amplitude or complete cessation, detectable with kinematic sensors) is an example of noticeable changes in tremor features occurring at the beginning of the voluntary movement: tremor features change and this is detectable by kinematic analysis. It is presumed that the voluntary command inhibits the central oscillator generating rest tremor. Since we are using both accelerometry and gyroscopes, the beginning of the finger-to-nose movement is clearly identified by changes in kinematics.The introduction and the legend of Figure 1 have been changed. Methods:“\"Mean age of the patients was 62 ± 20 years\". It would be more precise to describe the age of each individual subjects. Means are useful for high number of subjects. Same for other parameters.We have included a table detailing age and other parameters in the Methods of the revised article. Patients were followed on a regular basis during this European project. The clinical scores were pretty stable, although slight clinical fluctuations were observed (this is very common in the follow-up of neurological patients exhibiting a tremor in the upper limb). “\"After (1) hearing an acoustic signal, the patient (2) prepared themselves mentally for the execution of movement and (3) performed the task.\" It is not clear how long it takes this preparation. Was it induced by the experimenter instructions or it was a spontaneous strategy? How long does each of the phases take?”The text has been changed to the following:“Patients were told to keep the most relaxed attitude. After hearing an acoustic signal, they prepared themselves for the execution of movement by mental imagery of the movement. During a single run, the task was repeated about 10 times.” The preparation phase lasted typically between 2 and 8 sec. There was variability between trials depending on self-estimation by patients. This is typical in mental imagery. Some patients are “fast-imagers” and others are “slow-imagers”. “Is it necessary to indicate the files code? Otherwise please suppress it.”This is useful in making the figures more readable (see Figures 7, 9 and 10). “\"EEG signals were sampled at 256 Hz (re-sampling at 1000 Hz for synchronization purposes).\" It seems that the data were re-sampled to a higher frequency. Could it be considered an interpolation rather than a re-sampling? Anyway it would be better to sample at 1000 Hz, if needed.”Yes, we could consider an interpolation and/or a re-sampling. The rationale for the re-sampling at 1000 Hz is that it does not affect the spectral contents in the frequency sub-bands that are particularly of interest in our study. An interpolation technique is an alternative. “Figure 2 is very difficult to follow, maybe doing a composite figure with accelerometer, gyroscopes and magnetometers separated would be better. One example of tremor suppression would be appreciated.”We agree with the referee that this figure might be confusing. We have modified the figure in the revised article. The description of the sensors is reported in Gallego et al. (2010)Sensors are a combination of triaxial accelerometers, gyroscopes, and magnetometers. The low weight of IMUs makes them an optimal solution, as tremor changes its characteristics if a larger mass is attached to the limbs. Moreover, their small size does not interfere with user’s movements.The phenomenon of tremor suppression induced by the application of FES has been published earlier. Here is an example of essential tremor responding strongly to FES. This patient does not respond to any medication (including: propranolol, primidone, topiramate). Although refractory to conventional drugs, this patient was FES-sensitive. Figure from:Grimaldi G, Manto M: “Old” and Emerging Therapies of Human Tremor. Clinical Medicine Insights: Therapeutics. 2010; 2: 169-178. Publisher Full Text. Published under the Creative Commons CC-BY-NC 3.0 license. “\"The peaks (β/α and β²/α ratios) higher than a defined threshold were considered as indicators of a potential voluntary movement given that they represent the detection of the cortical motor preparation of the movement\" The authors are very confident with this option, but some information should be given to the non-specialist of BCI.”This method is now explained in the revised text. The following reference has been added: Pfurtscheller and Lopes da Silva (1999). This methodology is considered as a sound procedure for the detection of the preparation of movement.“\"Three time intervals were studied: pre-movement period, movement period, post-movement period. The pre-movement period (lasting 2 seconds) was defined according to the acoustic order given to the patients and the detection of the beginning of movement via the gyroscopes.\" Please give a more precise description of the time window analyzed - 2000 ms before the movement? 2000 ms after the auditory signal? In the middle? Was there always 2000 ms between auditory signal and movement?”The following sentence has been added: “by considering 2 seconds back from the point of detection of the beginning of movement. We decided to use a period of 2000 msec based on the available literature which considers that 2 seconds encompasses the preparation phase at the cortical level.” “\"The peaks (β/α and β²/α ratios) higher than a defined threshold were considered as indicators of a potential voluntary movement\" Was the threshold pre-defined a priori (which value?), or adjusted following the predictive value.”This has been studied earlier. The best thresholding process should be decided in a case-by-case scenario, as described in Giuliana, Mario and Yassin (2011); % of maximum ratio overtime; threshold considering mean and standard deviation of the ratios. “\"More than 800 possible EEG/EMG combinations (n=832)\", please clarify the origin of these 832 combinations.\"The number of combinations corresponds to 13 EEG channels (excluding the 2 channels for the eyes) and 64 EMG channels (16 EMG channels for each of the 4 muscles of the upper limb)13x64 = 832 combinations “\"Where p is the probability of movement and n the number of movements predicted\", please define more precisely the probability of movement.”The text has been changed to the following:“Where p is the probability of movement (true positives) and n the number of movements predicted, as previously described for the EEG QP.” “In general, it would be desirable to have an experimental protocol in which the subjects have the opportunity to decide if he/she wants to move or not. Or still better to go to a more ecological situation in which the subject is instructed to do the finger-to-nose movement at its own pace. And compare false positive and false negative predictions.”Thanks to the acoustic signal, the period of preparation of movement followed by the execution of the task is clearly defined. The patient knows that after the signal (1) he/she has to relax, (2) he/she has to prepare for the movement, and (3) he/she has to execute it. It is important to note that it is much easier for neurological patients to follow this sequence with an acoustic signal and not to include an additional internal evaluation to move or not to move. The introduction of an additional decision to move or not is a source of complexity, which by itself interferes with the severity of tremor. Some neurological patients have difficulties and show hesitations for self-paced movements. The task is easier with the acoustic cue. See also the reply to Yvonne Hoeller.Results:“\"The mean QP was 82±12% (median = 83.5%) for the β/α ratio and 79.5±10.4% (median = 80%) for the β²/α ratio.\" Please report individual subjects' values.\"This table shows the individual values. “Figure 3 reports values of QP much lower than the mean values reported in the text. Please clarify.”Figure 3 illustrates the effects of the selection of the sub-band beta upon the quality parameter. The figure illustrates values for one patient. “Which are the coherence values for Figure 4? The Y axis is missing.”The computed coherograms are shown using arbitrary units. “Figure 6: Is not the predicted predicting? Or, is not the movement pre-threshold predicting the post-threshold movement? Is that the reason why the Y channel is so good predictor of movement? Please clarify. If it is reduction of tremor, please show some examples.”There is very often one channel of the sensor which gives a much better predicting value. This rule can be applied to movement in general and to tremor in particular: one direction is much more informative (this is called the dominant axis). This explains why some groups consider that a single-axis sensor may provide relevant information. “The motivation of the simulation is not clear, because only the EEG can be simulated, not the movement. Please clarify the interest of the simulations study or remove it.”The simulation study is very important in order to have information about the strength of the ERS/ERD phenomenon. This parameter is important to state that a given patient having a strong enough ERS/ERD may be enrolled for a BCI-based therapy. This is a critical step, because some patients will benefit from a BCI-based management, whereas others not. We want to underline that we anticipate that in the near future this methodology will help to select patients for BCI programs. For the moment, some patients are enrolled and perform hours of training without success. An ERS/ERD-based decision to include patients in training programs will render the BCI-based management much more efficient. Discussion:“The discussion seems to clarify that the excellent predictor of reduction of tremor is produced by a different command than the voluntary control. But tremor should have a definite frequency, and this frequency property of the tremor seems not be taken in account in present report.”Tremor parameters have not been taken into account in this paper which is focused on the detection of voluntary movement. The overall concept of this European study was to predict the intentionality of movement and not to track the tremor parameters. FES is supposed to be active on the various forms of tremor. The point raised by the reviewer was addressed in detail in a previous European study (Manto et al., 2003). Tremor is a very dynamic process, but the major advantage of FES is to have a non-selective suppressive effect by acting on the peripheral nervous system."
}
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}
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https://f1000research.com/articles/2-282
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https://f1000research.com/articles/3-97/v1
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30 Apr 14
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{
"type": "Case Report",
"title": "Spinal cord stimulation for complex regional pain syndrome type 1 with dystonia: a case report and discussion of the literature",
"authors": [
"Caroline Voet",
"Bernard le Polain de Waroux",
"Patrice Forget",
"Ronald Deumens",
"Etienne Masquelier",
"Caroline Voet",
"Bernard le Polain de Waroux",
"Ronald Deumens",
"Etienne Masquelier"
],
"abstract": "Background: Complex Regional Pain Syndrome type 1 (CRPS-1) is a debilitating chronic pain disorder, the physiopathology of which can lead to dystonia associated with changes in the autonomic, central and peripheral nervous system. An interdisciplinary approach (pharmacological, interventional and psychological therapies in conjunction with a rehabilitation pathway) is central to progress towards pain reduction and restoration of function.Aim: This case report aims to stimulate reflection and development of mechanism-based therapeutic strategies concerning CRPS associated with dystonia.Case description: A 31 year old female CRPS-1 patient presented with dystonia of the right foot following ligamentoplasty for chronic ankle instability. She did not have a satisfactory response to the usual therapies. Multiple anesthetic blocks (popliteal, epidural and intrathecal) were not associated with significant anesthesia and analgesia. Mobilization of the foot by a physiotherapist was not possible. A multidisciplinary approach with psychological support, physiotherapy and spinal cord stimulation (SCS) brought pain relief, rehabilitation and improvement in the quality of life.Conclusion: The present case report demonstrates the occurrence of multilevel (peripheral and central) pathological modifications in the nervous system of a CRPS-1 patient with dystonia. This conclusion is based on the patient’s pain being resistant to anesthetic blocks at different levels and the favourable, at least initially, response to SCS. The importance of the bio-psycho-social model is also suggested, permitting behavioural change.",
"keywords": [
"Complex regional pain syndrome (CRPS) is a chronic pain disorder that usually affects the lower or upper extremities. Two types of CRPS have been described: CRPS type 1 (CRPS-1",
"reflex sympathetic dystrophy)",
"normally triggered by a painful trauma without any detectable associated nerve lesion and CRPS type 2 (causalgia)",
"involving a frank nerve injury. Both have similar symptomatology and are characterized by spontaneous pain that is disproportionate to the inciting event together with sensory",
"motor",
"autonomic and trophic changes1. Its incidence in the Netherlands is estimated at 26.2 per 100",
"000 persons per year",
"with women being more frequently impaired than men2. Physiopathogeny is complex",
"involving the central nervous system and peripheral neurogenic inflammatory processes3. CRPS is a multifactorial disorder associated with an aberrant host response to tissue injury4. Diagnostic criteria5 rely on the clinical presentation. Specific additional diagnostic tests do not appear to be useful and often are expensive6. CRPS is frequently associated with substantial disability",
"loss of quality of life and personal and societal economic burden4. A combined pharmacological",
"interventional and psychological approach",
"in conjunction with a rehabilitation pathway",
"has been proposed for the management of CRPS. Pain reduction and restoration of function form the mainstay of therapy7. Spinal cord stimulation (SCS) seems to be an effective and safe treatment of CRPS-18–11. Despite the diminishing effect of SCS over time",
"95% of patients with an implant would be willing to repeat the treatment for the same result (if it had not already been implanted)12. The success of SCS depends on the use of strict criteria for selecting patients that are likely to respond to this treatment8. Unfortunately",
"necessary re-intervention as a result of technical problems with the implant are frequent",
"especially during the first two years following implantation9",
"13. Nevertheless",
"SCS seems to be cost-effective10",
"12."
],
"content": "Introduction\n\nComplex regional pain syndrome (CRPS) is a chronic pain disorder that usually affects the lower or upper extremities. Two types of CRPS have been described: CRPS type 1 (CRPS-1; reflex sympathetic dystrophy), normally triggered by a painful trauma without any detectable associated nerve lesion and CRPS type 2 (causalgia), involving a frank nerve injury. Both have similar symptomatology and are characterized by spontaneous pain that is disproportionate to the inciting event together with sensory, motor, autonomic and trophic changes1. Its incidence in the Netherlands is estimated at 26.2 per 100,000 persons per year, with women being more frequently impaired than men2. Physiopathogeny is complex, involving the central nervous system and peripheral neurogenic inflammatory processes3. CRPS is a multifactorial disorder associated with an aberrant host response to tissue injury4. Diagnostic criteria5 rely on the clinical presentation. Specific additional diagnostic tests do not appear to be useful and often are expensive6. CRPS is frequently associated with substantial disability, loss of quality of life and personal and societal economic burden4. A combined pharmacological, interventional and psychological approach, in conjunction with a rehabilitation pathway, has been proposed for the management of CRPS. Pain reduction and restoration of function form the mainstay of therapy7. Spinal cord stimulation (SCS) seems to be an effective and safe treatment of CRPS-18–11. Despite the diminishing effect of SCS over time, 95% of patients with an implant would be willing to repeat the treatment for the same result (if it had not already been implanted)12. The success of SCS depends on the use of strict criteria for selecting patients that are likely to respond to this treatment8. Unfortunately, necessary re-intervention as a result of technical problems with the implant are frequent, especially during the first two years following implantation9,13. Nevertheless, SCS seems to be cost-effective10,12.\n\nWe here report a case of severe CRPS in a 31-year-old woman, who did not respond to the usual treatments, including anesthetic blocks. SCS brought relief of pain, allowing rehabilitation.\n\n\nCase description\n\nA 31-year-old white Caucasian woman suffering from repetitive ankle twisting underwent a second ligamentoplasty of the right ankle in 2008 for persisting instability. After intervention, she was treated with plaster-immobilization for seven weeks. Immediately after removal of the plaster, the patient reported intense pain and did not respond to standard painkillers (paracetamol, non-steroidal anti-inflammatory drugs and opioids), while physiotherapy became more and more difficult. Swelling of the right foot appeared, the scar re-opened and she presented with a dystonic posture of the right foot. Following an assessment by scintigraphy, possible CRPS was suspected. The diagnosis of CRPS-1 with dystonia was made and she was treated with several drugs, all of which were either without effect or poorly tolerated. A mobilization of the ankle was also performed under general anesthesia without long-lasting improvement.\n\nIn 2009, the patient was referred to a hospital where multidisciplinary management was started. Other treatments were tried, but without therapeutic benefits: dextropropoxyphene (600 mg/d), a buprenorphine patch (26.25 µg/h), hydromorphone (12 mg/d), clomipramine (50 mg/d), duloxetine (60 mg/d), pregabaline (300 mg/d), clonidine (0.30 mg/d), a lidocaine patch (2/day), intravenous pamidronate (30 mg/d) and methylprednisolone (64 mg/d). Three months later, the patient was referred to the Multidisciplinary Pain Center of CHU Mont-Godinne for administration of peridural and intrathecal anesthetic blocks (bupivacaine 5–15 mg). We noticed that the intrathecal block induced a contra-lateral motor block.\n\nWhen the patient was transferred to the Multidisciplinary Pain Center of the Cliniques Universitaires Saint-Luc in Brussels two months later, she presented with pain in the entire right foot, irradiating to just under the knee. The pain was permanent and reported on average at 8-9/10 on the VAS (Visual Analogue Scale). The patient described the pain as a burning feeling, which was enhanced by touch and mobilization. Walking required two canes, long distances necessitated a wheelchair. She also suffered from sleep and mood disturbances. Housekeeping was left to her husband and she took leave from her job at a child-care centre. Clinical examination showed a swollen foot and calf and tightened skin that was pale, gleaming and cold. The ankle was fixed in a dystonic equinus; only the toes presented active, though limited, movement of flexion-extension. The range of motion of the knee and hip were normal. Neurological assessment showed hypoesthesia like a sock on the right foot (up to the shin), hyperesthesia and hyperalgesia on the leg (on and above the knee- with allodynia (mechanical and dynamic rather than static). It was almost impossible for the patient to bear touch to the painful region, which meant that a complete sensory assessment could not be realized (Figure 1). Evoked somesthetic potentials indicated normal lemniscal pathways coming from the lower limbs. The presence of a peripheral injury could not be evaluated because of the pain in the right leg.\n\nIn our Multidisciplinary Pain Center, the patient underwent the following algological techniques in order to try starting rehabilitation:\n\nIntravenous ketamine (6 mg/h, increasing to 20 mg/h): no effect and handicapping side-effects (nausea, drowsiness).\n\nTwo anesthetic peripheral blocks of the right sciatic nerve at the knee, guided by echography (ropivacaine 115 mg, lidocaine 100 mg, clonidine 75 µg): no anesthesia and persistence of pain.\n\nPatches of clonidine (400 µg/day) and local anesthetic (lidocaine 5%) applied to the right foot: no effect.\n\nAnesthetic epidural block with a lateralized catheter. X-ray control with iohexol showed good epidural diffusion but injection of bupivacaine (25 mg) and lidocaine (100 mg) in combination with clonidine failed to produce any anesthesia or analgesia.\n\nNine anesthetic intrathecal blocks using bupivacaine (5 to 13 mg), clonidine (60 to 75 µg) or baclofen (50 µg): no effect was found with 5 mg of bupivacaine, a partial positive effect lasting a maximum 2h 15min for 10 to 13 mg of bupivacaine as reported by the patient. Treatment by continuous infusion and bolus of clonidine and bupivacaine respectively had a partial effect but had to be interrupted because of post-lumbar-puncture-syndrome. Baclofen had no effect.\n\nTranscutaneous electrical nerve stimulation (TENS): the stimulations were not perceived below the knee, this treatment was thus ineffective.\n\nTwo months later, almost one year after onset of CRPS, the patient discontinued all analgesics because of lack of therapeutic benefits and side effects on her cognitive functions and personality. Subsequently, she start noticing an improvement in her mood and cognitive functioning. Pain and neuro-orthopedic status of the right leg remained the same. As she started to feel better mentally, she progressively broadened her activities and started spinning and hydrotherapy.\n\nIn 2010, almost one year later, SCS (epidural electrode, Medtronic®, USA inserted percutaneously at lower lumbar level and pushed up to the T9 level) was trialled. During the testing period, we noticed a reduction in the intensity of hyperesthesia and allodynia of the right foot allowing touch and both active and passive mobilization of the foot, especially of the toes. Edema of the right foot decreased and vasodilation returned colouring the foot and warming it up. Importantly, the patient also experienced psychological relief. There was no effect on the equinus. After multidisciplinary discussion, it was decided to finally implant the pulse generator after about six weeks of testing.\n\nAt the time of writing this paper (2013), the patient is doing better. She has set up different strategies of coping, she is considering taking up her social life again and feels more optimistic about her future. Physiotherapy is now possible, but needs to be performed gently and progressively. Mobilization is performed during hydrotherapy. Orthopedic shoes have been manufactured in order to allow her to lean on her foot. Retrospectively, the Neuropathic Pain Symptom Inventory14 (NPSI), used to determine the quality of pain relieved by SCS showed a partial relief of both spontaneous pain and evoked pain (Figure 2). Laser evoked potentials (LEP) at one year after implantation of SCS showed a dysfunction of afferent small fibers (Aδ) from the right foot. No abnormalities where observed when stimulating the left foot and hand (Figure 3).\n\nIn blue, the spontaneous sensation is indicated, without the SCS functioning. The values for neuropathic pain when SCS is on are shown in red, indicating a global reduction of pain.\n\nNote the increased latency and reduced amplitude of LEP components in the affected foot as compared to the contralateral side. The vertical interrupted line represents the onset of the CO2 laser stimulus (duration 50 ms; surface area 79 mm2; intensity 9.7 mJ/mm2). Each side received 30 stimuli with an interstimulus interval of 8 to 15 s. The subject had to press a microswitch, held in her dominant hand, as fast as possible when perceiving the stimulus. To focus the patient’s attention, each stimulus was announced of 1.5 to 3 s beforehand, allowing her to fixe her gaze with open eyes during ±4 seconds to avoid eye movement artefacts.\n\n\nDiscussion\n\nAbout (9–49%) of patients with CRPS suffer from movement disorders, including loss of voluntary control, bradykinesia, dystonia, myoclonus and tremor. Dystonia occurs in approximately 20% of patients with CRPS and is characterized by fixed flexion postures of the fingers, wrist and feet that may vary in severity15. The prevalence of movement disorders increases as the disease duration lengthens15. The pathogenesis of CRPS and its relation to dystonia remain poorly understood. The central and peripheral nervous systems as well as immunological16, psychological4,15,17,18 and genetic19 factors seem to be implicated. We will only discuss neurological factors in this case report.\n\nThere is converging evidence for the role of the central nervous system in the physiopathogenesis of CRPS with dystonia. Central sensitization induced by tissue or nerve injury alters transmission and processing of peripheral sensorimotor input in the spinal cord. Associated with central disinhibition (both in the descending pathways and the brain itself), such changes set the stage for the development of movement disorders seen in CRPS15. Cortical involvement in CRPS is suggested by mislocalizations of tactile stimuli, changes of size and organization of the somatosensory map, changes in motor cortex representation and body perception disturbances20. The basal ganglia and parietal lobe seem especially related to some movement disorders such as dystonia and to hemineglect/inattention in CRPS21.\n\nRegarding the peripheral nervous system, large nerve fibers (proprioceptive afferents) do not seem to explain the underlying mechanisms of dystonia related to CRPS-1. Indeed, Van Rijn et al.22 found no differences in somatosensory-evoked potentials (SSEP) in CRPS-1 patients with dystonia compared to healthy controls after spatio-temporal stimulation (confirming the integrity of “cortical proprioceptive afferent processing”). In relation to our case report, we would like to highlight the role of small nerve fibers in CRPS (C and Aδ). Pathological studies on chronic CRPS-1 limbs show degeneration of small (C and Aδ) nerve fibers which serve nociceptive and autonomic functions4. However, this phenomenon does not seem to be specific to CRPS-1. Indeed, this degeneration is also seen in small-fiber-predominant polyneuropathies, which cause CRPS-like abnormalities23. Oaklander23 postulates that persistent CRPS-1 may represent a small-fiber-predominant mono- or oligoneuropathy that is initiated by a limb trauma. Moreover, dysfunction in small nerve fiber processing has been found by quantitative thermal testing in patients with CRPS-related dystonia24. On the other hand, patients with pure small-fiber polyneuropathies never develop dystonia, implying that neither small-fiber dysfunction nor its central consequences are a driving force behind dystonia25. Thus, it remains to be investigated whether nerve degeneration (i.e. dysfunction of small-diameter primary afferent nociceptor axons distal to trauma) causes CRPS-1 and/or dystonia4,17,24,25.\n\nAs such we do not have an unequivocal and clear explanation for the mechanism underlying dystonia in our CRPS-1 patient. However, LEP demonstrated dysfunction of small nerve fibers in the right foot is a possibility, as will be discussed in a later section of this article.\n\nA recent alternative neurological hypothesis proposed by Ethier et al.26, suggests a possible implication of the immune cells of the central nervous system, i.e. the microglia. These microglial could be activated in the brain as a result of a retrograde spread of neuroinflammation from the level of the spinal cord to the level of the motor cortex. As a consequence of microglial activation, functional changes may occur in the motor cortex. In predisposed individuals, these functional changes putatively trigger focal reduction of intracortical inhibition, a condition known to foster fixed dystonic postures. Further research in this area should help provide a better understanding of the mechanism underpinning CRPS-related dystonia.\n\nConventional therapies for the treatment of CRPS-1 with dystonia have poor efficacy. To our knowledge, there are no randomized controlled trials (RCT) of physical therapy, occupational therapy or pharmacotherapy in the treatment of movement disorders in CRPS27. Strategies that enhance the central inhibitory state may benefit these patients15. In some patients, the dystonia associated with CRPS responds markedly to intrathecal baclofen, a specific γ-aminobutyric acid (GABA) receptor agonist that inhibits sensory input to the neurons of the spinal cord28. A rehabilitation program associating laterality recognition, mental imaging and mobilization in front of a mirror29 is effective at reducing pain and increasing functioning by restoring sensory-motor integration6. Most of these treatment options (including mirror visual feed-back) had been previously tried out by our patient, without delivering a satisfactory effect on pain relief or quality of life.\n\nThe same problem was noticed with interventional treatment (e.g. SCS). Over the last decade, only one other case of CRPS with resistance to local anesthetic blocks has been reported, involving a 12-year-old girl suffering from CRPS-1 of the right ankle30. The mechanism of resistance to anesthetic blocks is currently unknown. The authors of this report, Maneksha et al.30, proposed that changes in the dorsal horn cells of the spinal cord, secondary to activation of N-methyl-D-aspartate (NMDA) receptors, may play a role in the pathophysiology of this pain syndrome. Our case provides further support for the importance of neural changes associated with CRPS-1. The implication, in various pain conditions, of voltage-gated sodium channels, mainly isoforms Na(v)1.7 and Na(v)1.8, but also others of the nine isoforms (Na(v)1-9), has been well demonstrated. For example, overexpression of different isoforms of Na(v) (at least 1.7) is suspected to play a key role in the physiopathology of radicular pain, post-herpetic neuralgia and trigeminal neuralgia31. In contrast, congenital deficiency of Na(v)1.7 is associated with inherited insensitivity to pain32. In severe CRPS-1 patients, these receptors are clearly upregulated in keratinocytes33. In the skin, their overexpression leads to neuronal hyperexcitability and pain, by increasing epidermal adenosine triphosphate (ATP) release and excessive activation of P2X receptors33. Moreover, it has been shown that mutation of Na(v)1.7, which increases excitability of sensory neurons, can lead to a decrease of sympathetic activity when expressed on sympathetic neurons32. A decrease in sympathetic activity, and a consequent increase of peripheral release of vasodilating peptides, leads to local changes such as erythema and edema, as seen in our patient. As a consequence, whether patients with CRPS-1 have an upregulation of Na(v) channels, at least Na(v)1.7, not only in keratinocytes, but also in the nervous system, may be worth investigating further.\n\nBased on the inefficacy of potent Na(v) blockers (i.e. local anesthetics), we can indicate that major neurologic changes were present in our severe CRPS-1 patient. On the basis of the observed lack of response to local anaesthetics at different levels, we suspect that these changes were not only present in the periphery, but also in the (sciatic) nerve trunk, and in the spinal cord.\n\nDespite her (relative) resistance to anesthetic blocks, our patient was a good responder to SCS. The mechanism of action of SCS is still incompletely understood and is frequently debated (Table 1). The results of several studies, mostly on animal models but also in patients, suggest that the effect of SCS is to a large part mediated via GABAB and muscarinic M4 receptors34–37. SCS induces GABA and acetylcholine (Ach) release in the spinal dorsal horn38–40 and activates descending serotoninergic pathways41, all of which inhibit spinal nociception processing. In parallel, Truin et al.42 demonstrated the role of NMDA receptors in the effect of SCS. Indeed, the combined use of SCS and sub-effective doses of intrathecal ketamine (an antagonist of NMDA receptors) resulted in a significant conversion of non-responders to SCS to responders to SCS. This effect could not be investigated in our patient as those treatments had not been given simultaneously. Besides, SCS is thought to affect peripheral vasodilatation via antidromic activation of spinal afferent neurons and inhibition of sympathetic efferents (small fibers). This effect seems to be mediated by calcitonin gene-related peptide (CGRP) and possibly nitric oxide (NO)43.\n\nA brief review of the literature, comparing studies on animals and on humans.\n\nGABA = γ-amino-butiric acid, Ach = Achetylcholine, NMDA = N-methyl-D-aspartic acid, CGRP = calcitonin gene-related peptide, NO = nitric oxide, TRPV1 = transient receptor potential vanilloïde 1, ERK = extracellular signal-regulated kinase.\n\nSmits44 suggests that the selection and subdivision by severity of mechanical allodynia may provide better pre-treatment predictions of the possible therapeutic benefits of SCS. These results match with those of Van Eijs13 suggesting that brush-evoked allodynia may be a significant negative prognostic factor of SCS treatment outcome after one year in chronic CRPS-1. The chances of achieving and maintaining successful pain reduction drop from 81% to 31% if allodynia is present13. However this notion partially conflicts with our observations. In our patient allodynia evoked by brushing or pressure on the painful area was present before SCS was tested and evoked pain scores were partially relieved by SCS, according to the NPSI (Figure 2).\n\nIt is important to emphasize that the reason someone with chronic pain gets better has as much to do with the nonspecific effects of treatment as with the treatment itself45. For instance, why did most of the invasive techniques undergone by our patient have no effect? And why was modulation of pain by SCS possible later on? Much of this has to do with the particular aspects of the human brain and the individual’s need to interpret pain46. Among those nonspecific treatment effects, factors that can impact on pain perception include patient-specific factors (degree of anxiety, desire to get better; improved coping etc.) and the interpersonal relationship between that person and their physician (perception of attention and caring47; major value of education, reassurance and counseling48; heightened expectations49). In the context of chronic pain patients who are challenging to treat, the importance of communication style is paramount50. It is important to remember that empathy, mutual respect and an open doctor-patient collaboration in the treatment are excellent skills in the interaction with patients51. Jamison45 suggests that the therapeutic quality of the practitioner’s manner and the role of the patient’s expectations of treatment are very powerful. We need to maximize those nonspecific effects of care in reducing the suffering of individuals with pain.\n\nThis case suggests the importance of behavioral change as part of the treatment of CRPS. First, the patient expected an explanation of her illness based on the bio-medical model52. This concept postulates that disease is fully accounted for by deviations from the norm of measurable biological (somatic) variables. A complex phenomenon is ultimately derived from a single primary principle (reductionism) and the mental component is separated from the somatic component (dualism, specifically psychophysic parallelism). Progressively, helped by a psychosocial accompaniment conducted by a clinical psychologist, her conception of illness converted into a bio-psycho-social model, accounting for human experiences besides the somatic abnormalities. That psychological work seemed to be necessary before trying out the SCS, even if, in this case, it remains, at least partially, hypothetical. Nevertheless, we propose that the bio-psycho-social model of rehabilitation in the treatment of CRPS would be an important step for achieving a change of behavior to acceptation of a disabling situation.\n\nFinally, the LEP realized on our patient suggest pathology of the small fibers (C and Aδ). Whether this is the cause or the result of CRPS is uncertain. To our knowledge, no article has been published exploring the link between LEP results and the outcome of treatment by SCS in CRPS-patients. This is an interesting topic for future research.\n\n\nConclusion\n\nThe resistance of pain to anesthetic blocks at different levels of the nervous system and the favorable response to SCS emphasizes the complexity of the pathophysiology of CRPS associated with dystonia in our case. This case is also notable because of its complex presentation: initial resistance to several treatments with subsequent reduction of pain by SCS. This poses the interesting question of the role of nonspecific treatment effects. Finally, it illustrates the importance of the bio-psycho-social model.\n\n\nConsent\n\nConsent for the publication of clinical details and images was obtained from the patient.",
"appendix": "Author contributions\n\n\n\nCV, BLPDW, PF and EM were involved in the patient’s care. CV and EM analyzed the patient’s data and discussed these with BLPDW, PF and RD. All the authors were involved in the manuscript preparation and approved the final version.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe authors would like to thank Prof. L. Plaghki for his contribution to this article. The execution and critical analysis of LEP were performed under his direction. The authors have no conflict of interest to declare.\n\n\nReferences\n\nWasner G, Schattschneider J, Binder A, et al.: Complex regional pain syndrome--diagnostic, mechanisms, CNS involvement and therapy. Spinal Cord. 2003; 41(2): 61–75. 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PubMed Abstract | Publisher Full Text\n\nMailis-Gagnon A, Furlan AD, Sandoval JA, et al.: Spinal cord stimulation for chronic pain. Cochrane Database Syst Rev. 2004; 3(3): CD003783. PubMed Abstract | Publisher Full Text\n\nKemler M, de Vet H, Barendse G, et al.: Effect of spinal cord stimulation for chronic complex regional pain syndrome Type I: five-year final follow-up of patients in a randomized controlled trial. J Neurosurg. 2008; 108(2): 292–98. PubMed Abstract | Publisher Full Text\n\nvan Eijs F, Smits H, Geurts J, et al.: Brush-evoked allodynia predicts outcome of spinal cord stimulation in complex regional pain syndrome type 1. Eur J Pain. 2010; 14(2): 164–69. PubMed Abstract | Publisher Full Text\n\nBouhassira D, Attal N, Fermanian J, et al.: Development and validation of the Neuropathic Pain Symptom Inventory. Pain. 2004; 108(3): 248–57. PubMed Abstract | Publisher Full Text\n\nvan Hilten J: Movement disorders in complex regional pain syndrome. Pain Med. 2010; 11(8): 1274–277. PubMed Abstract | Publisher Full Text\n\nde Rooij AM, Florencia Gosso M, Haasnoot GW, et al.: HLA-B26 and HLA-DQ8 are associated with Complex Regional Pain Syndrome with fixed dystonia. Pain. 2009; 145(1–2): 82–5. PubMed Abstract | Publisher Full Text\n\nLang A, Chen R: Dystonia in complex regional pain syndrome type 1. Ann Neurol. 2010; 67(3): 412–14. Publisher Full Text\n\nReedijk W, van Rijn M, Roelofs K, et al.: Psychological features of patients with complex regional pain syndrome type 1 related dystonia. Mov Disord. 2008; 23(11): 1551–559. PubMed Abstract | Publisher Full Text\n\nGosso M, de Rooij A, Alsina-Sanchis E, et al.: Systematic mutation analysis of seven dystonia genes in complex regional pain syndrome with fixed dystonia. J Neurol. 2010; 257(5): 820–24. PubMed Abstract | Publisher Full Text\n\nSwart C, Stins J, Beek P: Cortical changes in complex regional pain syndrome (CRPS). Eur J Pain. 2009; 13(9): 902–07. PubMed Abstract | Publisher Full Text\n\nLebel A, Becerra L, Wallin D, et al.: fMRI reveals distinct CNS processing during symptomatic and recovered complex regional pain syndrome in children. Brain. 2008; 131(Pt 7): 1854–879. PubMed Abstract | Publisher Full Text\n\nvan Rijn M, van Hilten J, van Dijk J: Spatiotemporal integration of sensory stimuli in complex regional pain syndrome and dystonia. J Neural Transm. 2009; 116(5): 559–65. PubMed Abstract | Publisher Full Text\n\nOaklander A, Fields H: Is reflex sympathetic dystrophy/complex regional pain syndrome type 1 a small-fiber neuropathy? Ann Neurol. 2009; 65(6): 629–38. PubMed Abstract | Publisher Full Text\n\nMunts A, van Rijn M, Geraedts E, et al.: Thermal hypesthesia in patients with complex regional pain syndrome related dystonia. J Neural Transm. 2011; 118(4): 599–603. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCooper M: Nerve injuries and the fixed dystonias of CRPS. Pain Med. 2011; 12(5): 842–43. PubMed Abstract | Publisher Full Text\n\nEthier C, Brizzi L, Giguere D, et al.: Corticospinal control of antagonistic muscles in the rat. Eur J Neurosci. 2007; 26(6): 1632–641. PubMed Abstract | Publisher Full Text\n\nGeertzen J, Perez R, Dijkstra P, et al.: EBGD Guidelines CRPS type I 2006. [Netherlands Association of Posttraumatic Dystrophy Patients]. Reference Source\n\nvan Hilten J, van de Beek W, Hoff J, et al.: Intrathecal baclofen for the treatment of dystonia in patients with reflex sympathetic dystrophy. N Engl J Med. 2000; 343(9): 625–30. PubMed Abstract | Publisher Full Text\n\nMoseley L: Graded motor imagery is effective for long-standing complex regional pain syndrome: a randomized controlled trial. Pain. 2004; 108(1–2): 192–98. PubMed Abstract | Publisher Full Text\n\nManeksha F, Mirza H, Poppers P: Complex regional pain syndrome (CRPS) with resistance to local anesthetic block: a case report. J Clin Anesth. 2000; 12(1): 67–71. PubMed Abstract | Publisher Full Text\n\nSiqueira S, Alves B, Malpartida H, et al.: Abnormal expression of voltage-gated sodium channels Nav1.7, Nav1.3 and Nav1.8 in trigeminal neuralgia. Neuroscience. 2009; 164(2): 573–77. PubMed Abstract | Publisher Full Text\n\nCummins T, Sheets P, Waxman S: The roles of sodium channels in nociception: implications for mechanisms of pain. Pain. 2007; 131(3): 243–57. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhao P, Barr T, Hou Q, et al.: Voltage-gated sodium channel expression in rat and human epidermal keratinocytes: evidence for a role in pain. Pain. 2008; 139(1): 90–105. PubMed Abstract | Publisher Full Text\n\nCui J, Meyerson B, Sollevi A, et al.: Effect of spinal cord stimulation on tactile hypersensitivity in mononeuropathic rats is potentiated by simultaneous GABA(B) and adenosine receptor activation. Neurosci Lett. 1998; 247(2–3): 183–86. PubMed Abstract | Publisher Full Text\n\nLind G, Meyerson B, Winter J, et al.: Intrathecal baclofen as adjuvant therapy to enhance the effect of spinal cord stimulation in neuropathic pain: a pilot study. Eur J Pain. 2004; 8(4): 377–83. PubMed Abstract | Publisher Full Text\n\nLind G, Schechtmann G, Winter J, et al.: Baclofen-enhanced spinal cord stimulation and intrathecal baclofen alone for neuropathic pain: Long-term outcome of a pilot study. Eur J Pain. 2008; 12(1): 132–36. PubMed Abstract | Publisher Full Text\n\nSong Z, Meyerson B, Linderoth B: Muscarinic receptor activation potentiates the effect of spinal cord stimulation on pain-related behavior in rats with mononeuropathy. Neurosci Lett. 2008; 436(1): 7–12. PubMed Abstract | Publisher Full Text\n\nCui J, O’Connor W, Ungerstedt U, et al.: Spinal cord stimulation attenuates augmented dorsal horn release of excitatory amino acids in mononeuropathy via GABAergic mechanism. Pain. 1997; 73(1): 87–95. PubMed Abstract | Publisher Full Text\n\nSchechtmann G, Song Z, Ultenius C, et al.: Cholinergic mechanisms involved in the pain relieving effect of spinal cord stimulation in a model of neuropathy. Pain. 2008; 139(1): 136–45. PubMed Abstract | Publisher Full Text\n\nSchechtman G, Wallin J, Meyerson B, et al.: Intrathecal clonidine potentiates suppression of tactile hypersensitivity by spinal cord stimulation in a model of neuropathy. Anesth Analg. 2004; 99(1): 135–39. PubMed Abstract | Publisher Full Text\n\nSong Z, Ultenius C, Meyerson B, et al.: Pain relief by spinal cord stimulation involves serotoninergic mechanisms: an experimental study in a rat model of mononeuropathy. Pain. 2009; 147(1–3): 241–48. PubMed Abstract | Publisher Full Text\n\nTruin M, Janssen SP, van Kleef M, et al.: Successful pain relief in non-responders to spinal cord stimulation: the combined use of ketamine and spinal cord stimulation. Eur J Pain. 2011; 15(10): 1049.e1–9. PubMed Abstract | Publisher Full Text\n\nPrager J: What does the mechanism of spinal cord stimulation tell us about complex regional pain syndrome? Pain Med. 2010; 11(8): 1278–283. PubMed Abstract | Publisher Full Text\n\nSmits H, Ultenius C, Deumens R, et al.: Effect of spinal cord stimulation in an animal model of neuropathic pain relates to degree of tactile \"allodynia\". Neuroscience. 2006; 143(2): 541–46. PubMed Abstract | Publisher Full Text\n\nJamison R: Nonspecific treatment effects in pain medicine. Pain Clinical Updates. 2011; XIX(2). Reference Source\n\nWiech K, Ploner M, Tracey I: Neurocognitive aspects of pain perception. Trends Cogn Sci. 2008; 12(8): 306–13. PubMed Abstract | Publisher Full Text\n\nJamison R, Raymond S, Levine J, et al.: Electronic diaries for monitoring pain: 1-year validation study. Pain. 2001; 91(3): 277–85. PubMed Abstract | Publisher Full Text\n\nBarsky A: Palliation and symptomatic relief. Arch Intern Med. 1986; 146(5): 905–09. PubMed Abstract | Publisher Full Text\n\nRoberts A, Kewman D, Mercier L, et al.: The power of nonspecific effects in healing: implications for psychological and biological treatments. Clin Psychol Rev. 1993; 13(5): 375–91. Publisher Full Text\n\nTam M, Su M: How to manage difficult patients. 2006. Reference Source\n\nElder N, Ricer R, Tobias B: How respected family physicians manage difficult patient encounters. J Am Board Fam Med. 2006; 19(6): 533–41. PubMed Abstract | Publisher Full Text\n\nEngel G: The need for a new medical model: a challenge for biomedicine. Science. 1977; 196(4286): 129–36. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "5167",
"date": "22 Jul 2014",
"name": "Frank van Eijs",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe an interesting but not infrequently occurring case of therapy resistant CRPS. The report is well described. Only few issues need to be addressed.Introduction\"involving a frank nerve injury\" may be replaced by \"involving a detectable large fiber nerve injury\"Case description“in 2008..” add the month “In 2009...” add the month \"the patient reported intense pain and did not respond to\"... replace and with which“Subsequently she start noticing...” replace start with startedDiscussionExplain P2X receptors“It is important to emphasize that the reason why someone with chronic pain gets better...”“factors that can impact...” replace can with may.In conclusion: although it's scientific value is of minor importance because of the nature of the article (being a case report), the report certainly is worth indexing.F. van Eijs, MD, PhD",
"responses": []
},
{
"id": "5717",
"date": "09 Sep 2014",
"name": "Michael Erdek",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThere are two comments I have regarding the submission:In the case description, the authors mention mobilization of the foot by a physiotherapist was not possible. Can they be more specific as to why that was the case?In the Discussion, you mention that 9-49% of CRPS patients suffer from movement disorders. What is your source for that figure?",
"responses": []
}
] | 1
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https://f1000research.com/articles/3-97
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https://f1000research.com/articles/2-239/v1
|
12 Nov 13
|
{
"type": "Research Article",
"title": "The net return from animal activity in agro-ecosystems: trading off benefits from ecosystem services against costs from crop damage",
"authors": [
"Gary W Luck"
],
"abstract": "Animals provide benefits to agriculture through the provision of ecosystem services, but also inflict costs such as damaging crops. These benefits and costs are mostly examined independently, rather than comparing the trade-offs of animal activity in the same system and quantifying the net return from beneficial minus detrimental activities. Here, I examine the net return associated with the activity of seed-eating birds in almond orchards by quantifying the economic costs and benefits of bird consumption of almonds. Pre-harvest, the consumption of harvestable almonds by birds cost growers AUD$57.50 ha-1 when averaged across the entire plantation. Post-harvest, the same bird species provide an ecosystem service by removing mummified nuts from trees that growers otherwise need to remove to reduce threats from fungal infection or insect pest infestations. The value of this ecosystem service ranged from AUD$82.50 ha-1–$332.50 ha-1 based on the replacement costs of mechanical or manual removal of mummified nuts, respectively. Hence, bird consumption of almonds yielded a positive net return of AUD$25–$275 ha-1 averaged across the entire plantation. However, bird activity varied spatially resulting in positive net returns occurring primarily at the edges of crops where activity was higher, compared to negative net returns in crop interiors. Moreover, partial mummy nut removal by birds meant that bird activity may only reduce costs to growers rather than replace these costs completely. Similar cost-benefit trade-offs exist across nature, and quantifying net returns can better inform land management decisions such as when to control pests or promote ecosystem service provision.",
"keywords": [
"ecosystem services",
"pest control",
"avian ecology",
"agriculture",
"cost-benefit trade-offs"
],
"content": "Introduction\n\nAnimals provide benefits to humans through ecosystem services including the provision of food and fibre, crop pollination, biological control, waste disposal, nutrient cycling and seed dispersal1–5. Animal behaviour also inflicts costs on humanity, particularly through damage to food crops grown for human consumption6–9. The monetary value of the benefits and costs of animal activity can be substantial. For example, Losey and Vaughan10 estimated that the annual value of wild pollinators to agriculture in the United States (US) was approximately US$3 billion, while natural pest control services were worth about US$13.6 billion annually. Conversely, crop damage caused by the European starling (Sturnus vulgaris) in the US costs around US$800 million each year11, while bird damage to horticultural production in Australia is estimated at AUD$300 million annually12.\n\nDespite the obvious cost-benefit trade-offs of animal activity, studies on the ecosystem services provided by animals and on the damage they cause have evolved largely independently13. Yet, to more accurately reflect the outcomes of animal activity for society, it is imperative to quantify and compare the costs and benefits of these activities in the same system. Cost-benefit trade-offs are most acute in agricultural systems14, which profit from a range of animal-based ecosystem services, but suffer also from substantial negative impacts from animal activity.\n\nThe net return associated with animal activity in a given system can be derived by subtracting the costs of this activity (damage inflicted) from the benefits (ecosystem services provided). Benefits and costs may be quantified in monetary terms or some other appropriate metric (e.g. net effect on crop yield). A net return can be either positive or negative depending on the difference between the value of the ecosystem service(s) and the value of the negative impact(s). This approach is fundamentally different to other cost-benefit trade-offs presented in the literature, such as trading off the cost of supporting ecosystem service providers (e.g. by planting or protecting their habitat) against the value of the services provided15, or comparing the cost of pest control strategies with the amount of damage inflicted by pests16. Conceptually, it is most similar to circumstances where particular animals inflict damage (e.g. insect pests), other animals help control these pests (e.g. insectivorous birds), and researchers compare crop yield with and without the ecosystem service providers (e.g. Mols and Visser2, and Kellerman3). Focussing on net return, however, shifts the emphasis to quantifying both the costs and the benefits of the activity of particular animals, subtracting one from the other, and includes cost-benefit trade-offs stemming from the activity of the same species or the same group of species.\n\nQuantifying net returns is applicable to many situations within agriculture and more broadly (Figure 1). For example, pollinating insects contribute substantially to the pollination of food crops17, but the same insects may also pollinate agricultural weeds18. In a given location, the net return of pollinator activity could be quantified by comparing crop yield from pollination vs. reductions in yield caused by competition from insect-pollinated weeds. Similarly, insectivorous animals may help control insect pests in crops2, but could also consume desirable insect species (e.g. pollinators)19. An analysis of insectivore activity, diet composition, and changes in crop yield with variation in animal activity and abundance could be conducted to quantify this cost-benefit trade-off.\n\nThese cost-benefit trade-offs are common across nature. A positive net return from animal activity occurs when benefits outweigh costs, with the converse resulting in a negative net return. Photo credits: FreeDigitalPhotos.net (top left: prozac1, top middle: sweetcrisis, top right: John White, 2nd row left: dan, 2nd row middle: artemisphoto, 2nd row right: xedos4, 3rd row middle: thawats, 3rd row right: Paul Brentnall; bottom row right: Dr Joseph Valks). Remaining photos are from thinstockphotos.co.uk.\n\nIn this study, I present the first ever field test of this net return approach by quantifying the economic costs and benefits of bird activity in almond orchards in southern Australia (Figure 2). Almonds are one of Australia’s fastest growing horticulture sectors. The area of almond plantations increased 5-fold between 2001 and 2011, from 5,900 ha to over 30,000 ha, and annual production is projected to reach 88,000 tonnes in 2016, up from 37,000 tonnes in 2011 (http://www.australianalmonds.com.au/industry). Australia will soon be the world’s second largest almond producer behind California, USA.\n\n(a) Almond orchards in north-west Victoria, Australia have expanded 10-fold in 10 years, now covering more than 20,000 ha. (b) A regent parrot (Polytelis anthopeplus) feeding on almonds. Regent parrots are one of 11 parrot and cockatoo species that have been recorded eating almonds. (c) Typical parrot damage to almonds. (d) A netted branch on an almond tree. Photo credits: Hugh McGregor and Shannon Triplett.\n\nThe expansion of the almond industry raises substantial production and conservation management challenges. In Australia, almond crops attract a number of native bird species, especially parrots, which eat almonds during the growing season and reduce crop yield20. However, the same bird species provide an ecosystem service to growers by eating residual nuts left on trees after the main crop has been harvested. These so-called ‘mummified nuts’ (mummy nuts) are susceptible to fungal infection, which may threaten future crop yields. Moreover, recent evidence shows that mummified nuts are used intensively by the carob moth (Ectomyelois ceratoniae) for food and breeding (http://australianalmonds.com.au/industry/conference_2012/proceedings). The carob moth is a pest of global significance, impacting the production of numerous crops worldwide including dates, figs, pistachio, citrus and pomegranate21–23. It is a major emerging threat to the Australian almond industry, as it feeds on almond kernels rendering them unsuitable for human consumption. Mechanical or manual removal of mummy nuts post harvest is one approach to controlling moth outbreaks and fungal infections. However, birds are already providing this service to Australian growers – the question addressed in my study is whether the monetary value of this service outweighs the costs of bird damage to almonds, resulting in a positive net return from bird activity.\n\n\nMethods\n\nMy study was conducted in almond plantations in north-west Victoria, Australia, centred on 34°45'00S 142°42'52E (Figure 2a). This was a two-phrase experiment based on the exclusion of birds from almond trees. The first phase quantified damage to ripening nuts – the ‘cost’ component of the cost-benefit trade-off. The second phase quantified the removal of mummy nuts by birds post-harvest – the ‘benefit’ component. Experiments were conducted in two almond blocks (~ 17 ha each) in a single plantation of even-aged trees (~ 8 years old) of the same almond variety (nonpareil). At least 13 bird species have been recorded feeding on almonds by local growers (Table 1), including the threatened regent parrot (Polytelis anthopeplus) (Figure 2b)20.\n\nTo quantify bird damage to ripening nuts, trees were netted using 15 mm diamond mesh bird exclusion netting during the growing season of 2010/11. Nets were placed in October following natural early abortion of nuts by trees, and remained on trees until harvest (~ March). A total of 120 trees were included in the experiment with 60 netted trees (treatment) and 60 open trees (control) divided evenly between the edge (exterior two rows) and interior (centre point furthest from the edge) of the almond blocks. An edge/interior contrast was included because bird activity tends to be highest close to block edges20. Control and treatment trees were assigned systematically to maximize interspersion31, and I maintained a minimum distance of four trees between each control and treatment in the same edge or interior row to avoid adjacency effects and spatial autocorrelation in damage impacts (e.g. netted trees impacting outcomes on non-netted trees). The purpose of netting trees was to record levels of nut loss attributable to factors other than birds (e.g. storm damage, tree health and natural abortion). This enabled me to partition out nut loss attributed to either birds or non-bird factors.\n\nIt was not possible to net entire trees without compromising the integrity of the experiment (e.g. nets ripping in strong wind); therefore, I netted a single, randomly chosen lateral branch on each treatment tree (Figure 2d; trees were divided into three height sectors (lower, middle and upper) and four quadrants based on cardinal N, S, E and W, and a height sector and quadrant was chosen at random to select target branches). A lateral branch on each control tree was also chosen at random and these were completely open to birds. At the beginning of the experiment, almonds on each lateral branch were counted (98 nuts branch-1, ± 1 SEM 3.5, n = 120). At the end of the experiment immediately prior to harvest, nuts were assigned to one of the following three categories: intact on branch; damage/loss attributed to birds; damage/loss attributed to non-bird factors.\n\nDamage to almonds by birds is readily identifiable through bite patterns20. While the hulls of nuts often remain on the tree after bird damage (see Figure 2c), some nuts may be completely removed by birds. In this case, it is difficult to attribute nut loss to birds or non-bird factors that may result in complete nut loss (e.g. storm damage). Nevertheless, I used data from netted trees to calculate mean percent nut loss from non-bird factors and adjusted values for open trees. Mean nut loss from netted trees was 5.9% (± SEM 0.82%, n = 60), so if, for example, 50 nuts were completely lost from an open tree, I considered that three nuts (5.9%) were lost due to non-bird factors and the remainder due to birds. I did not expect netting to affect non-bird related nut loss.\n\nThe monetary cost of bird activity was measured as reduced yield in the almond crop (damage or loss), converted to an AUD$ value based on the wholesale almond price for 2012 of $5.05 kg-1. The average weight of a raw, shelled almond is 1.3 grams (± SEM 0.005 grams, n = 500), so at a price of $5.05 kg-1 a single almond is worth approximately $0.007. Hence, a 10% yield loss from an open lateral branch with a starting crop of 100 almonds equates to $0.07 (10 nuts lost or damaged). I was able to extrapolate yield loss values from lateral branches to values per tree based on the mean number of nuts per tree (1268, ± SEM 43.6, n = 200), which was estimated as part of a related study on bird damage to almonds20. Based on the value of $0.007 nut-1, a 10% yield loss for an entire tree equates to $0.83 (± SEM $0.03). Per tree values were converted to per ha values using the recommended almond planting density of 250 trees ha-1 (http://www.dpi.vic.gov.au/agriculture/horticulture/fruit-nuts/nuts/almonds).\n\nPost-harvest, the same experimental design was employed to quantify the removal of mummy nuts left on trees (i.e. 60 netted and 60 control trees split evenly between the edge and interior of two almond blocks). Different trees and different lateral branches were used in this experiment, as I had to target trees and branches that retained mummy nuts. Nets remained on the trees for approximately 3 months, from March to June. Nuts were counted at the beginning and end of the experiment and assigned to one of the three categories listed above.\n\nTo quantify the monetary value of bird removal of mummy nuts, I used the replacement cost method. This method determines the economic value of an ecosystem service by calculating the cost of replacing that service via human-derived means. It has been used widely to estimate the replacement costs of ecosystem services provided by particular ecosystems (e.g. forests or wetlands; see for example Spangenberg and Settele32, Turner et al.33, and Zhang et al.34), but also for services provided by particular species1,35. While the use of this method under certain circumstances has been criticized (e.g. Winfree et al.36), it was the most appropriate approach in my study because mechanical or manual removal of mummy nuts by growers is the currently employed and least costly alternative to bird removal of mummy nuts.\n\nMummy nuts can be removed by shaking the almond tree using a large mechanical shaker (akin to a large tractor) or hand-poling, which involves a labourer knocking nuts from the tree using an elongated pole (manual labour). Mechanical removal, including sweeping and shredding of nuts, takes approximately 20 seconds per tree. The cost of this was converted to an hourly rate based on the hourly wage of a skilled mechanical-shaker driver plus a retail hire rate for large farm machinery ($38 hr-1). In Australia, the full-time minimum wage for adults in 2012 was $15.96 hr-1. I assumed skilled operators of large machines would attract a higher hourly rate than the minimum wage, and based my initial calculations on a nominal value of $20 hr-1 (see Results for cost-benefit trade-offs across different wage rates).\n\nHand-poling is more labour and time intensive than mechanical shaking, but requires less training. It can be used instead of or in addition to mechanical shaking if the latter method is unable to dislodge the majority of mummy nuts. The main cost of hand-poling is associated with the time taken to reduce the number of mummy nuts on an almond tree to an acceptable level (see Discussion). Assuming a minimum wage for hand-polers of $15.96 hr-1, 1 minute of hand-poling per tree costs $0.27 ($67.50 ha-1).\n\nI determined if there was a block or row location (edge/interior) effect on nut loss attributed to birds or non-bird factors pre- and post-harvest using a generalized linear model with a binomial response (nut damaged/lost vs. nut intact) where p ≤ 0.05 was considered statistically significant. Row location was nested within block for these analyses and both were treated as random factors. Analyses were conducted using SPSS Version 21.037.\n\n\nResults\n\nBird damage to almonds pre-harvest was generally low, averaging 2.8% (± 1 SEM 0.28%) of the crop. Damage was higher at the edge of almond blocks compared to the interior (χ12 = 16.18, P < 0.001; Figure 3), but there was no difference between almond blocks. Nut loss attributed to non-bird factors (e.g. storms, tree health, natural abortion) was more than two times greater than bird damage (5.9% ± SEM 0.82%) and differed between almond blocks (χ12 = 38.04, P < 0.001, higher in block 1), but not between row location (Figure 3). Post-harvest, mummy nut removal by birds was more than 10 times greater, on average, than bird damage pre-harvest (35.6% ± SEM 3.6%). Nut removal was much higher at block edges compared to the interior (χ12 = 224.5, P < 0.001; Figure 3) and also differed between almond blocks (χ12 = 5.89, P = 0.02, higher in block 1). Nut loss via non-bird factors post-harvest was 6.1% (± SEM 0.9%) and was higher at block edges (χ12 = 3.9, P = 0.05).\n\nThe percentage of almonds damaged or removed by birds, or lost through non-bird factors, at the edge and interior of almond blocks pre- and post-harvest. Error bars are ± 1 SEM; n = 120 both pre and post-harvest, split evenly among treatments.\n\nBased on an almond wholesale value of $5.05 kg-1, average bird damage pre-harvest (2.8%), across the entire plantations, cost growers $0.23 tree-1 or $57.50 ha-1 (± SEM $5; all amounts in AUD). Nut loss from non-bird factors cost more than double the loss attributed to birds ($122.50 ha-1, ± SEM $17.50). Yield loss from bird or non-bird factors represents 0.8% and 1.6%, respectively, of crop value ha-1 based on a ‘good’ yield of 1.5t ha-1 of almonds at current wholesale value (http://www.dpi.vic.gov.au/agriculture/horticulture/fruit-nuts/nuts/almonds).\n\nUsing a mechanical tree shaker, it costs growers $82.50 ha-1 to remove mummy nuts (incorporating salary ($20 hr-1) and machinery costs). A more generous salary of $30 hr-1 raises costs to $95 ha-1. Manual removal of mummy nuts using hand-poling is more expensive. Assuming only a 5-minute duration at each tree and a minimum wage of $15.96 hr-1, hand-poling costs $332.50 ha-1. If bird consumption reduces the number of mummy nuts to an acceptable level, negating the need for growers to remove nuts, the value of this ecosystem service outweighs the costs of crop damage by at least $25–$275 ha-1 (replacement costs of mechanical shaking and hand-poling, respectively). Hence, bird activity yields a positive net return (Figure 4).\n\nEcosystem service value is calculated using the replacement cost method based on the removal of mummy nuts via mechanical shaking or hand-poling. A positive net return (red) occurs when the ecosystem service value is greater than the cost of bird damage (blue).\n\nWhether a positive or negative net return occurs depends on the level of bird damage to crops, the market value of almonds, and the value of the ecosystem service. I calculate that a ‘break even’ point, where damage costs and ecosystem service value are about equal, occurs when bird damage is 4% and the value of mummy nut removal is $87.50 ha-1 based on the replacement cost of mechanical shaking ($63 hr-1; Figure 5a). Damage costs increase steadily beyond this point and always exceed ecosystem service value even when the replacement cost is high, yielding negative net returns for growers. However, if the value of the ecosystem service is based on the replacement cost of hand-poling mummy nuts, then the benefit outweighs the cost of bird damage (a positive net return) even if only 10 minutes or less is spent hand-poling each tree and bird damage rates equal 20% (Figure 5b).\n\n(a) Comparing the trade-off in the cost of bird damage (dashed line) vs. the value of the ecosystem service of mummy nut removal (solid line) with variation in damage rates and replacement cost estimates based on mechanical shaking. Higher rates of bird damage will generally result in negative net returns for growers even at relatively high replacement costs (ecosystem service value). (b) The same trade-off using replacement cost estimates from hand-poling (solid line) suggests that even high rates of bird damage (dashed line) will not outweigh ecosystem service value, consistently yielding a positive net return.\n\nThese averaged results are complicated by two factors. First, bird damage and mummy nut removal varied spatially – both being higher at the edge compared to the interior of almond blocks. Pre-harvest, bird damage at the edge of blocks averaged 3.5% equating to a cost to growers of $72.75 ha-1, while lower damage in the interior (2%) equals a cost of $41.50 ha-1. Second, the averaged values (across the entire plantation) assume that birds will remove most mummy nuts within a given time frame, negating the need entirely for mechanical or manual removal by growers. For the 3-month experiment, birds removed 36% of mummy nuts, on average, per tree. If ecosystem service value is calculated using a reduced cost estimate (rather than using replacement costs), then growers would save $30 ha-1 if only having to remove 64% of mummy nuts by mechanical shaking (i.e. $82.50 ha-1 to remove 100% of nuts minus $52.50 ha-1 to remove 64% of nuts), and $120 ha-1 by hand-poling.\n\nFor mechanical shaking, the ecosystem service value of partial mummy nut removal does not outweigh the cost of bird damage, but it does for hand-poling. Assuming a linear rate of mummy nut removal by birds over time, if growers are able to wait until the next almond flowering (approximately 5 months based on harvesting in February/March and flowering in July/August), then birds would have removed 60% of mummy nuts. In this instance, growers would save $52.50 ha-1 for mechanical shaking, making the ecosystem service value only marginally less than the cost of bird damage.\n\nHowever, at the edge of almond blocks, mummy nut removal over 3 months equalled 55%, equating to over 90% of nuts removed within 5 months assuming a linear rate of removal. In this instance, mechanical or manual removal may be completely unnecessary and even with the higher rate of bird damage there is still a positive net return from bird activity of at least $9.75 ha-1. This suggests that growers need to consider possible spatial variation in cost-benefit trade-offs. It is also important to note that my analysis assumes that a single visit per tree by the mechanical shaker is enough to remove the majority of mummy nuts. This is unlikely (see Discussion), resulting in a conservative estimate of ecosystem service replacement costs in this instance.\n\n\n\n\nDiscussion\n\nOn average, bird activity yielded a positive net return to almond growers, even with relatively low ecosystem service replacement costs. The replacement cost estimate of mechanical shaking is conservative because I assumed shaking will remove most mummy nuts in a single visit, but this may not be the case; mummy nuts occur because mechanical shaking during harvesting does not dislodge every almond from a tree24. Moreover, mechanical shaking may impact the following season’s yield if conducted just prior to bud development25. Therefore, it is likely that a combination of mechanical shaking and hand-poling, or hand-poling alone, is required to guarantee that most mummy nuts are removed. Owing to the higher costs of hand-poling, the ecosystem service value of mummy nut removal by birds always yielded a strong positive net return, even when damage rates pre-harvest were high, and even when ecosystem service value was calculated as the amount saved by growers if only having to remove ~ 60% of mummy nuts.\n\nNevertheless, it is important to recognise the spatial variation that occurred in bird activity and subsequent cost and benefit outcomes, and the fact that birds did not remove all mummy nuts. The value of the ecosystem service provided by birds relates to what is an ‘acceptable’ level of mummy-nut load remaining on trees post-harvest. In California (the world’s major almond growing region), it is generally considered that approximately two nuts per tree is an acceptable quantity of mummy nuts to reduce adverse impacts (http://www.ipm.ucdavis.edu/PMG/C003/m003dcmummynut.html). I am unaware of any similar guidelines for Australia, possibly because threats associated with mummy-nut retention (e.g. carob moth infestations) are relatively new, but it is important to recognise that bird activity is unlikely to remove all mummy nuts from trees before onset of the next crop. Therefore, bird activity may at best reduce costs for growers rather than replace these costs completely, and this reduction is likely to be highest at crop edges.\n\nInformation about the magnitude of positive or negative net returns can be used by agriculturalists to better inform crop management decisions, such as when and how to control pests. The trade-off between the monetary costs of pest damage vs. how much to invest in pest control is a common problem in agriculture16. Yet, such analyses would benefit immensely if they considered also the value of any services provided by the ‘pest’ species or other species in the system. If a positive net return from animal activity is recorded in a given agro-ecosystem, pest control activities may be unnecessary or even detrimental. Conversely, if a negative net return is recorded, the magnitude of the negative return could be used to guide pest control spending.\n\nIn almond plantations, birds are currently considered pests and are subject to control strategies such as shooting to scare. In my study, shooters were employed for an average of 120 days per season over the growing seasons of 2009/10 and 2010/11. Based on an 8-hr day and minimum wage ($15.96 hr-1), the cost of employing shooters is at least $15,322 season-1. Over a 15,500 ha plantation (the entire plantation estate for my study) this equates to about $1 ha-1, and appears to be a good investment if compared only to the cost of bird damage. For example, if shooting reduces bird damage by only 1% it would save growers $22.19 ha-1. Yet, in the context of an overall positive net return from bird activity, the investment in shooting may be completely unnecessary.\n\nIt could be argued that a positive net return was recorded in my study only because of current pest control strategies. However, our previous work20 and research more generally26,27 shows that shooting is largely ineffective at controlling bird behavior. Moreover, if season-long control strategies are effective in reducing bird use of almonds, they may disrupt the ecosystem service being provided post-harvest.\n\nAgriculturalists may also invest in improving service provision in addition to or instead of controlling pests. In my study system, birds appear to use almonds when alternative food resources are scarce20. Plantings of decoy crops (e.g. low value crops or stands of native forage plants)28,29 that provide food during the almond growing season could be used to reduce bird impacts. In my study area, native plants from genera such as Atriplex, Eremophila, Dodonaea and the family Amaranthaceae (e.g. chenopods) should be considered for decoy crops. Moreover, strategic planting coupled with plant phenology could ensure that food resources from decoy crops were near exhausted post almond harvest, resulting in birds moving into orchards to feed on mummy nuts. Parrot density in almonds is already higher late in the season, as more almonds have split hulls and kernels are easier to access20. This is why removal rates for mummy nuts were much higher than damage rates pre-harvest in my study (Figure 3). Hence, a single management action – planting decoy crops with appropriate phenology – could simultaneously reduce bird damage to almond crops while maintaining and possibly improving the service of mummy nut removal.\n\nAgricultural land-use management strategies must consider the cost-benefit trade-offs occurring across the spectrum of animal activities and their impacts. This approach is fundamentally different to current research that calculates only the benefit of an ecosystem service provided by animals, even when that service is the control of another animal pest. In these case studies, the benefit is usually measured as the difference in crop yield with and without the provision of the service (e.g. Mols and Visser2, Kellerman3,. Karp et al.38). While difference in yield could be considered the net outcome of the activities of the animal pests and their control agents, critical information is usually not considered such as the cost of any damage inflicted on crops by those species providing the ecosystem service or benefits provided by the ‘pest’ species. That is, it is important to acknowledge that an ecosystem-service provider may be a pest in a different context, and vice-versa for recognised pest species. This information is vital to guiding more strategic land management decisions regarding monetary investments in the control of pests or supporting the provision of ecosystem services.\n\nI’ve focussed on a small suite of species and a single activity to clearly illustrate the net return from animal activity. Analyses could be extended to encompass more species and more activities, dependent on available ecological knowledge. For example, I have recorded 35 insectivorous bird species using almond orchards in my study area that may contribute to controlling carob moth. Almond flowers require cross pollination to set seed, and in north-west Victoria, over 100,000 European honeybee (Apis mellifera) hives are trucked into almond plantations each year, costing growers more than $7 million annually. Yet, almost nothing is known of the potential contribution that native pollinators could make to almond pollination in this region30. A more complete analysis could consider the costs and benefits, and ultimately net return, of the activities of a range of species.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was supported by an Australian Research Council Linkage Grant (LP0883952) and Future Fellowship (FT0990436), and a grant from the Gould League of New South Wales to GL.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nI thank administrative staff and farm managers at Select Harvests for their help in completing the study. Shannon Triplett, Hugh McGregor and Katrina Lumb assisted with field work, and Kai Chan and Robin Naidoo provided valuable comments on a draft manuscript.\n\n\nReferences\n\nHougner C, Colding J, Söderqvist T: Economic valuation of a seed dispersal service in the Stockholm National Urban Park, Sweden. Ecol Econ. 2006; 59: 364–374. Reference Source\n\nMols CM, Visser ME: Great tits (Parus major) reduce caterpillar damage in commercial apple orchards. PLoS One. 2007; 2(2): e202. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKellermann JL, Johnson MD, Stercho AM, et al.: Ecological and economic services provided by birds on Jamaican blue mountain coffee farms. Conserv Biol. 2008; 22(5): 1177–1185. PubMed Abstract | Publisher Full Text\n\nWhelan CJ, Wenny DG, Marquis RJ: Ecosystem services provided by birds. Ann N Y Acad Sci. 2008; 1134: 25–60. PubMed Abstract | Publisher Full Text\n\nAizen MA, Garibaldi LA, Cunningham SA, et al.: How much does agriculture depend on pollinators? Lessons from long-term trends in crop production. Ann Bot. 2009; 103(9): 1579–1588. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNaughton-Treves L: Predicting patterns of crop damage by wildlife around Kibale National Park, Uganda. Conserv Biol. 1998; 12(1): 156–168. Reference Source\n\nConover MR: Resolving Human–Wildlife Conflicts:The Science of Wildlife Damage Management. (CRC Press, Florida). 2001. Reference Source\n\nPimentel D: (Ed) Encyclopedia of Pest Management. (Marcel Dekker, New York) 2002; 931. Reference Source\n\nPérez E, Pacheco LF: Damage by large mammals to subsistence crops within a protected area in a montane forest of Bolivia. Crop Prot. 2006; 25(9): 933–939. Publisher Full Text\n\nLosey JE, Vaughan M: The economic value of ecological services provided by insects. BioScience. 2006; 56(4): 311–323. Publisher Full Text\n\nPimentel D, Zuniga R, Morrison D: Update on the environmental and economic costs associated with alien-invasive species in the United States. Ecol Econ. 2005; 52: 273–288. Reference Source\n\nTracey JP, Bomford M, Hart Q, et al.: Managing Bird Damage to Fruit and Other Horticultural Crops. (Bureau of Rural Sciences, Canberra). 2007. Reference Source\n\nTriplett S, Luck GW, Spooner P: The importance of managing the costs and benefits of bird activity for agricultural sustainability. International Journal of Agricultural Sustainability. 2012; 10(4): 268–288. Publisher Full Text\n\nZhang W, Ricketts TH, Kremen C, et al.: Ecosystem services and dis-services to agriculture. Ecol Econ. 2007; 64(2): 253–260. Publisher Full Text\n\nCarvalheiro LG, Seymour CL, Veldtman R, et al.: Pollination services decline with distance from natural habitat even in biodiversity-rich areas. J Appl Ecol. 2010; 47(4): 810–820. Publisher Full Text\n\nZhang W, Swinton SM: Optimal control of soybean aphid in the presence of natural enemies and the applied value of their ecosystem services. J Environ Manage. 2012; 96(1): 7–16. PubMed Abstract | Publisher Full Text\n\nGaribaldi LA, Steffan-Dewenter I, Winfree R, et al.: Wild pollinators enhance fruit set of crops regardless of honey bee abundance. Science. 2013; 339(6127): 1608–1611. PubMed Abstract | Publisher Full Text\n\nHanley ME, Goulson D: Introduced weeds pollinated by introduced bees: cause or effect? Weed Biology and Management. 2003; 3(4): 204–212. Publisher Full Text\n\nRoulston TH, Goodell K: The role of resources and risks in regulating wild bee populations. Annu Rev Entomol. 2011; 56: 293–312. PubMed Abstract | Publisher Full Text\n\nLuck GW, Triplett S, Spooner PG: (In press) Bird use of almond plantations: implications for conservation and production. Wildlife Research.Reference Source\n\nMehrnejad M: Biology of carob moth E. ceratoniae, new pest on pistachio in Rafsanjan. Applied Entomology and Phytopathology. 2002; 60: 1–11.\n\nNay JE, Perring TM: Impact of ant predation and heat on carob moth (Lepidoptera: Pyralidae) mortality in California date gardens. J Econ Entomol. 2005; 98(3): 725–731. PubMed Abstract\n\nKishani-Farahani H, Goldansaz SH, Sabahi Q: A survey on the overwintering larval parasitoids of Ectomyelois ceratoniae in three regions in Iran. Crop Prot. 2012; 36: 52–57. Reference Source\n\nSafdari A, Ghassemzadeh HR, Abdollahpour SHA, et al.: Design, construction and evaluation of a portable limb shaker for almond tree. Australian Journal of Agricultural Engineering. 2010; 1(5): 179–183. Reference Source\n\nSibbett GS, Curtis CE, Gerdts M, et al.: Effect on yield from shaking almond trees for mummy nut removal. California Agriculture. 1983; 37(7): 20. Publisher Full Text\n\nBomford M: Review of research on control of bird pests in Australia. Proceedings of the 15th Vertebrate Pest Conference. Paper 9. 1992. Reference Source\n\nTracey J, Saunders G: Bird Damage to the Wine Grape Industry. (Bureau of Rural Sciences, Canberra). 2003; 192. Reference Source\n\nAlexander P: In National Bird Pest Workshop Proceedings,. eds Fleming P, Temby I, Thompson J. (Department of Conservation, Forests and Lands; Victoria and NSW Agriculture and Fisheries, Orange), 1990; pp 25–33. Reference Source\n\nHagy HM, Linz GM, Bleier WJ: Optimizing the use of decoy plots for blackbird control in commercial sunflower. Crop Prot. 2008; 27(11): 1442–1447. Publisher Full Text\n\nSaunders ME, Luck GW, Mayfield MM: (In review) Almond orchards with living ground cover host more wild insect pollinators. Biodivers Conserv. 2013; 17(5): 1011–1025. Publisher Full Text\n\nHurlbert SH: Pseudoreplication and the design of ecological field experiments. Ecol Monogr. 1984; 54: 187–211. Reference Source\n\nSpangenberg JH, Settele J: Precisely incorrect? Monetising the value of ecosystem services. Ecological Complexity. 2010; 7(3): 327–337. Publisher Full Text\n\nTurner RK, Morse-Jones S, Fisher B: Ecosystem valuation. Ann N Y Acad Sci. 2010; 1185: 79–101. PubMed Abstract | Publisher Full Text\n\nZhang B, Li W, Xie G: Ecosystem services research in China: progress and perspective. Ecol Econ. 2010; 69(7): 1389–1395. Publisher Full Text\n\nAllsopp MH, de Lange WJ, Veldtman R: Valuing insect pollination services with cost of replacement. PLoS One. 2008; 3(9): e3128. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWinfree R, Gross BJ, Kremen C: Valuing pollination services to agriculture. Ecol Econ. 2011; 71: 80–88. Publisher Full Text\n\nIBM Corp. IBM SPSS Statistics for Windows, Version 21.0. (Armonk, New York). 2012. Reference Source\n\nKarp DS, Mendenhall CD, Sandi RF, et al.: Forest bolsters bird abundance, pest control and coffee yield. Ecol Lett. 2013; 16(11): 1339–1347. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "2406",
"date": "09 Dec 2013",
"name": "Erik Nelson",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe general framework of the paper seems to be appropriate: 1- Calculate the monetary benefit of birds feeding in almond patches (“free” predation of mummified almonds by birds in lieu of costly human removal); 2- add up the cost of these birds in almond patches (predation of harvest-able almonds by birds); 3- subtract the value of lost harvest-able almonds from the cost of human and/or machine work avoided. If the resulting value is greater than 0 then birds on Australian almond patches are, in net, beneficial to society and not a pest in almond systems (assuming the investigated almond patch is representative of other Australian almond farms). Luck’s main conclusion is that birds are more likely to be a benefit to society and not a pest in aggregate IF 1) they cause the more expensive hand-poling (versus mechanical shaking) of trees to be avoided and 2) we focus on the birds’ impact on the edges of almond plots (in Luck’s experiment birds did not seem to penetrate to the interior of the almond patch). My observations and concerns with the methodology:What about the combination of bird and human control of mummified almonds? What is the optimal level of human input to mummified almond control given the free input provided by birds? For example, could farmers use GPS technology to determine where to best compliment beneficial bird activity? Is there a way to quickly and relatively cheaply determine where bird activity needs to be supported? Up to this point too much of the “ecosystem service on farms” literature has presented animal input as an either/or option (e.g., wild pollinators versus rented hives on animal pollination-limited crops). We need to start looking more and more at the OPTIMAL combination of human and natural input (e.g., farmers making this type of calculation when they decide how much nitrogen fertilizer to add to a field given a map of pre-existing nitrogen levels). How would we go about determining the optimal management of almond farms where birds are explicitly considered to be part of the system? How would the planting of decoy crops for birds on the periphery of the almond farm contribute to optimal management of farms? In the experiment Luck only nets branches and not whole trees. An experiment with whole trees netted would seem to be more appropriate. Luck’s explanation for not netting whole trees needs to be better defended in the text, and the potential issues with branch netting versus whole tree netting on experimental results need to be more fully considered. Luck relies a lot on linear extrapolation. For example, he found “mean nut loss from netted trees was 5.9%...so, if for example, 50 nuts were completely lost from an almond tree, I considered that three nuts (5.9%) were lost due to non-bird factors…” . First, he must mean that 5.9% were lost from the netted branches, not trees, as loss from branches was controlled, not whole trees. Second, is such a simple linear extrapolation from branch loss to tree loss appropriate? Luck thinks so, I am not so sure. I would like to see this issue investigated further. The market price of almonds can vary wildly year to year, just like any crop. Luck only uses market prices from one year to generate his conclusions. He needs to consider a greater range of prices in a sensitivity analysis. Luck needs to give us an idea of the relative use of manual hand-poling versus mechanical shaking of trees on almond farms now and in the future. Do most farmers mechanically shake trees? What is the trend? Is mechanical shaking becoming cheaper and more widespread over time? Information on this trend would seem to affect the direction of study into the impact of birds in the almond system. If mechanical shaking is becoming the norm than birds are likely to be, in net, a pest in the future and the most animal-friendly management of this pest should become the focus of research. Further, what are the future projections for almond prices? This would also affect conclusions on promising future research directions.",
"responses": [
{
"c_id": "781",
"date": "24 Apr 2014",
"name": "Gary Luck",
"role": "Author Response F1000Research Advisory Board Member",
"response": "I thank Dr Nelson for taking the time to review this paper, and for his insightful comments on the study. I have responded to each comment below and made changes to the paper where required to address relevant concerns.This is an excellent point – and, as the referee notes, a criticism that can be leveled at numerous studies of ecosystem services. I actually believe that I dealt with the issue of how both bird and human control of mummy nuts will likely be required when I highlighted the fact that birds are only removing a proportion of mummy nuts prior to the next almond flowering. Please see the last two paragraphs of the Results where I demonstrate that birds were removing only a certain percentage of mummy nuts and how this translated into reduced costs to growers. Implicit in this is that if growers want 100% of mummy nuts removed, they are going to have to employ mechanical or manual methods to supplement bird activity. I also raise this issue at various points in the Discussion, but maybe I wasn’t explicit enough (see new text below).I’m not sure I am comfortable in making pronouncements about a definitive ‘optimal’ combination of human and natural input. It is important to recognise that ecosystem service input will vary from one year to the next owing to changing environmental conditions. For example, bird numbers and activity in almond orchards can vary significantly with rainfall (Luck et al., 2013) Moreover, the cost of bird damage will vary with fluctuations in the market price of almonds, and the value of the ecosystem service will differ with fluctuations in replacement costs (e.g. change in salary rates).Hence, any optimal combination will vary from one year to the next. I argue that it is less about identifying an optimal combination (if it indeed exists) and more about understanding how cost-benefit trade-offs will change given different environmental conditions (affecting ecosystem-service delivery) and fluctuating monetary values (of both costs and benefits). Key to this is recognising what factors need to be measured or monitored each year so that growers can make informed decisions about orchard management that will minimise costs, maximise benefits, and not undermine current and future potential for ecosystem-service delivery.To highlight this issue properly, I have added the following text to the Discussion:\"The fact that birds did not remove all mummy nuts prior to the next almond flowering highlights the important point that ecosystem-service provision in this case does not replace completely the need for human input to achieve acceptable outcomes. A combination of human action and ecosystem-service provision is required, and I suggest this is likely to be the case in many different systems. It is important to determine, therefore, how the proportional contribution required from human action or other organisms changes with different environmental, social and economic conditions. For example, in my study, because bird activity was highest at crop edges, the proportional contribution by farm managers to mummy nut control may be minor in this location, but increase further from the edge.” I have added the following new text to the methods section (3rd paragraph under ‘Costs’).“Netting whole trees was problematic and could have compromised the integrity of the experiment in several ways. Netting the entire tree would have disrupted regular orchard maintenance such as selective pruning and the spraying of herbicides to control weed growth. Hence, netted trees would have differed from non-netted trees in more than just the presence of a net. Second, strong wind is locally common and can be particularly problematic at orchard edges, which have little protection from the elements. Given the structure of almond trees, and the closeness of plantings, there was a real threat of nets ripping during strong winds if placed over the entire tree. Third, the larger the area netted, the more likely that birds will find gaps in netting. It is extremely difficult to ensure that birds are completely excluded from an entire tree even when careful attention is paid to closing all visible gaps in the net. Owing to these concerns, I decided that netting a single branch was a more acceptable method to employ. I do not believe that this approach undermines the general conclusions of the study.” To avoid confusion, I have re-worded the relevant sentence as follows:“Mean nut loss from netted trees (i.e. netted branches) was 5.9% (± SEM 0.82%, n = 60), so if, for example, 50 nuts were completely lost from an open tree [branch], I considered that three nuts (5.9%) were lost due to non-bird factors and the remainder due to birds.”It is important to note that in this particular sentence I am not extrapolating values from branches to whole trees, but using nut loss from netted branches caused by non-bird factors (e.g. wind, tree health) to determine what percentage of nuts from open branches were likely to also have been lost owing to non-bird factors. Later in the paper, I do extrapolate yield losses per branch to whole trees based on values obtained from a related study (Luck et al., 2013) that measured the actual number of nuts across 200 trees. While this extrapolation is linear, it is the best estimate available given current knowledge of bird activity and damage in almond orchards. This is a valid point, but it is a criticism that can be leveled at every study that attempts to value costs or benefits of animal activity in crops in monetary terms. In this comment though, the referee only acknowledges one side of the equation in relation to the current study. Not only will market prices for almonds fluctuate, but so will the costs associated with replacing the ecosystem service such as the running costs of the mechanical shaker or the salary costs of orchard workers. Hence, any sensitivity analysis, should it be required, would need to consider both the costs of bird activity as market values of almonds change, and the ecosystem-service value of their activity with changes in the replacement costs of the service. This may or may not yield a substantially different net return. I had attempted to present this cost-benefit trade-off given differing costs of damage and value of the ecosystem service in Figure 5. This figure is, in essence, a sensitivity analysis of the cost-benefit and net return relationship given variation in monetary amounts, though I acknowledge that altering the market price of almonds was not part of the initial analysis.Therefore, to address this concern, I have added two new trend lines to Figure 5 which show how cost-benefit trade-offs vary under different market prices for almonds based on the expected 2013 price and a hypothetical 2014 price assuming the price increase from 2013-14 is equivalent to the increase from 2012-13. The purpose of my study was to argue for greater attention to be paid to cost-benefit trade-offs associated with animal activity in agriculture and to present an example of how these cost-benefit trade-offs could be examined. I would assume that if researchers in the future adopt my methods they will substitute the most relevant market prices and replacement costs etc. to calculate the most appropriate net return for their given situation.To address this issue further I have added the following text to the Discussion:“It is also important to note that the sign and the magnitude of the net return of animal activity will vary with fluctuations in the market prices for crops and the costs of replacing the ecosystem service (e.g. the running costs of machinery and the salary costs of orchard workers). An analysis of how cost-benefit trade-offs change with varying monetary values is provided in Figure 5, including trend lines based on different market prices of almonds. Researchers that adopt the approach of calculating a net return from animal activity should use the most recent and relevant monetary values for their specific situation.” Attempting to predict the future is always fraught with danger, so I do not believe it is appropriate to speculate on how almond prices or farm management will vary in coming years. The Australian almond industry is very reliant on the availability of water for irrigation, and in Australia, rainfall from year to year can be highly unpredictable and this will impact on farm management. Also, different farms are managed in different ways and it is not appropriate to generalise across numerous farms. Moreover, the threat of the carob moth has only recently been acknowledged and its presence in orchards may greatly alter orchard management into the future."
}
]
},
{
"id": "4503",
"date": "16 Apr 2014",
"name": "Stijn Reinhard",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe research seems to be performed well, but some remarks can be made with regard to the description and application of the research results.The objective of the paper is to quantify the costs and benefits of animal activity for society. Animal activity is related to ecosystem services. In this paper one ecosystem service is analysed: birds eating almonds in almond orchards in southern Australia. When birds eat pre-harvest almonds it is considered a cost and post-harvest consumption of almonds is regarded a benefit of bird activity. The benefits are associated with the prevention of fungal infection. Hence, the title is somewhat misleading. It should be more precisely: The net return from animal activity in agro-ecosystem services: trading off costs and benefits from ecosystem services. For an economic analysis, the context of the research objective should be clear. Are birds considered a pest by farmers (or the society) and are they looking for measures to reduce the nuisance. In that case the optimal measure to reduce the costs of bird eating almonds could be computed using a cost benefit analysis, taking into account various measures. A costs benefits analysis is usually estimated for the ex-ante evaluation of a project. The project is not clear in this paper. Costs are computed based on an analysis of branches of almond trees that are netted to prevent birds eating almonds before harvest. But although it is not explicitly stated in the paper, netting entire trees does not seem to be an attractive measure. Shooting to scare is mentioned as a measure, but this measure is not analysed (as a difference between shooting and no-shooting). It seems that shooting was practiced during the analysis, the impact of shooting on the results is not analysed explicitly. The benefits of almond removal by birds are calculated based on various assumptions on nut removal by the farmer (mechanically or by hand) and on the (labour) costs associated with this nut removal. Using actual (bookkeeping) information of the orchard (or similar orchards) would lead to a more robust analysis. The real benefit is the prevention of fungal infection. The author is not aware of Australian guidelines on the acceptable quantity of mummy nuts to prevent infection. His assumption that 2 nuts per tree is acceptable will affect the confidence interval of the results. The ‘generalized linear model with a binominal response’ used (page 5) needs to be elaborated in more detail to enable a review. Also the spatial configuration (based on edge and interior trees, and height sectors of trees) of the orchard needs to be described in more detail. The paper aims to more accurately reflect the outcomes of animal activity for society. It is not clear how these results can be applied by other orchards. Is the amount of birds feeding in the orchard representative for other orchards? Application of the results by farmers also depends on the assumptions used by the author. How sensitive are the results for the almond market price, the (increasing) acreage of almond plantations, variability in weather (and climate), the labour costs, the trend in mechanical shaking of trees? Do the results vary over years? The paper describes a partial analysis of ecosystem services of almond eating birds, e.g. other services these birds provide for society are not included. A more economically grounded approach will be necessary for a more complete analysis of these services to advice farmers or to inform the society on the value of nature.",
"responses": [
{
"c_id": "782",
"date": "24 Apr 2014",
"name": "Gary Luck",
"role": "Author Response F1000Research Advisory Board Member",
"response": "I thank Dr Reinhard for taking the time to review this paper, and for his constructive comments on the study. I have responded to each comment below and made changes to the paper where required to address relevant concerns. I believe the current title of my paper is most appropriate given the context of the study. The work I present is not an examination of trading off costs and benefits from ecosystem services. The animal activity in question (eating almonds) is only an ecosystem service in one context. In another context it inflicts costs to growers through damage to almonds pre-harvest. It is very important that this distinction remains clear rather than referring to the activity of eating almonds as just an ecosystem service. I set out the context of the study in the following paragraph: “The expansion of the almond industry raises substantial production and conservation management challenges. In Australia, almond crops attract a number of native bird species, especially parrots, which eat almonds during the growing season and reduce crop yield. However, the same bird species provide an ecosystem service to growers by eating residual nuts left on trees after the main crop has been harvested. These so-called ‘mummified nuts’ (mummy nuts) are susceptible to fungal infection, which may threaten future crop yields. Moreover, recent evidence shows that mummified nuts are used intensively by the carob moth (Ectomyelois ceratoniae) for food and breeding (http://australianalmonds.com.au/industry/conference_2012/proceedings). The carob moth is a pest of global significance, impacting the production of numerous crops worldwide including dates, figs, pistachio, citrus and pomegranate. It is a major emerging threat to the Australian almond industry, as it feeds on almond kernels rendering them unsuitable for human consumption. Mechanical or manual removal of mummy nuts post harvest is one approach to controlling moth outbreaks and fungal infections. However, birds are already providing this service to Australian growers – the question addressed in my study is whether the monetary value of this service outweighs the costs of bird damage to almonds, resulting in a positive net return from bird activity.” I argue that the results of a net return analysis should be used to guide the level of investment required in animal management in a given system. For example, if the net return of bird activity is positive then it would be unnecessary for growers to invest money in controlling bird activity. I raise this issue and provide an example in the context of the pest control strategy of shooting to scare as follows:“In almond plantations, birds are currently considered pests and are subject to control strategies such as shooting to scare. In my study, shooters were employed for an average of 120 days per season over the growing seasons of 2009/10 and 2010/11. Based on an 8-hr day and minimum wage ($15.96 hr-1), the cost of employing shooters is at least $15,322 season-1. Over a 15,500 ha plantation (the entire plantation estate for my study) this equates to about $1 ha-1, and appears to be a good investment if compared only to the cost of bird damage. For example, if shooting reduces bird damage by only 1% it would save growers $22.19 ha-1. Yet, in the context of an overall positive net return from bird activity, the investment in shooting may be completely unnecessary.” Please see my response to Point #2 from Dr Nelson's review, and the new text I have added in regards to the problems associated with netting whole trees. It was not my aim to analyse differences in bird activity with or without shooting to scare birds. It is well recognised that shooting to scare has minimal impact on bird activity (see citations 20, 26 and 27) as they quickly become habituated to this practice. Attempting a controlled experiment with and without shooting would be extremely difficult in my study area given shooting is conducted on an ad hoc basis and the sound of a gunshot travels large distances (and hence would compromise any ‘treatment’ sites supposedly free of shooting activity). Unfortunately, this detailed information is not recorded in a systematic way by growers and differs across different farms. Therefore, it was more appropriate to use the conservative estimate I included in my analysis, with the examination of how net returns vary under different cost and benefit scenarios (Figure 5 and related text). I discuss this issue at some length in the second paragraph of the Discussion, but I have added the following new text to clarify the issue further:“Nevertheless, if the acceptable quantity of mummy nuts that can be retained per tree is substantially higher than two, then the cost of mummy nut removal by growers is reduced even further.” I have expanded on the description of the edge/interior comparison and its related statistical analysis as follows:“A total of 120 trees were included in the experiment with 60 netted trees (treatment) and 60 open trees (control). Thirty netted trees and 30 open trees were located at the edge of almond blocks (the exterior two rows) and 30 netted trees and 30 open trees were located in the interior of almond blocks (the centre point furthest from the edge – approximately 300 m). An edge/interior contrast was included because bird activity tends to be highest close to block edges. Control and treatment trees were assigned systematically to maximize interspersion, and I maintained a minimum distance of four trees between each control and treatment in the same edge or interior row to avoid adjacency effects and spatial autocorrelation in damage impacts (e.g. netted trees impacting outcomes on non-netted trees).”“I determined if there was a block or row location (edge/interior) effect on nut loss attributed to birds or non-bird factors pre- and post-harvest using a generalized linear model with a binomial response. In this case, the binomial outcome was either nut damaged/lost vs. nut intact. That is, I compared nut outcome among almond blocks and edge and interior rows pre-harvest and post-harvest for open trees where the outcome could be either nut damaged/lost to bird activity or nut remained intact. I conducted the same analysis for netted trees pre- and post-harvest where nut outcome was affected by non-bird factors (e.g. wind). Row location was nested within block for these analyses and both were treated as random factors and p ≤ 0.05 was considered statistically significant.” Does the referee mean other almond orchards or other horticultural orchards? I don’t claim that the specific results I present are going to be transferable across a range of situations. That was never my intention. The aim of the paper is to present a case study of cost-benefit trade-offs from the activity of a given suite of animals. I do argue, however, that calculating the net return from animal activity in agro-ecosystems should be much more widely applied. Currently, research on ecosystem services mostly ignores the costs of animal activity, and research on animal pests largely ignores the benefits of animal activity. This has to change for a more holistic understanding of the outcomes of animal behaviour in different agricultural landscapes.I've added the following new text to the Discussion to make this clearer (second to last paragraph):“The specific results of the case study I present here may not be transferable across different contexts. Nevertheless, the need to calculate a net return from animal activity is broadly relevant because there are few situations in nature whereby the activity of animals in a given location is entirely beneficial or entirely detrimental – especially when considering a broad range of taxa.” See my response to Point #4 from Dr Nelson in regards to varying the market price of almonds including new text and new trend lines in Figure 5. Figure 5 shows trends in net returns given different labour costs, bird damage rates, time spent hand poling, different mechanical shaker rates etc. This is a great point and one I tried to highlight with the final paragraph of my Discussion as follows:“I've focused on a small suite of species and a single activity to clearly illustrate the net return from animal activity. Analyses could be extended to encompass more species and more activities, dependent on available ecological knowledge. For example, I have recorded 35 insectivorous bird species using almond orchards in my study area that may contribute to controlling carob moth. Almond flowers require cross pollination to set seed, and in north-west Victoria, over 100,000 European honeybee (Apis mellifera) hives are trucked into almond plantations each year, costing growers more than $7 million annually. Yet, almost nothing is known of the potential contribution that native pollinators could make to almond pollination in this region. A more complete analysis could consider the costs and benefits, and ultimately net return, of the activities of a range of species.”"
}
]
}
] | 1
|
https://f1000research.com/articles/2-239
|
https://f1000research.com/articles/3-81/v1
|
31 Mar 14
|
{
"type": "Case Report",
"title": "Case Report: retained gutta-percha as a cause for persistent maxillary sinusitis and pain",
"authors": [
"Benjamin L. Hodnett",
"Berrylin Ferguson",
"Benjamin L. Hodnett"
],
"abstract": "Dental sources of infection can produce acute and chronic maxillary sinusitis. In some cases, the source of the infection may be related to the presence of endodontic materials in the oral cavity. In this article, we report a case of retained gutta-percha in the maxillary sinus resulting in chronic sinusitis.",
"keywords": [
"Foreign bodies in soft tissues can often occur as a result of trauma. There are multiple case reports in the otolaryngology literature of projectiles that have become lodged in the paranasal sinuses1. Retained packing gauze from endoscopic sinus surgery has also been reported in the paranasal sinuses2. In the endodontic literature",
"many reports and reviews have described various materials that can cause disease of the maxillary sinuses3. In this case report",
"we present an interesting case of a patient who presented with recalcitrant maxillary sinusitis that was ultimately found to be related to retained endodontic material in her maxillary sinus."
],
"content": "Introduction\n\nForeign bodies in soft tissues can often occur as a result of trauma. There are multiple case reports in the otolaryngology literature of projectiles that have become lodged in the paranasal sinuses1. Retained packing gauze from endoscopic sinus surgery has also been reported in the paranasal sinuses2. In the endodontic literature, many reports and reviews have described various materials that can cause disease of the maxillary sinuses3. In this case report, we present an interesting case of a patient who presented with recalcitrant maxillary sinusitis that was ultimately found to be related to retained endodontic material in her maxillary sinus.\n\n\nCase report\n\nA 26-year-old Caucasian woman presented with a chief complaint of left-sided maxillary pain with intermittent, discoloured nasal drainage. Seven years prior to current presentation, the patient had reported a history of headaches, nasal congestion and bilateral discolored drainage refractory to prednisone, antibiotics, and endoscopic sinus surgery at an outside facility. Revision endoscopic sinus surgery one year after the initial surgery provided partial relief. Two years prior to current presentation, her left maxillary pain recurred, and a computed tomography (CT) scan revealed a left wisdom tooth projecting into her maxillary sinus. Following wisdom teeth extraction, she had marked improvement in pain and nasal drainage; however 8 weeks later, left maxillary pain returned and was associated with left-sided yellow nasal discharge. Her oral surgeon discovered an infection in the molar adjacent to the extracted wisdom tooth and performed a root canal.\n\nTwo weeks following the root canal, she presented with continued left maxillary tooth pain and left-sided discolored nasal discharge. Extraction of the molar failed to resolve her pain, and she was subsequently referred to the facial pain/headache clinic by her dentist where she was prescribed gabapentin 300 mg TID, which was ineffective. Her dentist also prescribed her multiple courses of clindamycin 150 mg TID and guaifenesin 600 gm QID, which would improve her pain and discoloured drainage. However, her symptoms would return after completing the antibiotics. On examination with rigid endoscopy, she had widely patent maxillary, ethmoid, and frontal sinus ostia with no purulence or polyposis. On flexible endoscopic examination of the floor of her left maxillary sinus, white to slightly yellow mucus was found. Aspiration of the mucus relieved the patient’s discomfort. Maxillary sinus cultures revealed few polymorphonuclear leukocytes, few mononuclear cells, and no microorganisms. Her most recent CT scan from two years prior to presentation at our facility was remarkable for minimal mucosal thickening of the floor of the left maxillary sinus was otherwise normal (Figure 1). An occult dental infection was considered high in the differential diagnosis; however, because no actual dental infection could be demonstrated, a medial maxillectomy was considered in order to facilitate topical washing of the left maxillary sinus. Follow-up appointments in the facial pain/headache clinic found “some features of migraine, but it is unclear whether headaches represent primary or secondary headaches with migraine features”. Repeat evaluations by her dentist found no evidence of a dental infection. Because of past improvement on an 8-week course of clindamycin 300 mg TID, she was prescribed a 12-week course of clindamycin 300 mg TDI before surgery was considered, and she was referred to an infectious disease specialist.\n\nOnly mild mucosal thickening of the floor of the left maxillary sinus and post-surgical changes related to endoscopic sinus surgery were noted.\n\nClindamycin initially improved her symptoms, but midway through the course her congestion returned. A bone scan was positive in the region of the left maxilla; however, a repeat indium scan was negative. Her CT scan was repeated and she was referred to an oral surgeon for evaluation (Figure 2–Figure 4).\n\nRetained gutta-percha (white arrow) noted in left maxillary sinus.\n\nThe CT scan was evaluated by an oral and maxillofacial surgeon who noted that there were two small remnants of the prior left maxillary root canal that had been performed two and half years ago (Figure 2–Figure 4). A combined endoscopic and Caldwell-Luc approach under computer-assisted navigation to drill out retained gutta-percha in the maxillary sinus resolved the patient’s pain and drainage immediately, without recurrence at her three month follow-up.\n\n\nDiscussion\n\nGutta-percha, a product of tropical rubber plants, has been used since the mid-1800s as an endodontic filling material following root canal procedures4. The chemical structure of gutta-percha is a trans-isomer of poly-isoprene, or natural rubber; however it is more crystalline than natural rubber and often is formulated with medications such as zinc oxide, iodoform, chlorhexidine and calcium hydroxide, which contribute to both the antibacterial and antifungal activity4–7. Animal studies have shown that gutta-percha becomes encapsulated by fibrous connective tissue with little inflammatory reaction8.\n\nMaxillary sinus complications from gutta-percha from root canals are rare. According to a previous case report, gutta-percha from a maxillary tooth root canal can migrate to and obstruct the maxillary ostium9. Gutta-percha has also been reported to migrate into the ethmoid sinus10. In the current patient, retained gutta-percha in the maxillary sinus resulted in chronic inflammation and a persistent sinusitis-type picture with nasal congestion, pain and drainage. Her symptoms preceding the sinus surgery may or may not have been related to dental infection, but she clearly improved following wisdom teeth extraction, and she relapsed due to the infection of the adjacent molar. The retained gutta-percha prevented the resolution of infection and symptoms, even following extraction of the affected tooth. Partial improvement with antibiotics directed to usual dental pathogens provided some evidence that the etiology of her symptoms was a dental infection. In patients with persistent sinusitis and a history of endodontic procedures, an evaluation for dental materials retained in or near the sinuses may be warranted to rule out an additional source of infection. Removal of these retained dental materials may require an external approach with drilling, which can be facilitated by endoscopic visualization through the Caldwell-Luc procedure.\n\nA review of the otolaryngology literature did not provide any additional case reports on retained gutta-percha. In fact, only two case reports were found in the oral and maxillofacial surgery literature that described retained gutta-percha in the paranasal sinuses. In the oral surgery and endodontic literature, there are multiple reports of aspergillosis occurring in the maxillary sinus as a result of overextension of root canals of maxillary teeth, especially using materials containing zinc oxide or formaldehyde3,11. One case series of aspergillosis of the maxillary sinus was found in the otolaryngology literature. In this series, 85 cases of aspergillosis of the maxillary sinus in non-immunosuppressed patients were reviewed. Of these, 94% presented evidence of a radio-opaque foreign body in the maxillary sinus, with 85% of the cases related to endodontic dental paste12.\n\nOur case report highlights the importance of investigating alternative sources of infection in cases of recalcitrant sinusitis. Dental sources of infection as well as retained or overextended endodontic materials should be investigated in patients with unexplained, chronic sinusitis.\n\n\nConsent\n\nWritten informed consent for publication of clinical details and clinical images was obtained from the patient.",
"appendix": "Author contributions\n\n\n\nBenjamin Hodnett wrote the manuscript. Berrylin Ferguson revised the manuscript. All authors approved the final manuscript for publication.\n\n\nCompeting interests\n\n\n\nNo competing interests were declared.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nYarlagadda B, Jalisi S, Burke P, et al.: Retrieval of projectile foreign bodies from the paranasal sinuses and skull base. Am J Rhinol Allergy. 2012; 26(3): 233–236. PubMed Abstract | Publisher Full Text\n\nGotwald TF, Sprinzl GM, Fischer H, et al.: Retained packing gauze in the ethmoidal sinuses after endonasal sinus surgery: CT and surgical appearances. AJR Am J Roentgenol. 2001; 177(6): 1487–9. PubMed Abstract | Publisher Full Text\n\nHauman CH, Chandler NP, Tong DC: Endodontic implications of the maxillary sinus: a review. Int Endod J. 2002; 35(2): 127–141. PubMed Abstract | Publisher Full Text\n\nFriedman CM, Sandrik JL, Heuer MA, et al.: Composition and mechanical properties of gutta-percha endodontic points. J Dent Res. 1975; 54(5): 921–925. PubMed Abstract | Publisher Full Text\n\nBodrumlu E, Alaçam T: The antimicrobial and antifungal activity of a root canal core material. J Am Dent Assoc. 2007; 138(9): 1228–1232. PubMed Abstract | Publisher Full Text\n\nTanomaru JM, Pappen FG, Tanomaru Filho M, et al.: In vitro antimicrobial activity of different gutta-percha points and calcium hydroxide pastes. Braz Oral Res. 2007; 21(1): 35–39. PubMed Abstract | Publisher Full Text\n\nBodrumlu E, Alaçam T: Evaluation of antimicrobial and antifungal effects of iodoform integrating gutta-percha. J Can Dent Assoc. 2006; 72(8): 733. PubMed Abstract\n\nSugawara A, Nishiyama M, Kusama K, et al.: Histopathological reactions of calcium phosphate cement. Dent Mater J. 1992; 11(1): 11–16. PubMed Abstract | Publisher Full Text\n\nCosta F, Robiony M, Toro C, et al.: Endoscopically assisted procedure for removal of a foreign body from the maxillary sinus and contemporary endodontic surgical treatment of the tooth. Head Face Med. 2006; 2: 37. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIshikawa M, Mizuno T, Yamazaki Y, et al.: Migration of gutta-percha point from a root canal into the ethmoid sinus. Br J Oral Maxillofac Surg. 2004; 42(1): 58–60. PubMed Abstract | Publisher Full Text\n\nGiardino L, Pontieri F, Savoldi E, et al.: Aspergillus mycetoma of the maxillary sinus secondary to overfilling of a root canal. J Endod. 2006; 32(7): 692–694. PubMed Abstract | Publisher Full Text\n\nLegent F, Billet J, Beauvillain C, et al.: The role of dental canal fillings in the development of Aspergillus sinusitis. A report of 85 cases. Arch Otorhinolaryngol. 1989; 246(5): 318–320. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "4316",
"date": "09 Apr 2014",
"name": "Rakesh K Chandra",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAn interesting case that creates awareness about odontogenic sources of maxillary sinusitis. the authors should describe any histopathology findings - what type of inflammation was seen - eg neutrophilic, eosinophilic, histiocytes, foreign body giant cell, etc.",
"responses": [
{
"c_id": "767",
"date": "11 Apr 2014",
"name": "Benjamin Hodnett",
"role": "Author Response",
"response": "No pathological specimen was available. The foreign material was embedded in the bone and drilled out by the oral surgeons."
}
]
},
{
"id": "4314",
"date": "09 Apr 2014",
"name": "Alkis Psaltis",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe value of single patient case reports to the scientific literature is debatable. Other than facilitating the documentation of rare conditions or experimental techniques, they are usually just articles of interest. Having said this, this well written article describing an odontogenic cause of refractory left maxillary sinusitis does highlight the importance of a broad differential when treating patients with sinus symptoms. My only criticism is the imaging provided and its purpose. The arrows used to highlight the foreign material, particularly in the coronal CT scan, appear to be pointing into space. Furthermore, there is little evidence of mucosal inflammation or osteitis, that one would expect to see in a patient with a foreign body reaction with symptoms of nasal discharge. Nevertheless, the significant improvement the patient experienced on removal of the retained gutta-percha does suggest that this was indeed the underlying cause of her symptoms.",
"responses": [
{
"c_id": "766",
"date": "11 Apr 2014",
"name": "Benjamin Hodnett",
"role": "Author Response",
"response": "This CT scan was performed at the completion of an antibiotics course, which may explain the lack of inflammation. The radiology reports minimal inflammation as well. The patient had been on multiple courses of antibiotics with symptomatic improvement for a period of time after each treatment. The lack of osteitis is interesting. However, as stated in the discussion, gutta-percha in animals becomes encapsulated by fibrous connective tissue with little inflammatory response. The mechanism by which an encapsulated foreign body with little surrounding inflammation may precipitate a chronic sinus infection is indeed unusual. We will also provide additional images which should better illustrate the foreign material."
}
]
},
{
"id": "4315",
"date": "16 Apr 2014",
"name": "Michael P. Platt",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting case that highlights the importance of identifying odontogenic causes for chronic maxillary sinusitis.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-81
|
https://f1000research.com/articles/3-96/v1
|
25 Apr 14
|
{
"type": "Review",
"title": "An analysis on the entity annotations in biological corpora",
"authors": [
"Mariana Neves"
],
"abstract": "Collection of documents annotated with semantic entities and relationships are crucial resources to support development and evaluation of text mining solutions for the biomedical domain. Here I present an overview of 36 corpora and show an analysis on the semantic annotations they contain. Annotations for entity types were classified into six semantic groups and an overview on the semantic entities which can be found in each corpus is shown. Results show that while some semantic entities, such as genes, proteins and chemicals are consistently annotated in many collections, corpora available for diseases, variations and mutations are still few, in spite of their importance in the biological domain.",
"keywords": [
"Annotated collections of documents are crucial components for developing new methods in text mining",
"such as extraction of named entities and relationships from the scientific literature. This lies in the fact that supervised learning systems need to rely on annotated documents to train the algorithms",
"and therefore",
"“learn” how to efficiently perform a certain task. Additionally",
"use of a standard collection of documents is practically the only way of performing an unbiased comparison between different systems for a particular task."
],
"content": "Introduction\n\nAnnotated collections of documents are crucial components for developing new methods in text mining, such as extraction of named entities and relationships from the scientific literature. This lies in the fact that supervised learning systems need to rely on annotated documents to train the algorithms, and therefore, “learn” how to efficiently perform a certain task. Additionally, use of a standard collection of documents is practically the only way of performing an unbiased comparison between different systems for a particular task.\n\nIn natural language processing (NLP), a corpus can be defined as a collection of documents which usually belongs to a particular topic and that has been annotated according to a pre-defined schema. When annotating semantic information, a schema is usually composed of some entities (e.g., genes, proteins), and optionally, relationships (e.g., protein-protein interactions, gene-disease relationships). The number of documents may vary from a couple of full text documents1,2, to hundreds of abstracts3 or thousands of sentences4.\n\nA schema can be composed of an arbitrary list of annotation types or based on terms pertaining to one or more ontologies. For example, the Variome1 and the CellFinder2 corpora contain annotations for a pre-defined list of entities, such as genes/proteins, cell lines, diseases and mutations. On the other hand, the CRAFT corpus5 includes annotations according to concepts in seven ontologies and terminologies to allow a better identification of the annotations and their interoperability with other biomedical resources. The annotation schema is usually part of a comprehensive guideline document in which more details of the annotation process are described, such as an overview of the concepts, the provenance of the documents and examples of situations where the annotation should (or should not) be carried out.\n\nCorpora are usually constructed for training or evaluation purposes during the development of a particular system (e.g., Gerner et al.6) but are often also created in the context of a challenge or shared task (e.g., Krallinger et al.7) to foster improvements on a particular task and allow comparison between different solutions. Corpora are usually manually annotated by human experts in a particular field or automatically derived using NLP techniques. When manually constructed by one or more annotators, it receives the denomination of a gold-standard corpus. In this process, annotators are required to carefully read the texts and manually annotate the text according to the pre-defined schema. This annotation process is usually supported by an annotation tool, such as Brat8 or Knowtator9, which provides a nice graphical user interface and ways to previously configure the annotation schema. A comprehensive survey of the annotation tools for the biomedical domain can be found in Neves et al.10. A good approach on corpus construction should include training for the particular annotation tool and the guidelines, an inter-annotator agreement and the construction of a consensus corpus derived from the later.\n\nFrequently, manual annotation is supported by text mining by providing automatically extracted annotations which are later validated by the annotators. This validation process should not only include checking the annotations which were automatically extracted by the text mining tools but also carefully reading the text to identify missing ones. Hybrid corpora in which part of the annotations correspond to non-validated automatic annotations and then manually annotated with others, such as relationships, can also be found. For instance, for the Drug-Drug Interaction corpus11, drugs were automatically annotated using the Metamap tool12 followed by the manual annotation of relationships by experts.\n\nFinally, corpora can also be completely derived from automatized methods and never manually validated by experts, the so-called silver-standard corpora. Despite the undeniable presence of wrong annotations and the absence of many others, previous works have demonstrated that these corpora can support development of semi-supervised or distant supervised systems for named-entity13 and relationship extraction14. As manual annotation or validation is not required in this case, such corpora tends to be much larger than the gold-standard ones. CALBC15 is an example of a silver-standard corpus derived from a community-based project which intended to automatically harmonize annotations generated from a variety of named-entity recognition tools.\n\nIn this work, I present a review on 36 corpora which are available for the biomedical natural language processing (BioNLP) domain and perform an analysis on the semantic types which they include. The motivation for this work is to provide the first comprehensive overview on BioNLP corpora and thus support choosing the most appropriate collection whenever necessary. Additionally, I show the impact of each corpora in the field and give insights for the construction of new corpora or for the extension of existing ones.\n\n\nCorpora and semantic types\n\nHere I present a comprehensive study on the semantic entities included in the gold-standard corpora which have been annotated for the named-entity recognition (NER), relationship extraction and event extraction tasks. Although there are corpora available for other BioNLP tasks, such as text classification16 and question answering17, these are not covered in this survey. I focus on gold-standard corpora which contain annotations for entity types, such as genes/proteins, chemicals and species. Thus, I also did not include corpora which have only text span annotations not related to a particular semantic entity, such as the Data Deposition corpus18 which contains annotations on data deposition statements. Given the focus on Biology, I did not consider corpora which were built with the medical domain in mind, such as BioText19 and Variome1. Silver-standard corpora, such as CALBC15, were also not included here. I also do not cover corpora which focused on the linguistic aspects rather on semantic annotations, such as the BioScope corpus20, which contains annotations for negations and speculations statements. Finally, only corpora which are still available for download were included.\n\nIn this section, I give an overview of 36 corpora made available on the BioNLP domain and describe how the semantic analysis of the corpora has taken place.\n\nHere I present the list of 36 corpora which have been considered in this study. For each of them, I include a brief description of its origin, which may include the type of documents it contains (abstracts and full texts), its annotation schema, tools which have been based on it, further extensions it has received and the number of citations its publications have received. Table 1 shows a summary of these corpora, including their first publications, year of release (according to the main publication), number of citations according to Google Scholar (as December-2013) and the corresponding URL. Some of the corpora I present here are included in the WBI repository (http://corpora.informatik.hu-berlin.de), which provides their full visualization using the Stav/Brat annotation tool8. The collections are presented in the alphabetical order.\n\nAIMed. The AIMed corpus21 contains annotation on proteins and protein-protein interactions (PPI) for 200 abstracts, which were selected from the documents for which curated annotations were found in the Database of Interacting Proteins (http://dip.doe-mbi.ucla.edu/dip/Main.cgi). The corpus is one of the five corpora widely used for the development of PPI extraction methods50 and thus, has been used for the development of a variety of PPI tools51.\n\nAnEM. The recently published AnEM corpus22 contains a total of 500 documents which contains annotations on the following anatomical entity types: organism subdivision, anatomical system, organ, multi-tissue structure, tissue, cell, developing anatomical structure, cellular component, organism substance, immaterial anatomical entity and pathological formation. It is probably the largest manually annotated corpus on anatomical entities and has been used for the development of the AnatomyTagger tool52.\n\nAZDC. The AZDC corpus23 contains almost 800 abstracts which includes the ones available in the EBI disease corpus (cf. below) and some from the Craven corpus (cf. also below). It contains annotations for diseases and normalization to UMLS unique concepts for some semantic subtypes and was used for the development of named-entity recognition tools for disease names, such as the recent DNorm system53.\n\nBacteria Gene Interaction. The Bacteria Gene Interaction (BGI) corpus24 was developed in the scope of the BioNLP Event Extraction Shared Tasks 2011 for assessing the extraction of genetic processes in Bacillus subtilis. It is derived from the LLL corpus (cf. below) for PPIs. This corpus has been extended for the Gene Regulation Network (GRN) task54 in the 2013 edition of the same challenge.\n\nBioCreative 2 Gene Mention. The BioCreative 2 Gene Mention4 corpus has been used in two editions of the BioCreative challenges (http://www.biocreative.org/) to foster improvements for gene/protein extraction. It is composed of sentences, opposed to documents, which were derived from Medline documents and contains annotation on gene and protein, though it does not make distinction between them. Given that it has been used in one of the most popular challenges in the BioNLP community, several studies have used this corpus for the development of gene/protein extraction systems, such as BANNER55.\n\nBioInfer. BioInfer25 is also one of the five popular corpora available for PPI50. It contains sentences derived from more than 800 documents and annotations are available for genes, DNA families or groups, proteins, protein complexes and protein families and groups. Just as the other five PPI corpora, the BioInfer corpus has been used for training and evaluation of several tools51.\n\nCellFinder. The CellFinder corpus2 was developed in the scope of the CellFinder database (http://cellfinder.de/) and includes annotations for six entity types (anatomical parts, cell lines, cell types, species and cell components) for 10 full text documents in the stem cell research field. This corpus has been mainly used for the evaluation of named-entity recognition approaches for the above entity types in Neves et al.2,56.\n\nCancer Genetics. The Cancer Genetics (CG) corpus26 was constructed for the Cancer Genetics task in the BioNLP Event Extraction Shared Task in 2013 and includes annotations on the development and progress of cancer. The corpus is composed of 600 abstracts split into three datasets and events are composed of anatomical and molecular entities, as well as annotations for organisms.\n\nCHEMDNER. The CHEMDNER corpus7 has been recently created in the scope of the CHEMDNER task in BioCreative IV for assessing performance of named-entity recognition tools for chemical compounds. It contains 10,000 abstracts split into training, development and test datasets and annotations for chemicals are classified in eight categories, such as systematic, formula or abbreviation.\n\nCRAFT. The CRAFT corpus5 is a recent and very comprehensive collection of 97 full text documents which has been annotated with concepts, such as gene/proteins, species, cells and chemicals, from nine ontologies and terminologies. The authors have reserved 30 of the full texts for a text mining challenge that is going to be carried out in the near future.\n\nCraven. The so-called Craven corpus27 is in fact a collection of three corpora which contains annotations on sub-cellular locations, PPIs and gene-disease associations. These corpora have been used for the development of methods for extracting the above binary relationships and support construction of knowledge bases.\n\nDrug-Drug Interaction. The Drug-Drug Interaction (DDI) corpus28 includes more than 700 documents derived from Medline and DrugBank, and includes annotations for drugs and binary relationships between them. It has been already evaluated on two shared tasks11,57 and thus, has been extensively used for both training and evaluation for NER and relatiosnhip extarction tasks.\n\nEBI Disease. The EBI Disease corpus29 is composed of 600 sentences selected from the Craven corpus (cf. above) which have been extended with associations to unique concepts in the UMLS terminologies.\n\nEDGAR. The EDGAR corpus30 contains annotations for genes, drugs and cells, including binary relationships between genes and drugs, genes and cells, and drugs and cells.\n\nEpigenetics and Post-translational Modifications. The Epigenetics and Post-translational Modifications (EPI) corpus31 was developed for the BioNLP Event Extraction Shared Task 2011 and contains 1,200 abstracts annotated with events related to epigenetic changes. Just like the Genia Event Extraction corpus (cf. below), it contains annotations for genes/proteins and annotations identified as “Entity” which might refer to a variety of entity types, such as cell locations or small molecules.\n\nEU-ADR. The EU-ADR corpus32 was constructed in the scope of the EU-ADR project, which aimed to automatically process health records. The corpus contains a total of 300 abstracts which are split into three groups, each containing annotations for two entity types and binary relationships: drug-target, drug-disease and target-disease.\n\nGeneReg. The GeneReg corpus33 is composed of 314 abstracts related to Escherichia coli and contains annotations of events for gene expression regulation. It has been created in order to allow its interoperability with the Genia corpus (cf. below) and other lexical resources, such as WordNet and the Specialist lexicon.\n\nGenia. The Genia corpus3 is probably one of the most popular corpora in the biomedical domain and has been used for the development of many named-entity tools, such as ABNER58, and also to assess systems in a shared task59. It contains 2,000 Medline abstracts with annotations based on the Genia ontology for DNA, RNA, proteins, lipids, cells, tissues, body parts and cell lines, among others.\n\nGenia Event Extraction corpora. The Genia Event Extraction (Genia EE) corpus34 has started from the annotation of 1,000 abstracts, half of the Genia corpus (cf. above), and was annotated with genes/proteins and biological events, such as gene expression and gene regulations. This version of the corpus was used for the BioNLP Event Extraction Shared Task which took place in 200960 and then extended with 15 full texts for the following edition of the challenge that took place in 201161. A new corpus composed of 34 full texts was constructed for the third edition of the shared task that took place in 201362. The corpora have been used for the development and comparison of a variety of systems for extracting events.\n\nGETM. The GETM corpus35 is composed of 150 abstracts derived from the development dataset of the Genia Event Extraction corpus (cf. above). Relationships were annotated between the gene expression events and the annotations for cells and anatomical locations which were present in the original corpus. It was used for the evaluation of a rule-based relationship extraction system on gene expression events in cell locations.\n\nGREC. The GREC corpus36 contains annotations for 240 Medline abstracts for events on gene regulation and expression related to ontologies, such as Gene Ontology and Sequence Ontology.\n\nHPRD50. The HPRD50 corpus37 has been created in the scope of the RelEx system and contains 50 abstracts and annotations for PPIs. The corpus is also one of the five PPI corpora50 and has been used for the development of a variety of PPI tools51.\n\nID. The ID corpus31 was developed for the BioNLP Event Extraction Shared Task 2011 and contains 30 full text documents annotated with biomolecular mechanisms of infectious diseases. The corpus is split into three datasets (training, development and testing) and events are related to annotations of proteins, chemicals and organisms.\n\nIEPA. The IEPA corpus38 is composed of more than 200 sentences extracted from Medline abstracts and is annotated with binary relationships between proteins. It is also one of the five popular corpora available for PPI50.\n\nLinnaeus. The Linnaeus corpus6 contains 100 full text documents annotated with annotations for organisms, all linked to identifiers in NCBI taxonomy (http://www.ncbi.nlm.nih.gov/taxonomy). It was built for the development of the Linnaeus system, one of the state-of-art tools for the annotation of organism names.\n\nLLL. The LLL corpus39 for PPI in Bacillus subtilis was release in the scope of the Learning Language in Logic (LLL) shared task and was later also included in the package of the five popular corpora available for PPIs50. The proteins are identified as agent or target in the relationships.\n\nMetabolites and Enzymes. The Metabolites and Enzymes corpus40 contains annotations for metabolites and enzymes names in almost 300 abstracts and was used for the evaluation of dictionary-based approaches for the recognition of these entity types.\n\nMutationFinder. The MutationFinder corpus41 is composed of 508 Medline abstracts annotated with mutations and it was used for the evaluation of the homonymous tool based on regular expression techniques.\n\nNagel. The Nagel corpus42 contains annotations for protein residues, species and mutations in 100 Medline abstracts which have been used for the evaluation of a system developed for the extraction of these triplets.\n\nNCBI Disease. The NCBI Disease corpus43 is composed of almost 800 abstracts derived from the AZDC corpus (cf. above) split into three datasets for training, development and blind testing. Annotations are classified into categories, such as modifier and specific disease, and it has been used for the development of the DNorm tool53.\n\nOSIRIS. The OSIRIS corpus44 contains abstracts annotated with genes and sequence variants and was used for the evaluation of a dictionary-based system developed for the extraction of the later. Annotations for genes are normalized to identifiers from the NCBI EntrezGene database (http://www.ncbi.nlm.nih.gov/gene).\n\nPathway Curation. The Pathway Curation (PC) corpus45 was created for the homonymous task in the BioNLP Event Extraction Shared Task 2013 in which participants were required to extract biomolecular events to support curation of pathways. It includes a total of 525 abstracts annotated with events which contain chemicals, gene, proteins, complexes and cellular components as arguments.\n\nPICAD. The PICAD corpus46 is another less popular PPI corpus composed of more that 1,000 sentences which were assembled in the scope of the development of a tool for this purpose.\n\nSCAI. The SCAI corpus47 includes 100 abstracts with annotations for chemicals and training and test datasets for the recognition of IUPAC names. This has been one of the most popular corpora for chemical named-entity recognition and has been used for the development of many tools, such as ChemSpot63.\n\nSNPCorpus. The SNPCorpus48 contains almost 300 abstracts and annotations for protein sequence and nucleotide sequence mutations and it has been used by the authors for extraction of these mentions from the text and their association to identifiers in biological databases.\n\nSpecies. The Species corpus49 has been recently built as an alternative to Linnaeus (cf. above). Instead of using full text documents, it aimed at providing more variability on the species names by using eight groups of 100 abstracts on the following categories: bacteriology, botany, entomology, medicine, mycology, protistology, virology, and zoology.\n\n\nSemantic analysis of corpora\n\nIn this section, I show an analysis of the semantic types of the annotations present in the corpora discussed above. This analysis has been carried out based on the publications associated with the corpora and sometimes by checking the annotation types for the corpora which are available at the WBI Corpora repository. Here I only consider those annotations which are meaningful enough to be associated with one of the pre-defined semantic types under consideration (cf. below). For instance, I do not consider the “Entity” annotations in the Genia Event Extraction corpus60.\n\nSix top level semantic types were decided based on the annotations available in the corpora and on the UMLS semantic types (http://semanticnetwork.nlm.nih.gov/SemGroups/SemGroups.txt). The following are the types along with their mapping to the UMLS sematic groups and types:\n\ngene/protein: semantic group \"Genes & Molecular Sequences\" (GENE), as well as the types T116 (Amino Acid, Peptide, or Protein) and T114 (Nucleic Acid, Nucleoside, or Nucleotide);\n\nvariant/mutation: semantic type T045 (Genetic Function);\n\ndrug/chemical: semantic group \"Chemicals & Drugs\" (CHEM), except for the types T116 and T114 which were considered gene/proteins (cf. above);\n\ncell/anatomy: sematic group \"Anatomy\" (ANAT);\n\ndisease: semantic group \"Disorders\" (DISO);\n\norganisms: semantic group \"Living beings\" (LIVB).\n\nThe gene/protein category covers a wide range of small molecules and includes gene, proteins, protein complexes, gene complexes, protein families/groups, RNA, DNA families/groups, regulons, etc. Most of the corpora which include these entities do not make a distinction between them, such as the BioCreative Gene Mention4. In the cell/anatomy semantic type, I include all kinds of cellular and anatomical locations, whether in vivo or in vitro, as follows: cell lines, cell types, cell components, sub-cellular locations, developing anatomical structures, anatomical systems, organs and tissues. Drugs and chemicals were put together in the same group as some corpora include both of them, although these are sometimes classified into categories. Variants and mutations were assembled in a single group and, finally, one category for diseases and one for species, which are more homogeneous groups and whose annotations are not usually classified in distinct categories in corpora.\n\n\nComparison and discussion\n\nIn this section I present an analysis of the semantic types for the named-entities present in 36 corpora. Figure 1 shows an overview of which annotations are available for each corpora, as well as which corpora contain annotations for a particular semantic type. It also gives an idea of the similarities between corpora in terms of the entity types they share.\n\nThe closer a corpus is to the center of the figure, more distinct semantic types it contains. The CRAFT corpus is the collection which contains the higher number of semantic entities, namely, gene/proteins, species, chemicals and cells, but still lacks annotations for disease, variants/mutations and anatomical parts. The Cancer Genetics (CG), CellFinder, EDGAR, EU-ADR, Infectious Disease (ID), Pathway Curation (PC) and Nagel corpora are the ones which come closer to the CRAFT corpus, each containing annotations for three different types, but with a great variability on which of these three types are considered.\n\nOn the other hand, the farther a corpus is to the center of the figure, less distinct semantic types it contains and most of the corpora fall into this situation. There are 12 corpora which contain only annotations for gene/proteins, three for diseases, two for variants or mutations, two for species or organisms and three for chemical or drugs. Curiously, no corpus contains annotations only for cell anatomical entities, except the AnEM corpus, which was also placed in the disease semantic types because it contains annotations on pathological formations.\n\nGenes and proteins are the most popular entities in biomedical corpora: in a total of 26 collections. However, these have different purpose and number of documents. Early initiatives, such as the BioCreative Gene Mention corpus, were based on sentences instead of documents, but following corpora have annotated the abstracts instead. Recently developed corpora include annotation of full text documents, such as CellFinder, CRAFT and the Genia Event Extraction, in order to allow systems to make use of the complexities of the languages which can only be found in the full text but not in the abstracts64. Most of the corpora classified in this group make no distinction between genes, proteins, complexes, or families, except for Genia and the Bacteria Gene Interaction corpora. Corpora whose annotations are mapped to identifiers in a database, e.g., EntrezGene, such as CRAFT and OSIRIS, allow their use for the development of gene/protein normalization tools65. Finally, the high number of corpora available for gene/protein corpora is due to the importance of these entities for the molecular biology domain and to the research in the last years on PPIs and biological events.\n\nCorpora which contained annotations for chemicals and drugs were few until the release of the SCAI corpus, which focused initially on the IUPAC nomenclature. But this has become a hot topic in the last couple of years and following corpora have provided annotations also for drugs and their interactions (DDI corpus), as well as anotations on full text documents (CRAFT corpus). The CHEMDNER corpus classifies chemicals in some predefined categories and was used in the one of the shared tasks in last BioCreative challenge, which attracted the participation of many teams. Relationships of chemical compounds with other semantic entities can be found in the EU-ADR and also for more complex events, such as in the shared tasks of Cancer Genetics and the Infectious Disease in the BioNLP Event Extraction Shared Tasks.\n\nDuring many years, the Linneaus corpus and tool have been the state-of-art resources for benchmarking and extraction of species annotations, respectively. The simplicity of the nomenclature and the high performance of Linnaeus has not encouraged further research in this line. However, the release of the Species corpus some months ago aims to provide more variety on the annotations for organism, by choosing a higher number of abstracts, as opposed to few full text documents in the Linnaeus corpus. Additionally, abstracts are grouped on eight categories of organisms (bacteriology, botany, entomology, medicine, mycology, protistology, virology, and zoology), thus, ensuring the diversity of annotations. Other recent full text corpora which contains annotations for organisms are the CellFinder and CRAFT corpora.\n\nAnnotations for cell and anatomical parts have since many years been limited to the cell lines and cell types in the Genia and EDGAR corpora. However, the recent release of the AnEM corpus, which include a careful classification of these entities based on many ontologies, along with the AnatomyTagger tool52, will certainly encourage new solutions in this area. Other recent corpora for cell annotations are the full text documents of the CRAFT corpus, including mapping to the Cell Ontology, as well as the annotations for cell lines, cells types and anatomical parts in the CellFinder corpus.\n\nMost corpora which contain annotations for diseases exclusively are somehow related to each other as all of them contain documents which have been selected from the AZDC and the Craven corpora. The recent release NCBI Disease corpora aims to improve research in this field by classifying mentions based on some pre-defined categories, followed by the release of the DNorm tool53. Associations of diseases with other entity types are still scarce and only present in the EU-ADR and Craven corpora.\n\nFinally, variations and mutations have also received little attention from the BioNLP community, and the four available corpora are composed only of abstracts. Co-occurrence of these entities in the text is available for genes in the OSIRIS corpus, however, no explicit relationships was annotated between them. Such relationships are only available with genes and species in the Nagel corpus, but its small size (100 abstracts) hinders text mining solutions based on machine learning methods, being only suitable for evaluation purposes.\n\nFrom Figure 1, it is straightforward to observe which corpora are available according to the entity types of interest. The aim of this study is to encourage the use of less popular corpora which are already available and whose suitability for the text mining tasks has been scientifically evaluated. However, when choosing to use more than one corpora, the text miners will probably need to deal with more than one format for the documents and annotations, and write specific parsers for each of them. This is a problem that the BioC initiative66 is aiming to solve with the recent introduction of the BioC XML format. Indeed, many of the corpora shown here have already been converted to this format using the Brat2BioC tool67 and made available in the WBI Corpora repository. Given that most of the corpora are available under a flexible license, this review will also serve as a starting point for further updates on the repository and allow not only their availability for visualization but also for download in the BioC format.\n\n\nConclusions\n\nIn this survey I presented an overview on the semantic entity types available for 36 corpora in the biomedical domain. The annotations were classified in six categories (gene/protein, drug/chemical, cell/anatomy, variant/mutation, species and disease) and an overview on which corpora contain each of these semantic types has been shown. I hope that this review can be of help when choosing the best corpora for developing a named entity recognition tool and also to encourage re-use (re-annotation) of existing corpora instead of building a new one.",
"appendix": "Competing interests\n\n\n\nThe author declares no competing interests.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nI would like to thank Prof. Ulf Leser for hosting the WBI Corpora repository and Dr. Antonio Jimeno Yepes for discussions on the UMLS semantic types.\n\n\nReferences\n\nVerspoor K, Jimeno Yepes A, Cavedon L, et al.: Annotating the biomedical literature for the human variome. Database (Oxford). 2013; 2013: bat019. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNeves M, Damaschun A, Kurtz A, et al.: Annotating and evaluating text for stem cell research. In Proceedings of the Third Workshop on Building and Evaluation Resources for Biomedical Text Mining (BioTxtM 2012) at Language Resources and Evaluation (LREC), Istanbul, Turkey. 2012; 16–23. Reference Source\n\nKim JD, Ohta T, Tateisi Y, et al.: GENIA corpus--semantically annotated corpus for bio-textmining. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nCaporaso JG, Baumgartner WA Jr, Randolph DA, et al.: MutationFinder: a high-performance system for extracting point mutation mentions from text. Bioinformatics. 2007; 23(14): 1862–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNagel K, Jimeno-Yepes A, Rebholz-Schuhmann D: Annotation of protein residues based on a literature analysis: cross-validation against UniProtKb. BMC Bioinformatics. 2009; 10(Suppl 8): S4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDoğan RI, Lu Z: An improved corpus of disease mentions in pubmed citations. In Proceedings of the 2012 Workshop on Biomedical Natural Language Processing, BioNLP ’12, Stroudsburg, PA, USA, 2012. Association for Computational Linguistics. 2012; 91–99. Reference Source\n\nFurlong LI, Dach H, Hofmann-Apitius M, et al.: OSIRISv1.2: A named entity recognition system for sequence variants of genes in biomedical literature. BMC Bioinformatics. 2008; 9(1): 84. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOhta T, Pyysalo S, Rak R, et al.: Overview of the pathway curation (pc) task of bionlp shared task 2013. In Proceedings of the BioNLP Shared Task 2013 Workshop, Sofia, Bulgaria, August 2013. Association for Computational Linguistics. 2013; 67–75. Reference Source\n\nBell L, Zhang J, Niu X: Mixture of logistic models and an ensemble approach for protein-protein interaction extraction. In Proceedings of the 2nd ACM Conference on Bioinformatics, Computational Biology and Biomedicine. BCB ’11, New York, NY, USA, ACM. 2011; 371–375. Publisher Full Text\n\nKolárik C, Klinger R, Friedrich CM, et al.: Chemical names: Terminological resources and corpora annotation. In Proc. of the Workshop on Building and Evaluating Resources for Biomedical Text Mining. 2008; 51–58. Reference Source\n\nThomas PE, Klinger R, Furlong LI, et al.: Challenges in the association of human single nucleotide polymorphism mentions with unique database identifiers. BMC Bioinformatics. 2011; 12(Suppl 4): S4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPafilis E, Frankild SP, Fanini L, et al.: The SPECIES and ORGANISMS Resources for Fast and Accurate Identification of Taxonomic Names in Text. PLoS One. 2013; 8(6): e65390. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPyysalo S, Airola A, Heimonen J, et al.: Comparative analysis of five protein-protein interaction corpora. BMC Bioinformatics. 2008; 9(Suppl 3): S6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTikk D, Thomas P, Palaga P, et al.: A comprehensive benchmark of kernel methods to extract protein–protein interactions from literature. PLoS Comput Biol. 2010; 6: e1000837. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPyysalo S, Ananiadou S: Anatomical entity mention recognition at literature scale. Bioinformatics. 2014; 30(6): 868–75. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLeaman R, Islamaj Dogan R, Lu Z: DNorm: disease name normalization with pairwise learning to rank. Bioinformatics. 2013; 29(22): 2909–17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBossy R, Bessières P, Nédellec C: Bionlp shared task 2013 – an overview of the genic regulation network task. In Proceedings of the BioNLP Shared Task 2013 Workshop, Sofia, Bulgaria, August 2013. Association for Computational Linguistics. 153–160. Reference Source\n\nLeaman R, Gonzalez G: BANNER: An executable survey of advances in biomedical named entity recognition. Pacific Symposium of Biocomputing. 2008; 652–63. PubMed Abstract\n\nNeves M, Damaschun A, Mah N, et al.: Preliminary evaluation of the CellFinder literature curation pipeline for gene expression in kidney cells and anatomical parts. Database (Oxford). 2013; 2013: bat020. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSegura-Bedmar I, Martínez P, Zazo MH: Semeval-2013 task 9: Extraction of drug-drug interactions from biomedical texts (ddiextraction 2013). In Second Joint Conference on Lexical and Computational Semantics (*SEM), Volume 2: Proceedings of the Seventh International Workshop on Semantic Evaluation (SemEval 2013), Atlanta, Georgia, USA Association for Computational Linguistics. 2013; 341–350. Reference Source\n\nSettles B: Abner: an open source tool for automatically tagging genes, proteins and other entity names in text. Bioinformatics. 2005; 21(14): 3191–2. PubMed Abstract | Publisher Full Text\n\nKim JD, Ohta T, Tsuruoka Y, et al.: Introduction to the bio-entity recognition task at jnlpba. In Proceedings of the International Joint Workshop on Natural Language Processing in Biomedicine and its Applications, JNLPBA ’04, Stroudsburg, PA USA, Association for Computational Linguistics. 2004; 70–75. Reference Source\n\nKim JD, Ohta T, Pyysalo S, et al.: Extracting bio-molecular events from literature — the bionlp’09 shared task. Computational Intelligence. 2011; 27(4): 513–540. Publisher Full Text\n\nKim JD, Nguyen N, Wang Y, et al.: The Genia Event and Protein Coreference tasks of the BioNLP Shared Task 2011. BMC Bioinformatics. 2012; 13(Suppl 11): S1. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKim JD, Wang Y, Yasunori Y: The genia event extraction shared task, 2013 edition overview. In Proceedings of the BioNLP Shared Task 2013 Workshop, Sofia, Bulgaria, August 2013, Association for Computational Linguistics. 8–15. Reference Source\n\nRocktäschel T, Weidlich M, Leser U: ChemSpot: A hybrid system for chemical named entity recognition. Bioinformatics. 2012; 28(12): 1633–40. PubMed Abstract | Publisher Full Text\n\nBretonnel Cohen K, Johnson H, Verspoor K, et al.: The structural and content aspects of abstracts versus bodies of full text journal articles are different. BMC Bioinformatics. 2010; 11(1): 492. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMorgan AA, Lu Z, Wang X, et al.: Overview of BioCreative II gene normalization. Genome Biol. 2008; 9(Suppl 2): S3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nComeau DC, Doğan R, Ciccarese P, et al.: Bioc: a minimalist approach to interoperability for biomedical text processing. Database (Oxford). 2013; 2013: bat064. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYepes AJ, Neves M, Verspoor K: Brat2bioc: conversion tool between brat and bioc. In BioCreative IV. 2013. Reference Source"
}
|
[
{
"id": "4561",
"date": "06 May 2014",
"name": "Roman Klinger",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper “An analysis on the entity annotations in biological corpora” is a review of several corpora which are available to the research community and which consist of textual data with annotation. These annotations are either on an entity level or with relations between entities.The author makes very clear what this paper is about, which corpora are discussed and where the border is drawn to resources not discussed here. The comprehensive list of corpora is enriched with citation counts to make clear which corpora are already commonly used and which might be under-explored by the community. She makes clear that she would like to motivate researches to make use of these corpora as well.In general, I think that this paper is a very valuable review of resources available to the community. The author does not define strictly what the motivation for this paper is, what the audience should be and who can specifically benefit from it. I could think of at least two scenarios:1. A researcher would like to improve or develop a method for a specific domain/entity class (like drug-drug-interaction or recognition of chemical names, for instance). Then the author can get an overview of available corpora and be quite sure that she or he does not miss a corpus of the specific domain when the class is mentioned in this review.2. A researcher would like to evaluate a method and needs resources, he or she does not necessarily care so much about the specific domain and can select from the variety of discussed resources in this review.I propose that such or similar motivations are added to the introduction.Title: I am not sure if a corpus can be “biological” — or dealing with classes from the bio(medical) domain.Abstract: Should indicate for whom this review might be of value. Some examples of cases for use would be great, I think.Introduction: A schema does not consist of entities only, but also of entity classes. Maybe it would be interesting to discuss the issue of having a bias in the annotation towards the automatic tool when annotations are only validated.“Such corpora tends…” —> “corpora tend” or “such corpus tends”“I show the impact of each corpora” —> “each corpus”The author cites the paper on the 5 reviews commonly used to study PPI. Are there no other such reviews?Corpora and semantic types:List of corpora:“I also did not include corpora which have only text span annotations not related to a particular semantic entity…” — I do not understand that. Several of the corpora the author is discussing do not have links to database or ontology IDs. This should be made clear. (examples are the BC2 GM corpus, the SCAI corpus, I think AZDC as well.)Abner is at least evaluated on BioCreative data as well. I am not sure it has only been trained on GENIA.Semantic Analysis of Corpora“I only consider those annotations which are meaningful enough to be associated with one of the pre-defined semantic types under consideration” I think that formulation could be improved to make clearer what ‘meaningful’ means.Comparison and Discussion“On the other hand” — without “On the one hand”“different number of documents” — “numbers”?“relationships was” -> “relationships were”“corpora which contains” -> “contain”",
"responses": []
},
{
"id": "5114",
"date": "14 Jul 2014",
"name": "Paloma Martínez",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article shows a review of 36 publicly annotated corpora in the biological domain. The analysis is performed taking into account different features such as type of text (abstracts, full text publications, etc.), types of entities, types of relationships (if any), number of documents, if automatic or manual annotation, if annotations are related to other resources (such as ontolologies), etc.It is an interesting and useful work that can help researchers to find a corpus to perform evaluations of their machine learning methods. As the author describes in the introduction sections, annotated corpora are valuable resources that allow to train and test algorithms and also to compare state-of-the art works each other.I think that the paper is a good contribution to the journal. Below I give several suggestions in order to improve the article as well as some data to correct a couple of mistakes.In the Introduction section, it would be beneficial to introduce to the reader the “inter-annotator agreement” as well as the main ways to calculate it. Before introducing the annotation schema it is necessary to give a definition of “Schema” (is it a conceptual schema? What are the elements a schema should contain) In the paragraph about DDI corpus, some remarks should be included: in the first version of the corpus used in the DDIExtraction 2011 task the drugs were automatically annotated by Metamap tool1 but in the new version (used in DDI Extraction 2013 shared tasks), every annotation was manually revised by two pharmacists) At the end of this section, I would like to see something about the precision experts have annotating entities and relationships. This would also help to know what is the limit systems are able to manage recognizing entities and relations. In section “Corpora and semantic types” it is a good idea to give the number of citations from Google Scholar, although the most recent corpora have almost no citations. The DDI corpus has 0 citations because the reference is from October 2013; Segura-Bedmar et al. (2011)1is another reference that the author could include in the article corresponding to the first version of the corpus. Concerning the figures of DDI corpus, it consists of 792 texts selected from the DrugBank database (DDI-DrugBank dataset) and other 233 Medline abstracts (DDI-MedLine dataset) on the subject of DDIs. The corpus was manually annotated with a total of 18,502 pharmacological substances and 5028 DDIs, including both pharmacokinetic (PK) as well as pharmacodynamic (PD) interactions. Concerning drugs, there are two corpora that do not appear in the article: (a) PK Corpus and PF DDI Corpus2 with approx. 600 abstracts about clinical pharmacokinetics and pharmacogenetics, in-vitro and in-vivo drug-drug interactions. (b) PK DDI corpus3 consisting of 64 FDA drug labels with annotations for drugs with their precipitant or object roles in drug-drug interaction. Instead of having a paragraph for each corpus, I suggest including a tabular representation of corpora and characteristics. For instance, with a column for each feature (document type, annotation tool, categories of entities, number of mentions, format, availability, etc.). This representation would help to compare different corpora. I also suggest mentioning that linguistic phenomena such as co-reference resolution are also required in annotation task, especially in the detection of entities. For instance, how are words such as “drug”, “disease”, “medication” and others annotated in these corpora? Some typos: In the section: List of biological corpora - Drug-Drug Interaction: “...has been extensively used for both training and evaluation for NER and relatiosnhip extarction tasks.”",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-96
|
https://f1000research.com/articles/3-83/v1
|
03 Apr 14
|
{
"type": "Opinion Article",
"title": "Recommendations to enable drug development for inherited neuropathies: Charcot-Marie-Tooth and Giant Axonal Neuropathy",
"authors": [
"Lori Sames",
"Allison Moore",
"Renée J.G. Arnold",
"Sean Ekins",
"Lori Sames",
"Allison Moore",
"Renée J.G. Arnold"
],
"abstract": "Approximately 1 in 2500 Americans suffer from Charcot-Marie-Tooth (CMT) disease. The underlying disease mechanisms are unique in most forms of CMT, with many point mutations on various genes causing a toxic accumulation of misfolded proteins. Symptoms of the disease often present within the first two decades of life, with CMT1A patients having reduced compound muscle and sensory action potentials, slow nerve conduction velocities, sensory loss, progressive distal weakness, foot and hand deformities, decreased reflexes, bilateral foot drop and about 5% become wheelchair bound. In contrast, the ultra-rare disease Giant Axonal Neuropathy (GAN) is frequently described as a recessively inherited condition that results in progressive nerve death. GAN usually appears in early childhood and progresses slowly as neuronal injury becomes more severe and leads to death in the second or third decade. There are currently no treatments for any of the forms of CMTs or GAN. We suggest that further clinical studies should analyse electrical impedance myography as an outcome measure for CMT. Further, additional quality of life (QoL) assessments for these CMTs are required, and we need to identify GAN biomarkers as well as develop new genetic testing panels for both diseases. We propose that using the Global Registry of Inherited Neuropathy (GRIN) could be useful for many of these studies. Patient advocacy groups and professional organizations (such as the Hereditary Neuropathy Foundation (HNF), Hannah's Hope Fund (HHF), The Neuropathy Association (TNA) and the American Association of Neuromuscular and Electrodiagnostic Medicine (AANEM) can play a central role in educating clinicians and patients. Undertaking these studies will assist in the correct diagnosis of disease recruiting patients for clinical studies, and will ultimately improve the endpoints for clinical trials. By addressing obstacles that prevent industry investment in various forms of inherited neuropathies, we can envision treatment options for these rare diseases in the near future.",
"keywords": [
"There are currently no approved treatments for any of the forms of inherited neuropathy (IN)",
"which are each considered rare diseases. Charcot-Marie-Tooth (CMT) Disease 1A is the most common form of IN. The underlying disease mechanisms are unique in the different forms of CMT (e.g. CMT1A",
"CMT2",
"etc.)1. To date",
"at least 51 causal genes have been implicated in CMT2. CMT Type 1 diseases are demyelinating and Type 2s are axonal neuropathies3. Based on the prevalence of all CMTs being 1:2500 and the recent census figures of over 300 million people in the United States of America (USA)",
"it is likely that more than 75",
"000 Americans have CMT1A",
"which is caused by the peripheral myelin protein 22 (PMP22) duplication gene phenotype",
"which represents 68.7% of all CMT Type 1s in the USA4. Symptoms of the disease often present in the first two decades of life5",
"with patients having reduced compound muscle and sensory action potentials",
"slow nerve conduction velocities",
"sensory loss",
"progressive distal weakness",
"foot and hand deformities",
"decreased reflexes",
"bilateral foot drop and about 5% becoming wheelchair bound. Of the axonopathies",
"CMT2A is the most common form. CMT2A is primarily caused by mutations in the gene encoding mitofusin (MFN2)6. Mutations or rearrangements in PMP22",
"myelin protein zero (MPZ) and gap junction β1-protein (GJB1) or MFN2 account for 90% of molecular diagnoses7. CMT2A has been described to severely affect infants and children",
"with most affected individuals non-ambulatory by age 20",
"and having profound proprioception loss in addition to weakness6."
],
"content": "Introduction\n\nThere are currently no approved treatments for any of the forms of inherited neuropathy (IN), which are each considered rare diseases. Charcot-Marie-Tooth (CMT) Disease 1A is the most common form of IN. The underlying disease mechanisms are unique in the different forms of CMT (e.g. CMT1A, CMT2, etc.)1. To date, at least 51 causal genes have been implicated in CMT2. CMT Type 1 diseases are demyelinating and Type 2s are axonal neuropathies3. Based on the prevalence of all CMTs being 1:2500 and the recent census figures of over 300 million people in the United States of America (USA), it is likely that more than 75,000 Americans have CMT1A, which is caused by the peripheral myelin protein 22 (PMP22) duplication gene phenotype, which represents 68.7% of all CMT Type 1s in the USA4. Symptoms of the disease often present in the first two decades of life5, with patients having reduced compound muscle and sensory action potentials, slow nerve conduction velocities, sensory loss, progressive distal weakness, foot and hand deformities, decreased reflexes, bilateral foot drop and about 5% becoming wheelchair bound. Of the axonopathies, CMT2A is the most common form. CMT2A is primarily caused by mutations in the gene encoding mitofusin (MFN2)6. Mutations or rearrangements in PMP22, myelin protein zero (MPZ) and gap junction β1-protein (GJB1) or MFN2 account for 90% of molecular diagnoses7. CMT2A has been described to severely affect infants and children, with most affected individuals non-ambulatory by age 20, and having profound proprioception loss in addition to weakness6.\n\nOther than braces or other orthopedic devices, there are no effective therapies for any of the various forms of CMTs8. Unfortunately, CMT1A is also a textbook case of how ground-breaking fundamental discoveries by academic scientists are not being rapidly translated into therapeutics quickly enough. For example, the causal gene duplication defect for the most common form of CMT (PMP22) was identified in 19919,10 and confirmation that the resulting elevated gene dosage is responsible for the pathology of the disease was established in 1993, yet the first high throughput screen (HTS) to search for a small molecule therapeutic was not published until 201211. This represents a 21-year gap in translation from gene to screen. The tide is now slowly changing and a small pipeline of molecules currently exists for CMT1A. We are aware of at least two companies with preclinical and clinical compounds. Recently, a press release from Addex Therapeutics reported that the preclinical compound Addex ADX71441 reduces PMP22 expression in a dose-dependent manner comparable to baclofen in a pre-clinical transgenic mouse model of CMT1A disease. This drug is a new molecular entity that will require extensive preclinical testing, thus it is many years (possibly a decade) from US Food and Drug Administration (FDA) approval. Pharnext is a French pharmaceutical company which has just completed a Phase II trial (manuscript in preparation) for a combination therapy (baclofen, naltrexone and sorbitol) for CMT1A12. All of these components are already FDA-approved, so if the Phase III clinical trial (proposed for 2014, Dr. Daniel Cohen, Pharnext personal communication) is successful, this could be the first treatment to market for any form of IN. In addition, the coming five years will likely be an explosive time in CMT2A research as the long-awaited, very difficult to engineer, CMT2A knock out mouse model is being characterized by Dr. David Pleasure (University of California, Davis). Preliminary data reveal that this model will be useful for better understanding the mechanisms behind the disease (molecular targets) — and will represent a putative model for testing the efficacy and safety of investigational therapeutics.\n\nRecent clinical studies of other agents targeted for the treatment of CMT1A have not been successful, including the use of high-dose ascorbic acid, which exemplified the need for better therapeutic options13. Further, a 5-year longitudinal study also using ascorbic acid in subjects with CMT1A, failed to identify a physiologically significant biomarker14 in patients vs controls. Indeed, “at the end of the study, 30 patients (68%) and 2 controls (8%) stated that their physical condition had deteriorated during the 5-year study period”14. Verhamme et al. hypothesize that early weakness, summarized as pes cavus, lack of reserves, etc., contribute to increased disability in patients, as they aged at the same rate as controls (same decrease in muscle strength and compound Muscle Action Potential [CMAP])14. The Inherited Neuropathy Consortium Rare Diseases Clinical Research Network (RDCRN) website (as of October 30th 2013) lists the following six studies: 6601: Natural History Evaluation of Charcot Marie Tooth Disease; 6602: Genetics of CMT- Modifiers of CMT1A, New Causes of CMT2; 6603: Development of CMT Peds Scale for Children with CMT; 6606: An Analysis of the Symptomatic Domains Most Relevant to Charcot Marie Tooth Neuropathy (CMT) Patients; 6608: Survey of Current Management of Orthopedic Complications in Charcot Marie Tooth Disease Patients; 6604: Development and Validation of a Disability Severity Index for Charcot Marie Tooth Disease. All but studies 6604 and 6606 are listed as still recruiting. These clinical studies are also listed on clinicaltrials.gov with “unknown” status, indicating further the sub optimal progression of research studies in these diseases to date.\n\nIn contrast to CMT, which can vary in severity and is relatively prevalent, GAN is the most severe form of IN. GAN is an “ultra-rare” disease with tens of patients globally (with many likely to be undiagnosed), but neurologists suspect that some CMT Type 2 patients whose causal gene remains unknown may actually have GAN15 greatly expanding the patient population. GAN generally appears in early childhood and progresses slowly as the neuronal injury becomes more severe. GAN is a single-gene, autosomal recessive disorder that is fatal by the late teens or early 20s. The GAN gene encodes the protein gigaxonin16. Gigaxonin is an intracellular protein needed for long-term nerve survival. It is a predicted E3 ligase adaptor protein believed to flag substrates for ubiquination on the proteasome17. Recent studies also suggest that disturbed cytoskeletal regulation, likely involving the proteasome degradation pathway18, is responsible for the formation of aggregates of some type 3 and type 4 intermediate filament proteins, which is a morphological characteristic of this disease, and many others.\n\nBased on histopathological analysis of large axonal swellings, “giant axons” are filled with neurofilament (NF) triplet proteins (neurofilament light chain [NF-L], neurofilament medium chain [NF-M] and neurofilament heavy chain [NF-H]) in both the central and peripheral nervous systems of GAN patients19. The knockout GAN -/- mouse has a similar histological phenotype, with increased levels of NF proteins, α-internexin, peripherin, as well as vimentin, with mRNA transcription levels remaining normal. Additionally, histological analysis of brain sections from the GAN -/- mouse has revealed accumulations of NF-H and of α-internexin in the cortex (negative for other NF subunits)19.\n\nUsing three cellular models, Mahammad et al.18 showed that restoring functional gigaxonin in cultured cells had a direct impact on vimentin, peripherin and NF-L. Using GAN patient fibroblasts, they were able to clear the vimentin aggregates by expressing wild type (WT) gigaxonin. Using a rat PC12 cell line stably expressing WT gigaxonin, they were also able to show the loss of peripherin in these cells. Lastly, using a human neuroblastoma cell line, SH-SY-5Y that stably expresses WT gigaxonin, they revealed clearance of NF-L protein by both immunoblotting and immunofluorescence. Since their manuscript18 was accepted for publication, the Goldman and Opal laboratories have obtained evidence that gigaxonin also regulates the turnover of glial fibrillary acidic protein (GFAP) in astrocytes (Dr. Robert Goldman, Northwestern University Feinberg School of Medicine, personal communication).\n\nLikewise, Mussche et al. were able to demonstrate proof-of-concept of GAN gene delivery in vitro and in vivo by clearing IF aggregates. They showed that the restoration of WT gigaxonin with adeno-associated virus vector serotype 2 (AAV2) clears vimentin IF aggregates in the fibroblasts of GAN patients. Using an AAV9 viral vector, (the same vector being used in the GAN gene delivery clinical trials that HHF is sponsoring), GAN knockout mice received an intracisternal injection of an AAV9/GAN vector that allowed the delivery of the GAN gene to the brainstem and spinal cord. The authors observed that “the treated mice showed a nearly complete clearance of peripherin IF accumulations in the brainstem and spinal cord at 3 weeks post-injection”20. These studies demonstrated that gigaxonin gene transfer can reverse the cellular IF aggregate pathology associated with GAN. Based on this and other published studies (described earlier), it is fair to speculate that vimentin, peripherin, NF-L, NF-M, NF-H, α-internexin and GFAP could be used as putative surrogate biomarkers of disease severity.\n\nAs GAN progresses, patients typically become quadriplegics, dependent on a feeding tube and ventilator, and usually die in the second or third decade. The majority of GAN patients that have been identified to date have also had extremely kinky hair. However, there are two confirmed GAN cases with straight hair, further suggesting CMT as a differential diagnosis for GAN21. Some pathological factors in GAN, like NF-L and peripherin accumulation, are also hallmarks of many other diseases22 including Alexander disease (AxD), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, Parkinson’s disease, dementia with Lewy bodies, neuronal IF inclusion disease (NIFID), diabetic neuropathy, spinal muscular atrophy (SMA), and some forms of CMT (such as CMT 2E)18,23. Thankfully, basic cellular biology research has caught up with clinical research for GAN. Patient cerebrospinal fluid (CSF) samples that have been collected over the past three years can finally be used in pilot studies now that putative surrogate biomarkers have finally been elucidated18.\n\nOver the past five years, HHF, through grassroots efforts, has funded the development and study of a novel GAN gene delivery investigational new biologic (INB) for the treatment of GAN. The gene delivery product is an AAV9 capsid with a self-complementary genome containing the WT-gigaxonin transgene (Dr. Steven Gray, UNC personal communication). This group has achieved broad distribution of the vector throughout the central nervous system (CNS) of both non-human primates and pigs with transduction of the transgene found even deep into the brain parenchyma20. National Institute of Neurological Disorders and Stroke (NINDS) is sponsoring the Phase I trials that will take place at the National Institutes of Health (NIH) Clinical Center in Bethesda, MD, USA. US Recombinant DNA Advisory Committee (RAC) approval was obtained during a public hearing in Washington, DC that took place on June 12, 2013. Assuming FDA and Investigational Review Board (IRB) approvals, a Phase I trial is expected to begin in the second quarter of 2014.\n\nWith research reaching such advanced stages we propose that some efforts should be made to fill the various gaps in clinical studies, in order to translate therapeutics to patients more rapidly for the related diseases CMT1A, CMT2A and GAN. An important component will be to train clinicians, identify the diseases and provide information to hereditary neuropathy patients to help them manage their condition. In addition, there need to be continued efforts to bring new patients to the attention of researchers for clinical studies and energize pharmaceutical companies and investors to pursue treatments for hereditary neuropathies.\n\nWe propose that in order to translate these preclinical advances to the clinic and result in successful therapeutics there should be 1) alternative outcomes measures for CMT; 2) demonstration that potential treatments are having an effect on the QoL of patients; 3) genetic testing panels to facilitate identification of patients with CMT and GAN for future clinical trials; 4) validation of putative CSF biomarkers for GAN; 5) use of the GRIN database to capture patients for future clinical trials and 6) education of clinicians and patients as to therapeutic options for these diseases. Each of these topics is discussed further below.\n\nThe development of therapies for patients critically depends on the outcomes measures used in clinical trials. There are limited efforts to find additional outcomes measures for CMT, including in vivo confocal microscopy of Meissner corpuscles24, Anterior Tibialis CMAP amplitude25, CMT neuropathy score (second version)26 and patient identified QoL27. We now propose that a promising technique called electrical impedance myography (EIM) could be validated as an outcomes measure for CMT1A and CMT2A disease progression, facilitating the execution of therapeutic CMT clinical trials in patients.\n\nSkulpt, Inc. (previously Convergence Medical Devices, Inc.) has developed a handheld device specifically for performing EIM measurements in patients. EIM measures the impedance of skeletal muscle over a frequency range between 1 kHz and 10 MHz. The impedance is measured at each frequency by applying low-intensity electrical current via surface electrodes and measuring the resulting voltage signals using a second set of surface electrodes. EIM involves only minimal risk. The estimated testing duration for an individual subject is 15 to 30 minutes, depending on the number of muscles tested. The data obtained with this handheld EIM device have shown marked alterations between healthy individuals and patients. Data from both human subjects and animal disease models, including ALS, SMA, Duchenne muscular dystrophy (DMD) and neurogenic mouse models, show that EIM may be sensitive to a variety of pathological states28–31. We envisage that EIM will help quantify the severity of the disease affecting various muscle groups as well as measure changes in the progression of the disease over time, which are critical aspects to determine if a therapeutic has any value. A recent DMD EIM pilot study enrolled 61 boys with DMD and 31 healthy controls and validated whether EIM allowed detection of diseased muscle tissues vs control tissues28. Further, EIM was helpful in quantifying the change in muscle quality over time (Dr. Eugene Williams, Dart Therapeutics, personal communication). This was a single day evaluation study that showed strong inter- and intra-rater reliability. It was also a cross sectional study, which used boys over a wide age range, and allowed to correlate EIM readings with age and measures of disease progression28. While this is not technically showing changes over time (a true “longitudinal study”), it does provide similar information and presents some valuable preliminary data.\n\nThus, EIM represents a novel, cutting-edge technology that deserves to be evaluated as to its validity in CMT and other INs.\n\nAlthough CMT is typically a slowly progressive disease, it may have a profound negative impact on patients’ functional, emotional and social abilities, resulting in reduced QoL. A number of studies of the influence of CMT on QoL have been published in the past several years; however, they have been either in small numbers of patients, with non-validated instruments, and/or specific to non-US locations, which reduce their generalizability to US populations27,32–34. These studies have employed generic QoL instruments, such as the EQ-5D32,35, SF-3633,34,36, the Child Health Questionnaire37,38, and qualitative interviews using a semi-structured approach and open-ended questions27. Moreover, different evaluations have produced opposing results. For example, a study of 20 Italian patients with follow-up at two years found worsening strength of distal muscles of upper limbs and proximal muscles of lower limbs, sensory function and walking disability, but no worsening of QoL, as measured by the SF-3633. Due to the small number of patients, it was not possible to stratify according to disease severity, which may have confounded the results. The researchers hypothesized that this may have been due to adaptive mechanisms of the patients because of the slow progression of disease or lack of sensitivity of the QoL instrument to changes specific to CMT. All of these authors33 called for the development of CMT1A disease-specific QoL instruments (adult and child) as there is currently no disease-specific QoL instrument for CMT. Disease-specific instruments are more responsive to small, but important, changes in health than are generic instruments39. A disease-specific instrument for CMT1A would have enhanced validity (having it measure what was intended), reliability (the extent to which it discriminates between individuals in a population in a consistent manner), sensitivity to change (the ability to measure true change regardless of whether it is relevant or meaningful to the patient or clinician) and responsiveness (the ability to detect change that is important to the patient)40.\n\nJohnson and colleagues27 recently completed a study using a comprehensive qualitative interview method to identify symptoms and themes that had the greatest impact on CMT1A-related QoL in 16 adult patients. The themes mentioned most frequently were difficulty with mobility and ambulation, specific-activity impairment and emotional distress, while the most frequently noted individual symptoms in this study included impaired walking, falls and tripping. It is likely, given the small number of patients, these did not correlate with CMT Neuropathy Score (CMTNS) as an indication of disease severity.\n\nPadue et al. recently described a survey of CMT patients and caregivers and their perspectives and perceptions of efficacy and needs41. This cross sectional study used the Rehabilitation Access Questionnaire, the SF36 QoL questionnaire and the family APGAR. This observational survey of 123 patients enrolled through clinical and genetic testing, suggested that patients believe it is important to feel better after physical therapy. There was a discrepancy between the perception of benefit from rehabilitation for the patient vs the caregiver.\n\nBarrett and Birdsall42 also completed an online qualitative study of adults (N=82) that used a modified van Kaam phenomenological method to explore (until saturation) thoughts, perceptions and feelings, including physical, emotional, and social effects. In addition, the study explored their sense of Barrett’s power as knowing participation in change—involving their ability to make informed choices with the intention of freely involving themselves in creating desired changes. The extent of the impact varied with severity, attitude, coping methods, resources, available interventions and treatments. Lack of awareness in the medical community was a predominant theme, as well as extensive description of the physical, emotional, and social impacts. Regarding personal power, searching for ways to educate themselves regarding their disease, to access quality of care, to feel fulfilled despite the severity of symptoms and ways to improve QoL were themes that occupied awareness (A). Regarding choices (C), many patients reflected the ways they were coping with physical limitations and stress, choosing protective and beneficial actions, as well as choosing attitudes taking advantage of available resources, taking charge of health decisions, giving up destructive habits, finding hobbies, altruistic actions, bringing information to physicians, and making necessary career changes. Regarding freedom to act intentionally (F), the range was from most who felt no freedom to those feeling absolutely free in relation to CMT. This was most often related to available and adequate medical treatment by physicians who “cared”. Regarding involvement in creating change (I), being able to find the best and affordable medical care and research findings often allowed people to make major changes. Each of these four power dimensions (A, C, F, I) represent areas for different groups (e.g. HNF or others) to address when planning interventions to guide patients to on how they can be empowered to improve their QoL. In addition, 13 QoL quantitative questions were explored by Barrett and Birdsall42 using descriptive statistics.\n\nIn summary, variable results have been found in terms of QoL domains affected, with some studies showing that social and emotional aspects were significantly affected by the disease34, and others showing no long-term influence on social and emotional functioning, but instead demonstrating impacts on physical and mental health33. Moreover, the impact of disease progression on QoL is unclear, as some researchers have found paradoxical outcomes of worsening disease with no or only mild deterioration in QoL27,33. This is further confounded by a research study in children with CMT1A, where physical signs and symptoms were found to be of significantly greater value than psychological aspects of QoL, as relayed by their parents37. Since there is no clear delineation of disease progression symptoms, relationship to disease severity, longitudinal follow-up, determination of best methods of measuring QoL or disease-specific instrument, future studies should evaluate changes in impact of factors. These factors should include improved medical care, education through the Internet, advocacy support from CMT organizations and other dynamics, on patients’ QoL over the past ten years. Also newer instruments, such as NIH’s Patient Reported Outcomes Measurement Information System (PROMIS®) and Neuro-QOL, need to be used in comparison to more established tools to determine if any of these tools is/are responsive enough to capture the impact of these diseases on patients’ health-related QoL (HRQoL). Neuro-QOL is a new, standardized approach to measuring HRQoL across common neurologic diseases and has been validated in multiple neurological conditions, both in adult and pediatric patients43. PROMIS® is a tool defined as “a system of highly reliable, precise measures of patient–reported health status for physical, mental, and social well–being”44. Further psychometric testing within CMT and between CMT and GAN will help evaluate the generalizability of these new tools. Ultimately, there should be separate development and validation of CMT1A (disease-specific) QoL instruments for adults and children for use in future clinical trials.\n\nINs are widely known as diverse in both their genetic causes and clinical phenotypes. INs are also very frequently underdiagnosed or misdiagnosed45. In 2013 a large prospective study found that nearly half of the patients referred to specialist peripheral neuropathy clinics have an unrecognized inherited cause46. We are aware that the sequencing company Athena/Quest Diagnostics has sequenced more than 100,000 CMT patients and claims to have the largest variant database in the world. Previous discussions have suggested that published prevalence data are outdated, and that CMT is much more prevalent than previously estimated (Dr. Stephen L. Vincent, Director of Business Development, Quest Diagnostics and Athena Diagnostics, personal communication). It is therefore important that updated prevalence data be communicated to illustrate the large and underserved patient population in order to motivate the pharmaceutical industry to develop treatments for CMT. There should therefore be coordinated efforts for the genetic diagnosis of this community with initial focus on CMT1A, CMT2A and GAN in order to have well-defined cohorts available for upcoming clinical studies.\n\nTo date, 2000 variants have been identified on the causal gene in cystic fibrosis and 18 causal variants have been identified (7). Perhaps CMT should be considered in the same light as the Cystic Fibrosis Foundation/Vertex Pharmaceuticals apparent view of cystic fibrosis, in which each genotype (e.g. mutations G551D, Delta F508 etc.) is treated as a different disease, requiring separate drugs for patients with different mutations. This ultimately represents an approach to individualized therapy. For INs, a long journey awaits as the field is working to elucidate causal genes in many cases, not to mention the many variants on a given gene that mandate a unique therapeutic approach. This is in stark contrast to the belief and understanding of most neurologists who are content in not investigating the genetic diagnosis of patients because no treatments currently exist. This paradigm must change in order to better understand the variability in the different forms of IN. The genotype must be identified and non-disease causing variants evaluated, as this can lead to the development of therapeutic targets. With the advancements in other personalized medicine approaches such as antisense oligonucleotides, we must endeavor to obtain a thorough understanding of the genetics behind the various forms of IN; otherwise it will be difficult to obtain effective therapies. In our view the field of IN is at least 20 years behind cystic fibrosis, largely because genetic testing has not been a priority. We have coined the phrase “diagnostic complacency” to describe this situation. Just because most forms of IN are not fatal and no treatments exist, it does not mean that CMT and idiopathic neuropathy patients do not deserve to be genetically diagnosed and have the opportunity to contribute to research by sharing their phenotype/genotype relationships.\n\nAthena/Quest Diagnostics currently provides a variety of CMT next-generation sequencing options and plans on adding newly identified genes to these test panels in early 2015 (Dr. Stephen L. Vincent, Quest Diagnostics and Athena Diagnostics, personal communication). In addition, soon they may be able to provide disease-specific exome sequencing. This will allow them to test all known CMT genes in a single test in the same timeframe that it takes to run a single test today. We are not aware of other companies doing sequencing for CMTs specifically, therefore Athena’s/Quest’s CMT next-generation sequencing test offerings may provide a rapid result and cost savings for idiopathic CMT subjects in contrast to running multiple single or small test panels over an extended period of time. Prevention Genetics (a US company) provides services to sequence the GAN gene.\n\nThe development of new CMT panels that include the GAN gene and other newly-identified CMT Type 2 genes that have a phenotype and electrophysiological findings similar to GAN will be important now that a Phase I trial is planned for GAN.\n\nIn order to assist in Phase I and Phase II trials of a first-in-human gene delivery vector delivered to the spinal cord there should be validated putative surrogate biomarkers of GAN disease progression. CSF has long been exploited as a relatively non-invasive way to biopsy the CNS47. Subjects with GAN will likely be the first disease community in the world to receive a therapeutic gene delivered to the spinal cord, paving the way for other related neurological disorders such as SMA which, according to the National Organization for Rare Disorders (NORD), is the leading genetic cause of death in children.\n\nAs it has been reported that gigaxonin regulates GFAP in astrocytes48, quantifying the levels of GFAP in body fluids may be useful to determine the severity and/or clinical progression of GAN. Normally, low levels of GFAP are seen in body fluids, and will thus require the development of very sensitive detection methods, such as sandwich ELISA assays49. CSF GFAP levels have been shown to be elevated in a number of neurological conditions, reflecting a wide range of etiologies (reviewed in50). In several situations GFAP is evaluated relative to other biomarkers linked to neuronal damage, such as neurofilaments51,52. Generally, the levels of GFAP are modest in the slowly progressive conditions such as dementia and multiple sclerosis (MS), and in contrast, higher but transient, in vascular accidents, trauma, and infection. The level of GFAP in CSF may have a prognostic value53–55 which is worth exploring. For instance, according to a study on patients with subarachnoid hemorrhage, six days after the event the GFAP level was approximately 8-fold higher in non-survivors compared to survivors55. It has been described that GFAP levels in CSF seem to drop in response to treatment in patients with neuromyelitis optica given corticosteroid therapy56.\n\nAlexander disease (closely related to GAN) is caused by dominantly-acting gain of functional mutations in GFAP. These mutations lead to increased levels of expression, measured either by mRNA or protein. GFAP has been measured in the CSF from three Alexander disease patients57, and was elevated in them all. Since mutations in GFAP are the root cause of Alexander disease, and increased levels of GFAP within astrocytes are thought to be central to the pathogenesis58, GFAP measurements might prove especially valuable for assessing either severity or progression of this particular condition. For instance, the highest levels of GFAP in this study were found in a patient who, although intermediate in terms of age of onset, was rapidly deteriorating at the time of biopsy. Recent studies in the mouse model of Alexander disease also showed a positive correlation between the levels of protein present in brain parenchyma and the levels of protein in CSF, with an increased expression correlating with the severity of phenotype59.\n\nIt is perhaps not surprising that Alexander disease and GAN show interesting similarities. The hallmark pathological feature in Alexander disease is the presence of stress protein aggregates (known as “Rosenthal fibers”) within the cytoplasm of astrocytes, along with variable degrees of leukodystrophy. Indeed, the presence of Rosenthal fibers has also been described in several patients with GAN60–62. We can therefore speculate that loss of gigaxonin may affect GFAP causing accumulation and aggregation of the protein and may produce an Alexander disease–like pathology.\n\nA recent review on the MRI features of leukodystrophies reported some similarities between GAN and Alexander disease but focused only on lesions in the white matter. We could predict that GFAP levels will be increased in the CSF of GAN patients and will likely rise with the progression of the disease. We further hypothesize that GFAP levels will stabilize and potentially fall in response to treatment, thus serving as a useful biomarker for the GAN gene delivery clinical trial. It will be interesting to see if the abundance of GFAP, NF-H, NF-M, NF-L, vimentin, α-internexin and peripherin (all proteins implicated in GAN morphology) in CSF, changes over time and correlates with disease severity in the future trial. It is also intriguing to think that correlating NF-L abundance in CSF to disease progression in GAN may also translate to CMT2E, which is caused by dominant point mutations in NF-L. Most intriguing is the prospect that the markers for this devastating disorder in the pilot study could potentially be valuable surrogate endpoints demonstrating efficacy of the first-in-human AAV9 GAN gene delivery trials.\n\nA patient registry is a place to store detailed information about patients with a specific disease or syndrome. In general, collection, analysis and dissemination of data on disease progression and patient responses to long-term disease management strategies represents a valuable way to improve understanding of the disease and keep medical professionals up to date on the latest advances. The current Inherited Neuropathy Consortium (INC) has a very basic contact registry only for CMT1A, CMT1B, CMT2A, CMTX, CMT4, other known CMT peripheral neuropathy and other unknown CMT peripheral neuropathy. In contrast, the HNF & HHF launched the Global Registry for Inherited Neuropathies (GRIN, Figure 1) – in 2013 with the primary objective of identifying patients with INs to gain a better understanding of disease phenotypes, which vary among all the different types and sub-types of CMT and related INs. This registry is far more extensive (Supplemental Figure 1–Supplemental Figure 10) than anything else available for INs such as basic contact registries, as it covers all INs and is managed by Patient Crossroads, which is a professional organization that has developed registries for more than 250 diseases. These registries are used by participants in more than 75 countries. GRIN can be used to connect patients, healthcare providers, researchers, clinical investigators, regulators, policy-makers, industry and beyond.\n\nThe GRIN collects data on patients who have either been clinically diagnosed by a doctor or genetically diagnosed with a form of IN (Figure 2). Establishing the registry addresses three critical needs. First, the scientists who study various forms of IN need accurate information to understand the specific forms of neuropathy; second, the scientists who are conducting preclinical studies may need tissue samples from registrants who have already agreed to be contacted by GRIN if samples are needed to advance research for their form of IN. Additionally, clinicians, pharmaceutical and biotechnology companies ready to begin trials would need the GRIN administrator to distribute and advertise the study and clinical-trials.gov study links. The Patient Crossroads platform on which the database is built provides robust query functionality and allows researchers to narrow in on specific phenotypes and genotypes. If a patient looks like a good match for research, the scientist will contact HNF. The foundation or its agent will then contact the IN family. Scientists will not contact the families directly and will only be given de-identified patient data.\n\nAlthough there is no direct benefit to any patients participating in the registry, by collecting information on patients in the registry, scientists can:\n\nStudy why individuals have different symptoms.\n\nLearn about phenotype/genotype relationships (how a particular mutation type can lead to different or unique symptoms).\n\nLearn how future treatments work or don’t work for a given patient population.\n\nObtain tissue samples from registrants who agreed to be contacted for such purpose.\n\nHelp medical professionals improve how they treat patients with IN, for example, pain management.\n\nSpeed up research in INs by collecting information that scientists can use.\n\nLet patients (or their families) know when they may be eligible for clinical research studies (clinical trials).\n\nLet pharmaceutical companies know the number of patients with a particular form of IN who would be willing to consider volunteering for experimental trials.\n\nThis longitudinal database of patient-contributed data includes the ability for patients to create an account that allows them to login and update information, complete questionnaires or provide patient-reported outcomes measures and track results, compare their responses to the de-identified responses of other registry participants, and upload medical records for use in identifying confirmed diagnosis of hereditary neuropathy patients. GRIN also provides the ability to monitor registration metrics, respond to registrant inquiries, add researcher-specific questions, and export de-identified data in CSV format that is widely accepted by statistical and scientific software.\n\nGRIN currently collects data on meaningful characteristics such as pes cavus, absent-to-poor reflexes, toe walking, mobility devices on various levels of disability deficits, etc., management, and outcomes of disease progression with and without palliative treatments. Another critical component of the registry is to provide a better understanding of disease progression in the general population of both diagnosed and undiagnosed patients. Environmental factors and contraindicated drugs have been confirmed to exacerbate CMT and/or present onset of symptoms in asymptomatic patients64. This obviously can alter disease progression and may be relevant to the natural history and clinical outcomes of treatments.\n\nThe registry has several objectives:\n\n1. Offer additional data to enable the pharmaceutical industry to judge the risk – reward of the patient population for pursuing future clinical trials.\n\n2. Obtain a better understanding of natural history from the perspective of the patient. Currently the patient, caregiver or parent of a minor, answers the questions, and when tests (needle electromyography, nerve conduction velocities or genetic tests) are available, patients and/or physicians upload the results to the registry or contact HNF directly.\n\n3. Evaluate safety, efficacy, and influence of therapy on patients’ QoL, which is not currently a feature of any IN registry.\n\n4. Record patient experience in a period prior to baseline as a control for future clinical trials.\n\n5. Help to establish relationship of genotype – phenotype on disease progression.\n\n6. Help to define data that are clinically meaningful.\n\nGRIN includes a comprehensive series of questions covering many areas and samples of these are now provided for: diagnosis/assessments, genetic test results, general health, neurology, orthopedic, alternative/complimentary therapies, skin/hair, clinical trials, and research/bio-repository (Supplemental Figure 1–Supplemental Figure 10). GRIN will help close the data gap to enable a more accurate representation of the patients’ experience. The registry contains well thought out questions and was beta tested by patients, caregivers and medical professionals. GRIN is also IRB-approved through the Chesapeake IRB. HNF’s diverse team of medical advisors, industry leaders, and the data used from focus groups conducted and analyzed through the National CMT Resource Center in 2010 helped to develop the purpose, objective, feasibility and the robust registry. GRIN is being marketed globally through aggressive pay-per-click campaigns (via a Google grant) to actively search out patients with CMT and other neuropathies. These marketing efforts ask them to join the registry and encourage them to be seen at one of the currently-funded RDCRN CMT Centers, Muscular Dystrophy Association Centers or by medical professionals trained to recognize, diagnose and treat CMT where patients are not economically able or are too severely affected to travel.\n\nIf we are to develop new technologies and drugs for IN then we need to continually train clinicians to be aware of the state of the art knowledge of the diseases such as the growing list of known neurotoxins that must be avoided. At the same time we need to educate patients about their disease and what is in the research and clinical pipeline. The training of future physician-scientists is therefore critical to accelerating research in the hereditary neuropathies field. There is also the responsibility of foundations like HNF to educate the entire medical community to be aware of hereditary neuropathies. This would lead to faster and more accurate patient diagnosis that could have an impact on medical expenses and patient QoL.\n\nClose affiliations with other patient advocacy organizations focused on INs is also important to educate all members of the community. As indicated throughout this document, key advocates include: HNF, HHF, The Neuropathy Association, Neuropathy Action Foundation, and NORD. To date HNF has actively supported the efforts of the RDCRN Inherited Neuropathies Consortium by including new studies for recruitment in monthly newsletters to over 7,000 members and aggressive social media outreach. Platforms used include Facebook, Twitter and the CMT Inspire Community. With well over 1,000 patients, the Inspire CMT community has dramatic branding and is a highly targeted and engaged population with valuable content for patients and clinicians. Inspire offers access to members for clinical studies that include market research and awareness campaigns where each member receives daily or weekly e-mails from Inspire — all aimed at getting the community to take action and move research forward. The mission of the Neuropathy Association is to bring help, hope, and healing to the estimated over 20 million people living with over 100 various forms of neuropathy. A significant segment of this population has an idiopathic neuropathy. Experts in the field have found that there is a high likelihood that many of these are actually unrecognized INs. The Neuropathy Association’s extensive network of patients, through their membership base (over 50,000), extensive online and social media networks (35,000–45,000 unique website visitors per month and 12,000 followers on Facebook) and the 15 designated Centers of Excellence at prominent academic and medical institutions across the USA, are critical to engaging clinicians – and their patients - to participate in future clinical trials. The Neuropathy Association and the HNF recently hosted a monthly Facebook Chat (July 2013), focusing on hereditary neuropathies, with the goal of giving participants a chance to hear from — and ask questions to — health experts in the field, the Association and Foundation staff, collaborating patient advocacy groups, as well as each other. These examples show that by actively engaging the advocacy groups and their networks through social media we can reach patients which, in previous years, would have been impossible without extensive media expenditures.\n\nThe Neuropathy Association has awarded grants for scientific research on hereditary neuropathies and the supported research projects have yielded significant results. For example, Landsverk et al. identified the gene causing a hereditary neuropathy (hereditary neuralgic amyotrophy [HNA]) that attacks the muscles responsible for controlling the movement of shoulders and arms65. In addition, Florian Thomas MD (St. Louis University, St. Louis, USA) and his international team have uncovered three different genetic mutations specific to dominant intermediate CMT neuropathy type C, a form of CMT disease66. Organizations like The American Association of Neuromuscular and Electrodiagnostic Medicine (AANEM) could be funded to support awarding of fellowships that focus on rare neuromuscular disease research. This could have a significant impact on training and education for the IN clinical community.\n\nAdditionally, a regular Hereditary Neuropathies Workshop could serve as a forum to promote awareness of IN, encourage patient recruitment for future clinical trials and GRIN, and provide a means for encouraging more participation by other researchers and companies in the field, as well as provide a learning platform to benefit new researchers; this would encourage the researchers’ development in this area and promote their interest in pursuing additional research projects in the field of hereditary neuropathies.\n\n\nConclusion\n\nIn conclusion, we have recognized the following gaps in translation and obstacles to future clinical studies for IN via lack of: a validated QoL instrument that is sensitive enough to capture disease impact; validated biomarkers that correlate with severity of disease progression; a large, robust, clinical patient registry that accurately captures severity and range of symptoms, along with genotypes; physician education and awareness about IN; accurate genetic diagnoses; patient and physician education regarding the need for an early diagnosis; prevalence data; and data regarding patient willingness to consider volunteering for experimental trials for various forms of IN. We have suggested some recommendations that could perhaps remedy this situation--positively impacting patients with CMT, GAN and other INs.",
"appendix": "Author contributions\n\n\n\nAll authors conceived and contributed to the literature review, and the writing of the article. All authors read and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nL.S. is founder of Hannah’s Hope Fund and CEO BioGan Therapeutics, LLC. A.M. is founder of Hereditary Neuropathy Foundation and president of BioGan Therapeutics. S.E. works for Collaborations in Chemistry, and Consults for Hannah’s Hope Fund, the Hereditary Neuropathy Foundation and Arnold Consultancy & Technology, LLC.\n\n\nGrant information\n\nSome of this work was funded by the Centers for Disease Control and Prevention (CDC) Grant # U38DD000713-0.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nWe gratefully acknowledge all of the scientists, patient advocacy groups and other organizations we collaborate with (some of whom have kindly provided unpublished information) and the individuals and companies who have so generously supported our foundations. These include: Thomas H. Brannagan, III MD (Columbia University); Adrienne Rhodes (Blue Fountain Media); Steven Gray, PhD (University of North Carolina); Lisa M. Phelps (NORD); Victoria H. Lawson, MD (Ohio State University); Florian P. Thomas, MD (Saint Louis University); Pragna Patel, PhD (University of Southern California); Jose Bohorquez, PhD (Skulpt, Inc); Kyle Brown (PatientCrossroads); Carsten G. Bönnemann, MD (NIH); Daniel Cohen, MD, PhD (Pharnext); Shirlyn A. Adkins, JD, (AANEM); Stanley S. Young, PhD (NISS); Xavier Badia MD, MPH, PhD (University of Barcelona); Dominick V. Spatafora (Neuropathy Action Foundation); Michael Serada MD, (Max-Planck Institute of Experimental Medicine); Stephen L. Vincent, PhD, (Quest Diagnostics and Athena Diagnostics); Leslie Hinyard, PhD, MSW (Saint Louis University); Eugene Williams (Dart Therapeutics), Mickaël Guedj, PhD (Pharnext); Elizabeth A.M. Barrett, PhD (Hunter College); Natacha T. Pires, MBBS (The Neuropathy Association); Albee Messing PhD, (University of Wisconsin-Madison), Gerry Shaw PhD, (University of Florida); David Pleasure MD, (University of California-Davis); Tina M. Tockarshewsky (The Neuropathy Association); Yonglong Zhou PhD, (Arnold Consultancy & Technology); Katrina A. Gwinn MD, (NIH); Robin Conwit MD, (NIH); and Courtney Hollett (Hereditary Neuropathy Foundation).\n\n\nSupplementary materials\n\nFigures relating to the Global Registry for Inherited Neuropathies.\n\n\nReferences\n\nLee SM, Chin LS, Li L: Protein misfolding and clearance in demyelinating peripheral neuropathies: Therapeutic implications. Commun Integr Biol. 2012; 5(1): 107–10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShy ME: Lessons from London. J Neurol Neurosurg Psychiatry. 2012; 83(8): 767–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBort S, Sevilla T, Vílchez JJ, et al.: [The diagnosis and prevalence of locus CMT1A duplication in Charcot-Marie-Tooth disease type 1]. Med Clin (Barc). 1995; 104(17): 648–52. PubMed Abstract\n\nNaef R, Suter U: Many facets of the peripheral myelin protein PMP22 in myelination and disease. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nMahammad S, Murthy SN, Didonna A, et al.: Giant axonal neuropathy-associated gigaxonin mutations impair intermediate filament protein degradation. J Clin Invest. 2013; 123(5): 1964–1975. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDequen F, Bomont P, Gowing G, et al.: Modest loss of peripheral axons, muscle atrophy and formation of brain inclusions in mice with targeted deletion of gigaxonin exon 1. J Neurochem. 2008; 107(1): 253–64. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMussche S, Devreese B, Nagabhushan Kalburgi S, et al.: Restoration of cytoskeleton homeostasis after gigaxonin gene transfer for giant axonal neuropathy. Hum Gene Ther. 2013; 24(2): 209–19. PubMed Abstract | Publisher Full Text\n\nRoth LA, Johnson-Kerner BL, Marra JD, et al.: The absence of curly hair is associated with a milder phenotype in Giant Axonal Neuropathy. Neuromuscul Disord. 2014; 24(1): 48–55. PubMed Abstract | Publisher Full Text\n\nOpal P, Goldman RD: Explaining intermediate filament accumulation in giant axonal neuropathy. Rare Diseases. 2013; 1: e25378. Publisher Full Text\n\nFabrizi GM, Cavallaro T, Angiari C, et al.: Charcot-Marie-Tooth disease type 2E, a disorder of the cytoskeleton. Brain. 2007; 130(Pt 2): 394–403. PubMed Abstract | Publisher Full Text\n\nAlmodovar JL, Ferguson M, McDermott MP, et al.: In vivo confocal microscopy of Meissner corpuscles as a novel sensory measure in CMT1A. J Peripher Nerv Syst. 2011; 16(3): 169–74. PubMed Abstract | Publisher Full Text\n\nKomyathy K, Neal S, Feely S, et al.: Anterior tibialis CMAP amplitude correlations with impairment in CMT1A. Muscle Nerve. 2013; 47(4): 493–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMurphy SM, Herrmann DN, McDermott MP, et al.: Reliability of the CMT neuropathy score (second version) in Charcot-Marie-Tooth disease. J Peripher Nerv Syst. 2011; 16(3): 191–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJohnson NE, Heatwole CR, Ferguson M, et al.: Patient identification of the symptomatic impact of charcot-marie-tooth disease type 1A. J Clin Neuromuscul Dis. 2013; 15(1): 19–23. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRutkove SB, Darras BT: Electrical impedance myography for the assessment of children with muscular dystrophy: a preliminary study. J Phys Conf Ser. 2013; 434(1): pii: 012069. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRutkove SB, Caress JB, Cartwright MS, et al.: Electrical impedance myography as a biomarker to assess ALS progression. Amyotroph Lateral Scler. 2012; 13(5): 439–45. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRutkove SB, Gregas MC, Darras BT: Electrical impedance myography in spinal muscular atrophy: a longitudinal study. Muscle Nerve. 2012; 45(5): 642–7. PubMed Abstract | Publisher Full Text\n\nRutkove SB, Shefner JM, Gregas M, et al.: Characterizing spinal muscular atrophy with electrical impedance myography. Muscle Nerve. 2010; 42(6): 915–21. PubMed Abstract | Publisher Full Text\n\nCalvert M, Pall H, Hoppitt T, et al.: Health-related quality of life and supportive care in patients with rare long-term neurological conditions. Qual Life Res. 2013; 22(6): 1231–1238. PubMed Abstract | Publisher Full Text\n\nPadua L, Pareyson D, Aprile I, et al.: Natural history of Charcot-Marie-Tooth 2: 2-year follow-up of muscle strength, walking ability and quality of life. Neurol Sci. 2010; 31(2): 175–178. PubMed Abstract | Publisher Full Text\n\nTaniguchi JB, Elui VM, Osório FL, et al.: Quality of life in patients with Charcot-Marie-Tooth disease type 1A. Arq Neuropsiquiatr. 2013; 71(6): 392–396. PubMed Abstract | Publisher Full Text\n\nAjay A, Walters WP, Murcko MA: Can we learn to distinguish between “drug-like” and “nondrug-like” molecules? J Med Chem. 1998; 41(18): 3314–3324. PubMed Abstract | Publisher Full Text\n\nWare JE Jr, Sherbourne CD: The MOS 36-item short-form health survey (SF-36). I. Conceptual framework and item selection. Med Care. 1992; 30(6): 473–483. PubMed Abstract | Publisher Full Text\n\nBurns J, Ramchandren S, Ryan MM, et al.: Determinants of reduced health-related quality of life in pediatric inherited neuropathies. Neurology. 2010; 75(8): 726–731. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLandgraf JL, Abetz L, Ware J: The Child Health Questionnaire (CHQ): A User’s Manual. 1999, HealthAct: Boston, MA. Reference Source\n\nWiebe S, Guyatt G, Weaver B, et al.: Comparative responsiveness of generic and specific quality-of-life instruments. J Clin Epidemiol. 2003; 56(1): 52–60. PubMed Abstract | Publisher Full Text\n\nBryant D, Guyatt G, Arnold R: Patient-reported outcome measures. In Pharmacoeconomics: from theory to practice, R. Arnold, Editor, CRC Press (Taylor and Francis Group, LLC): Boca Raton, 2010; 149–162. Publisher Full Text\n\nPadua L, Pazzaglia C, Schenone A, et al.: Rehabilitation for Charcot Marie tooth: a survey study of patients and familiar/caregiver perspective and perception of efficacy and needs. Eur J Phys Rehabil Med. 2014; 50(1): 25–30. PubMed Abstract\n\nBarrett E, Birdsall C: What It’s Like to Live with CMT? Manuscript in preparation. Reference Source\n\nAndersson T, Miners JO, Veronese ME, et al.: Diazepam metabolism by human liver microsomes is mediated by both S-mephenytoin hydroxylase and CYP3A isoforms. Br J Clin Pharmacol. 1994; 38(2): 131–137. PubMed Abstract | Free Full Text\n\nAnthony LB, Bennett RE, Deegan PM, et al.: Cytochrome P-450 in vitro activation of cyclophosphamide (CPA) and Ifosfamide (IFOS). Clin Pharmacol Ther. 1996; 59: 199. Publisher Full Text\n\nDyck PJ, Oviatt KF, Lambert EH: Intensive evaluation of referred unclassified neuropathies yields improved diagnosis. Ann Neurol. 1981; 10(3): 222–6. PubMed Abstract | Publisher Full Text\n\nKlein CJ, Duan X, Shy ME: Inherited neuropathies: clinical overview and update. Muscle Nerve. 2013; 48(4): 604–22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nConly JM, Ronald AR: Cerebrospinal fluid as a diagnostic body fluid. Am J Med. 1983; 75(1B): 102–108. PubMed Abstract | Publisher Full Text\n\nLiem RK, Messing A: Dysfunctions of neuronal and glial intermediate filaments in disease. J Clin Invest. 2009; 119(7): 1814–1824. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlbrechtsen M, Sørensen PS, Gjerris F, et al.: High cerebrospinal fluid concentration of glial fibrillary acidic protein (GFAP) in patients with normal pressure hydrocephalus. J Neurol Sci. 1985; 70(3): 269–274. PubMed Abstract | Publisher Full Text\n\nLiem RK, Messing A: Dysfunctions of neuronal and glial intermediate filaments in disease. J Clin Invest. 2009; 119(7): 1814–1824. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBlennow M, Sävman K, Ilves P, et al.: Brain-specific proteins in the cerebrospinal fluid of severely asphyxiated newborn infants. Acta Paediatr. 2001; 90(10): 1171–1175. PubMed Abstract | Publisher Full Text\n\nTullberg M, Rosengren L, Blomsterwall E, et al.: CSF neurofilament and glial fibrillary acidic protein in normal pressure hydrocephalus. Neurology. 1998; 50(4): 1122–1127. PubMed Abstract | Publisher Full Text\n\nBlennow M, Rosengren L, Jonsson S, et al.: Glial fibrillary acidic protein is increased in the cerebrospinal fluid of preterm infants with abnormal neurological findings. Acta Paediatr. 1996; 85(4): 485–489. PubMed Abstract | Publisher Full Text\n\nNorgren N, Sundström P, Svenningsson A, et al.: Neurofilament and glial fibrillary acidic protein in multiple sclerosis. Neurology. 2004; 63(9): 1586–1590. PubMed Abstract | Publisher Full Text\n\nPetzold A, Keir G, Kerr M, et al.: Early identification of secondary brain damage in subarachnoid hemorrhage: a role for glial fibrillary acidic protein. J Neurotrauma. 2006; 23(7): 1179–1184. PubMed Abstract | Publisher Full Text\n\nMisu T, Takano R, Fujihara K, et al.: Marked increase in cerebrospinal fluid glial fibrillar acidic protein in neuromyelitis optica: an astrocytic damage marker. J Neurol Neurosurg Psychiatry. 2009; 80(5): 575–577. PubMed Abstract | Publisher Full Text\n\nKyllerman M, Rosengren L, Wiklund LM, et al.: Increased levels of GFAP in the cerebrospinal fluid in three subtypes of genetically confirmed Alexander disease. Neuropediatrics. 2005; 36(5): 319–323. PubMed Abstract | Publisher Full Text\n\nMessing A, Brenner M, Feany MB, et al.: Alexander disease. J Neurosci. 2012; 32(15): 5017–5023. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJany PL, Hagemann TL, Messing A: GFAP expression as an indicator of disease severity in mouse models of Alexander disease. ASN Neuro. 2013; 5(1): e00109. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKretzschmar HA, Berg BO, Davis RL: Giant axonal neuropathy. A neuropathological study. Acta Neuropathol. 1987; 73(2): 138–144. PubMed Abstract | Publisher Full Text\n\nKumar K, Barre P, Nigro M, et al.: Giant axonal neuropathy: clinical, electrophysiologic, and neuropathologic features in two siblings. J Child Neurol. 1990; 5(3): 229–234. PubMed Abstract | Publisher Full Text\n\nThomas C, Love S, Powell HC, et al.: Giant axonal neuropathy: correlation of clinical findings with postmortem neuropathology. Ann Neurol. 1987; 22(1): 79–84. PubMed Abstract | Publisher Full Text\n\nvan der Knaap MS, Valk J: Magnetic resonance of myelination and myelin disorders. Berlin: Springer Vol. Third, 2005. Publisher Full Text\n\nMcDonald CM: Clinical approach to the diagnostic evaluation of hereditary and acquired neuromuscular diseases. Phys Med Rehabil Clin N Am. 2012; 23(3): 495–563. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLandsverk ML, Ruzzo EK, Mefford HC, et al.: Duplication within the SEPT9 gene associated with a founder effect in North American families with hereditary neuralgic amyotrophy. Hum Mol Genet. 2009; 18(7): 1200–1208. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJordanova A, Thomas FP, Guergueltcheva V, et al.: Dominant intermediate Charcot-Marie-Tooth type C maps to chromosome 1p34-p35. Am J Hum Genet. 2003; 73(6): 1423–1430. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "4344",
"date": "07 Apr 2014",
"name": "Flávia L. Osório",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the present article the authors review the literature on inherited neuropathies - especially Charcot-Marie-Tooth and Giant Axonal Neuropathy - based on recent clinical evidence. The authors then offer several suggestions to overcome gaps in this area of knowledge, with criticism and argumentation.I approve this article for indexation. My only suggestion is that the authors remove figures 1 and 2 from the main body of the article and include them in the Supplementary materials instead.",
"responses": [
{
"c_id": "773",
"date": "16 Apr 2014",
"name": "Sean Ekins",
"role": "Author Response",
"response": "We thank the reviewer for their positive feedback. We have made the suggested changes and removed the two figures to the supplemental materials."
}
]
},
{
"id": "4493",
"date": "14 Apr 2014",
"name": "Jean-Michel Vallat",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a quite interesting paper which stresses that genetic treatments for neuropathies should be further investigated by neuroscientists, MD’s who have such patients in their charge, and pharmaceutical companies. On the other hand, we all agree that discoveries in molecular biology should induce increasingly personalized medicine.Nevertheless, some points have to be explained or clarified:The general classification of ‘inherited neuropathies’ includes a great number of disorders. The title of this paper has to be explained in the introduction: it is clearly understandable why the authors have chosen CMT, as it is the most frequent genetic neuropathy. Now it should be discussed why they also included GAN; maybe because it is one of the rarest hereditary neuropathies? But many patients with other inherited neuropathies - such as: porphyria, Chédiak-Higashi syndrome, storage disorders with neuropathy etc. - could argue that they have been forgotten. It could be useful to present a simplified classification of inherited neuropathies in the introduction (perhaps in the form of a table?). The frequency of GAN is discussed and I agree is clearly underestimated: see a recent paper in Neurology1 (which could be mentioned). It states: “Giant axonal neuropathy is a rare (worldwide 50 families reported) autosomal recessive disorder”. Actually, in our neuropathology laboratory we have about 13 GAN nerve biopsies, all from Algeria. I am not sure that GAN is “the most severe form of IN” - even though, of course, it is very severe - leukodystrophies and storage disorders induce very severe encephalopathy. It should be explained that many symptoms of GAN are mostly explained by CNS involvement (impaired cognition, cerebellar dysfunction) - which is not at all the case in most CMT - so an efficient drug for this disease should also be able to cross the blood brain barrier. I disagree with this sentence: “CSF has long been exploited as a relatively non-invasive way to biopsy the CNS”. It is true that CSF data can be very important in CNS disorders, but in no case can it be treated as a biopsy! Actually the preliminary results of the Pharnext phase II trial have been published.2 I agree that, at the present time, QoL should be the primary objective in therapeutic trials in CMT. I am not sure that Neuro-Qol would be a good tool as it takes into account pain and sensory signs which are not usually the main symptoms of CMT. Anyway, it will take a long time to validate such scales (PROMIS also) in CMT. It is correct that EIM may be a very interesting tool to follow CMT progression; so, it has to be validated - which will take some time. The possibility of utilizing this test in the coming CMT therapeutic trials could be discussed. Rutkove et al.3 have recently shown that EIM data correlates with standard measures of ALS severity.",
"responses": [
{
"c_id": "772",
"date": "16 Apr 2014",
"name": "Sean Ekins",
"role": "Author Response",
"response": "We thank the reviewer for their positive feedback. We have made most of the suggested changes. Each of the following bulletpoints refers to their comments. We do not think the title needs any explanation as it just focuses on CMT and GAN as examples of inherited neuropathies (INs). These two diseases also represent the extremes. Yes we acknowledge there are other INs but two of the authors’ families suffer from these diseases hence our interest in them specifically. We have added the reference for GAN as suggested. We have edited out “most” to leave “severe”. We have added “As GAN progresses (through effects on the CNS),…” in the text. We agree that ability to cross the BBB will be important for a GAN small molecule but we do not describe small molecule work on GAN, only gene therapy which is close to human trials. Therefore we found no appropriate place to add this in the text. We hope these comments will suffice. We replaced “biopsy” with “sample”. We have added this Pharnext reference to a meeting abstract, but the full paper is still forthcoming. Thank you for agreeing that QoL is important for CMT trials. As neither Neuro QoL or PROMIS have been used to our knowledge with CMT, we feel they should at least be tried. Thank you for agreeing that EIM may be a very interesting tool to follow CMT progression. We have added a further reference on ALS and EIM as you suggested."
}
]
}
] | 1
|
https://f1000research.com/articles/3-83
|
https://f1000research.com/articles/3-28/v1
|
28 Jan 14
|
{
"type": "Research Article",
"title": "Astrocytes require insulin-like growth factor I to protect neurons against oxidative injury",
"authors": [
"Laura Genis",
"David Dávila",
"Silvia Fernandez",
"Andrea Pozo-Rodrigálvarez",
"Ricardo Martínez-Murillo",
"Ignacio Torres-Aleman",
"Laura Genis",
"David Dávila",
"Silvia Fernandez",
"Andrea Pozo-Rodrigálvarez",
"Ricardo Martínez-Murillo"
],
"abstract": "Oxidative stress is a proposed mechanism in brain aging, making the study of its regulatory processes an important aspect of current neurobiological research. In this regard, the role of the aging regulator insulin-like growth factor I (IGF-I) in brain responses to oxidative stress remains elusive as both beneficial and detrimental actions have been ascribed to this growth factor. Because astrocytes protect neurons against oxidative injury, we explored whether IGF-I participates in astrocyte neuroprotection and found that blockade of the IGF-I receptor in astrocytes abrogated their rescuing effect on neurons. The protection mediated by IGF-I against oxidative stress (H2O2) in astrocytes is probably needed for these cells to provide adequate neuroprotection. Indeed, in astrocytes but not in neurons, IGF-I helps decrease the pro-oxidant protein thioredoxin-interacting protein 1 and normalizes the levels of reactive oxygen species. Furthermore, IGF-I cooperates with trophic signals produced by astrocytes in response to H2O2 such as stem cell factor (SCF) to protect neurons against oxidative insult. After stroke, a condition associated with brain aging where oxidative injury affects peri-infarcted regions, a simultaneous increase in SCF and IGF-I expression was found in the cortex, suggesting that a similar cooperative response takes place in vivo. Cell-specific modulation by IGF-I of brain responses to oxidative stress may contribute in clarifying the role of IGF-I in brain aging.",
"keywords": [
"Oxidative stress is usually considered a mechanism of brain aging1. However",
"contradictory data2 and lack of firm evidence3 makes it difficult to firmly establish its actual significance in this process (see López-Otín et al.4 for a recent review). One important aspect that requires further clarification in this regard is the relationship between oxidative stress and insulin peptides",
"a well conserved family of hormones firmly linked to aging. Extensive work in vertebrates and invertebrates indicates that the insulin-like growth factor I (IGF-I)/insulin signalling (IIS) pathway has a negative impact on aging. It has been argued that this detrimental action is mediated by reducing cell defences to oxidative stress5–7 which",
"in turn is harmful for neuronal survival1. However",
"IGF-I has been shown to be largely neuroprotective8",
"even in conditions such as ischemic injury or brain trauma where oxidative stress is most likely a major pathogenic mechanism9. Thus",
"it is unclear whether or not IGF-I protects the brain against oxidative stress as the current evidence is contradictory."
],
"content": "Introduction\n\nOxidative stress is usually considered a mechanism of brain aging1. However, contradictory data2 and lack of firm evidence3 makes it difficult to firmly establish its actual significance in this process (see López-Otín et al.4 for a recent review). One important aspect that requires further clarification in this regard is the relationship between oxidative stress and insulin peptides, a well conserved family of hormones firmly linked to aging. Extensive work in vertebrates and invertebrates indicates that the insulin-like growth factor I (IGF-I)/insulin signalling (IIS) pathway has a negative impact on aging. It has been argued that this detrimental action is mediated by reducing cell defences to oxidative stress5–7 which, in turn is harmful for neuronal survival1. However, IGF-I has been shown to be largely neuroprotective8, even in conditions such as ischemic injury or brain trauma where oxidative stress is most likely a major pathogenic mechanism9. Thus, it is unclear whether or not IGF-I protects the brain against oxidative stress as the current evidence is contradictory.\n\nA possible explanation for these apparently contradictory observations may be that modulation of the cellular response to oxidative stress by IGF-I is cell-dependent10. Until now, only neurons have been studied in this regard. However, astrocytes, a major cellular element of the brain, are essential contributors to neuronal homeostasis and are coupled to neurons in the response to oxidative stress in order to help protect them11. It is thus possible that IGF-I participates in the response of astrocytes to oxidative stress as part of the overall brain response. Contrary to what we previously observed in neurons12, we report here that IGF-I protects astrocytes against oxidative stress and, very significantly, also co-operates with astrocytes to protect neurons.\n\n\nMethods\n\nWe used postnatal rats and mice for in vitro cultures (P0-3 days for astrocytes and P7 for neurons) and 3 month old mice for in vivo experiments. P2 Wistar rats (8 g ± 0.04 body weight, n=240, Harlan, Spain), P3 (2 g ± 0.03, n=36, Harlan) and 3 months old (27.6 g ± 0.812; n=24) C57BL6 mice and P7 GFP transgenic mouse pups (4.25 g ± 0.22, n=126; in-house colony) were used. Pups used were of both sexes and no attempt to sex them was done. Adult mice were male. Rat tissue was used in all in vitro experiments except when using GFP cells derived from transgenic mice. In vivo experiments were done in mice for future comparisons with transgenic mice. All efforts were made to minimize suffering and reduce the number of animals. Animals were kept under light/dark, (12h/12h) conditions following EU guidelines (directive 86/609/EEC) and handled according to institutionally-approved procedures (CSIC bio-ethics subcommittee; project code SAF2010-1703). Animals were fed ad libitum with laboratory rodent chow (Teklad Global 2018S) and kept in standard laboratory cage conditions with 4 animals/cage.\n\nAntibodies used in this study are detailed in Table 1. The different drug inhibitors used in the study are given in Table 2. Hydrogen peroxide (H2O2) and the calcium chelator BAPTA-AM were purchased from Sigma (Steinheim, Germany). IGF-I and SCF were purchased from Prospec-Tany Technogene, (Israel).\n\npECE-FOXO3 and pECE-FOXO3-TM (triple mutant T32A/S253A/S315A, herein called MFOXO3) were kindly provided by ME Greenberg (Harvard Medical School, Boston, USA). p6xDBE-luc (reporter luciferase plasmid with six copies of the DAF16 family protein-binding element) and pRL-TK (TK-Renilla luciferase) were a kind gift of BM Burgering (University Medical Centre, Utrecht, The Netherlands). Dominant negative IGF-IR expression plasmid was kindly donated by D. Le Roith (Mt Sinai, New York, USA). Plasmids expressing shRNA for TXNIP1 were purchased from Origene (USA). Txnip1 plasmid was purchased from Thermo Scientific Open Biosystems (Waltham, USA).\n\nCerebellar granule cultures were produced from either P7 rat or GFP transgenic mouse cerebella as previously described13. In brief, cells were plated onto 6 or 12-well dishes coated with poly-l-lysine (1 μg/ml) at a respective final density of 1.5×106/well or 0.45×106/well. Cells were incubated at 37°C/5% CO2 in Neurobasal (Gibco, USA) medium supplemented with 10% B27 (Gibco), glutamine (5 mM) and KCl (25 mM). All experiments were carried out in 2–7 day old cultures, with neurons showing neurite extensions. Different times in vitro were used to analyze time-dependent parameters such as cell survival. On the day of the experiment, medium was replaced with Neurobasal + 25 mM KCl. Two hours later, IGF-I (10-7 M) and/or hydrogen peroxide (H2O2) at doses of 50–150 µM were added. Inhibitory drugs were given 45 min before treatments. We used H2O2 as an oxidant stimulus because it is an endogenously produced reactive oxygen species (ROS) that serves as a precursor to hydroxyl radicals and possesses signalling capacities14. Astrocyte cultures were prepared from P3 rat or GFP mouse forebrain, as previously described15 after animals were sacrificed by decapitation. Cells were grown on Dulbecco’s modified Eagle's medium F12 (DMEM-F12) supplemented with 10% fetal calf serum. After 12 days astrocytes were seeded at 2.5×105 or 1.25×105 cells/well in 6-well and 12-well culture plates, respectively. On the day of the experiment cells were treated with IGF-I (10-7 M), H2O2 (50–200 µM) and/or inhibitors, as above. For transfection, astrocytes were seeded at 2.5×105 or 1.25×105 cells/well in 6-well and 12-well culture plates respectively, and after 16h constructs were mixed with Fugene HD (Roche, Switzerland) in a 1:3 ratio, and added following the manufacturer’s instructions. Alternatively, astrocytes were electroporated (2×106 astrocytes with 2 µg DNA or shRNA) before seeding using an astrocyte Nucleofector Kit (Lonza, Switzerland). After electroporation, cells were plated to obtain a final cell density on the day of the experiment similar to that obtained with the transfection method. All experiments were performed after 48h. The transfection efficiency was 20–30% and 60–80% for electroporation, as assessed with a GFP vector. At least three independent experiments were done in duplicate wells.\n\nCell viability was determined by different methods. When using transfected astrocytes, a GFP-pCMV vector and the different constructs under evaluation were used in a 1:5 ratio. In this case, GFP+ astrocytes were scored prior to treatment to determine baseline survival (time 0) and at different times as indicated in the results. Alternative viability assays for astrocytes included measuring cell metabolism with fluorescein diacetate (0.1 µg/ml FDA) or number of propidium iodide (PI) cells12 as specified in the results section. For the latter, cells were stained with 2 µg/ml PI as a marker of dead cells plus DAPI staining as a marker of total cell number. PI+ and DAPI+ cells were counted under a Leica CTR 6000 fluorescence microscope. Percentage of viable cells indicates the number of PI+ cells related to total cell number. The experiments were done in triplicate and a total of three independent experiments were done. For neuronal-specific viability assays, cerebellar neurons from GFP mice were seeded on 12-well plates (4.5×105 cells) coated with poly-L-Lysine and grown with Neurobasal medium plus B27, 4 mM glutamine and 25 mM KCl. After 4–5 days, cultures were treated with 100 nM IGF-I in the presence or absence of 50–100 µM H2O2. Pictures of GFP+ cells (green fluorescence) were taken every 24 hours up to 3 days in an IncucyteTM 2010A Rev2 system (Essen BioScience, USA). For co-cultures, 1.25×105 wild type mouse astrocytes/well were seeded on 12-well plates and grown with DMEM-F12 plus 10% FBS. After 48–72 hours, GFP neurons were isolated and plated onto astrocytes. We used forebrain astrocytes and cerebellar neurons because in our experience the forebrain and cerebellum yielded very high numbers of astrocytes and neurons, respectively (thus minimizing animal use). Furthermore, in this study we were interested in exploring general, rather than region-specific neuroprotective characteristics of astrocytes. Nevertheless, we also carried out co-cultures with neurons and astrocytes from the same region (forebrain) and the results obtained were identical than when using cells from differing regions (see Figure 2 in results). Culture medium was changed to DMEM-F12 plus B27, 4 mM glutamine and 25 mM KCl (the latter only in the case of neurons). Two days later, co-cultures were treated with 100 nM IGF-I ± 50–100 µM H2O2. Pictures were taken every 24 hours up to 5–7 days as above. For protein silencing or overexpression, 2×106 astrocytes were electroporated in a Nucleofector®II (Amaxa Biosystems Lonza, Switzerland) and seeded at 1.25×105/well. Co-cultured neurons were seeded as described above. Viability of neurons was assessed by counting the number of cells expressing GFP using Incucyte software with a set cell size threshold to avoid including GFP+ cell debris and dying cells. Viability is expressed as percentage of GFP+ cells at the beginning of the experiment (time 0). At least three independent experiments were done.\n\nWestern blotting was performed as described13. Neurons were washed once with ice-cold PBS and lysed with 1% NP-40, 150 mM NaCl, 20 mM Tris, pH 7.4, 10% glycerol, 1 mM CaCl2, 1 mM MgCl2, 400 μM sodium vanadate, 0.2 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml aprotinin and 0.1% phosphatase inhibitor cocktails I and II (Sigma-Aldrich). To normalize for protein load, membranes were reblotted (Re-Blot, Chemicon, USA) and incubated with an appropriate control antibody (see Results). Levels of the protein under study were expressed relative to protein load. Different exposures of each blot were collected to ensure linearity and to match control levels for quantification.\n\nDensitometric analysis was performed using Analysis Image Program (Bio-Rad, USA). A representative blot is shown from a total of at least three independent experiments. IGF-I levels in culture medium were measured using Quantikine ELISA for mouse/rat IGF-1 (R&D Systems, USA). In brief, cells were treated as described above and 1 ml of culture medium was collected after 24 hours, spun to eliminate cell debris, and stored at -80ºC. Samples were lyophilized overnight and resuspended in 150 µl of calibrator buffer. After vortexing, samples were centrifuged 10 min/14,000 rpm (Hettich, Germany) and assayed according to manufacturer´s instructions. A total of three and four independent experiments were done for neurons and astrocytes, respectively.\n\nLuciferase assays were done as previously reported12. In brief, cells were transfected with a reporter construct bearing six canonical FOXO binding sites (6×DBE-luciferase) and co-transfected with different constructs, as indicated in each experiment. Transfections were performed in triplicate dishes. Luciferase counts were normalized using TK-Renilla luciferase. At given times, neurons were lysed in passive lysis buffer (PLB) and luciferase activity was analysed using a luminometer and dual luciferase assay kit according to the manufacturer (Promega, USA). Background luminescence was subtracted. Luciferase activity was expressed as fold of increase over control levels. At least three independent experiments were done.\n\nAfter 18h of exposure to H2O2, cell death was assessed. Cells were detached using 0.25% Trypsin-1.3 mM EDTA (Invitrogen) during 5–10 minutes, centrifuged (200×g, 5 min/4ºC), and resuspended in cold PBS. Propidium iodide (PI 5 µg/ml; Sigma) in PBS was added prior to flow cytometry analysis using a FACSAria cytometer (BD Biosciences). Fluorescence intensity, forward scatter (FSC), and side scatter (SSC) were collected in logarithmic scale. The emission filter used was 600–620 nm band pass (FL3). A fluorescence blank was measured and subtracted from the fluorescence of the sample. Dead cells were identified as red fluorescence positive events with low FSC (small PI permeable cells). Debris was always excluded from the analysis. At least three independent experiments were conducted.\n\nMitochondrial O2- production levels were measured by using the fluorescent probe MitoSOXTM Red (Life Technologies, USA). Briefly, astrocytes were pre-treated overnight with IGF-I and then 200 µM H2O2 were added during 1 hour. Cells were incubated with 1.5 µM MitoSOXTM Red in DMEM-F12 for 10 min/37ºC and washed 3 times with PBS. Astrocytes were then trypsinized and fluorescence was measured by flow cytometry (510 nm excitation/580 nm emission) using the cytometer, as described15. A total of six independent experiments were done. Alternatively, ROS generation was assessed in astrocytes cultured on coverslips with the fluorogenic marker carboxy-H2DCFDA (Molecular Probes, USA) during 30 min/37ºC, protected from the light. When using this ROS marker it is not possible to distinguish endogenous ROS from exogenously applied H2O2. Nevertheless, we compared this method to the oxidation of luminol (which detects superoxide anions) that distinguishes H2O2 from other ROS and we obtained identical results with either method (data not visualized). The reason we used carboxy-H2DCFDA is because we could obtain both qualitative (cell images) and quantitative (fluorimetry assay) measurements within the same assay. After incubation with carboxy-H2DCFDA, cells were gently washed 3 times with warm DMEN, and mounted, or, alternatively, assayed for fluorimetry. Pictures were taken at 40× magnification using a Leica fluorescence microscope (Germany). A representative picture is shown. Fluorescence intensity in lysed cells was measured using a FluoroStar fluorimeter.\n\nAn RT2 ProfilerTM PCR Array (SABiosciences, USA) was used to screen a battery of growth factors following the manufacturer´s recommendations. After treatment, astrocytes were lysed and RNA extracted using Trizol (Life Technologies, USA). The resulting cDNA synthesis reaction was diluted in water, mixed with the qPCR master mix, and loaded in a 96 well PCR Array plate. PCR was performed following manufacturer´s instructions.\n\nThree-month old male mice (4–6 per group) were anesthetized with 3% isoflurane (in 70% N2O, 30% O2) for induction and with 2% isoflurane for maintenance. Rectal temperature was maintained at 36.5 C with a heating pad. The frontal branch of the medial cerebral artery (MCA) was exposed and occluded permanently by suture ligation as previously reported, with modifications16. Briefly, an incision perpendicular to the line connecting the lateral canthus of the left eye and the external auditory canal was made to expose and retract the temporalis muscle. A burr hole was drilled, and frontal and parietal branches of the MCA were exposed by cutting and retracting the dura. The frontal branch of the MCA was elevated and ligated with a suture nylon monofilament 8/0. Following ligation, a sharp decrease of blood flow was evidenced with a laser Doppler flowmetry (Järfalla, Sweden). Following surgery, mice were returned to their cages, kept at room temperature and allowed free access to food and water. All physiological parameters measured: rectal temperature, mean arterial pressure and blood glucose levels were not different between groups. Sixteen hours after medial cerebral artery occlusion (MCAO), animals were killed by neck dislocation by an experienced researcher to assess infarct outcome. The brain was removed and the infarcted area isolated and processed for RNA and protein isolation.\n\nTotal RNA isolation from cell lysates or brain tissue was carried out with Trizol. One µg of RNA was reverse transcribed using High Capacity cDNA Reverse Transcription Kit (Life Technologies) according to the manufacturer’s instructions. For the quantification of specific genes, total RNA was isolated and transcribed as above and 62.5 ng of cDNA was amplified using TaqMan probes for Txnip1, IGF-I or SCF and 18S as endogenous control (Life Technologies). Each sample was run in triplicate in 20 μl of reaction volume using TaqMan Universal PCR Master Mix according to the manufacturer’s instructions (Life Technologies). All reactions were performed in a 7500 Real Time PCR system (Life Technologies). Quantitative real time PCR analysis was carried out as previously described17. Results were expressed as relative expression ratios on the basis of group means for target transcripts versus reference 18S transcript. At least three independent experiments were done.\n\nData are expressed as mean ± SEM. Differences among groups were analyzed by one-way ANOVA followed by a Newman-Keul’s or t-test using Graph Pad Prism softwareA p<0.05 was considered significant.\n\n\nResults\n\nWhereas neurons cultured without astrocytes are very sensitive to acute oxidative insult elicited by H2O2 (Figure 1A), when cultured with astrocytes, neurons become very resilient (Figure 1A). To determine whether IGF-I participates in the neuroprotective effects of astrocytes against oxidative stress we first confirmed that it is endogenously produced by these cells. As shown in Figure 1B, not only astrocytes but also neurons (albeit at much lower levels) secrete IGF-I into the culture medium. In response to H2O2 astrocytes secrete lower, but still substantial, amounts of IGF-I, and so IGF-I may still participate in neuroprotection by astrocytes. To directly test this possibility we blocked IGF-I signalling in astrocytes with a dominant negative (DN) IGF-IR18 (Figure 1C) and determined their ability to protect neurons against oxidative challenge. As shown in Figure 1D, a significantly greater percentage of neurons co-cultured with mock-transfected astrocytes survived compared to neurons cultured with astrocytes transfected with DN IGF-IR. Similar results were obtained after H2O2 challenge (not visualized).\n\nA) Neurons are protected from oxidative stress in the presence of astrocytes whereas when cultured alone they rapidly die. Viability of GFP neurons was measured as the number of green (GFP+) cells two days after H2O2 treatment in the presence or absence of wild type astrocytes (F=41.85; ***p<0.001 vs. neurons alone). B) Both astrocytes and neurons secrete IGF-I, although astrocytes produce much higher levels (*p<0.05 vs. neurons). H2O2 lowers IGF-I secretion. C) Astrocytes were transfected with a dominant negative IGF-IR (IGF-IR DN) and the ratio pAkt/Akt (histograms) was measured as an index of IGF-I signalling. In the presence of DN IGF-IR, signalling by IGF-I was markedly reduced. Representative blots and quantitative histograms are shown (*p<0.05 vs. IGF-I alone). D) Blockade of IGF-IR function with dominant negative IGF-IR compromises neuroprotection by astrocytes. GFP neurons were seeded on top of wild type astrocytes transfected with an IGF-IR DN construct or mock-transfected (control). Viability of GFP neurons was measured after 5 days (***p<0.001 vs. control). Experiments were done at least 3 times in this and all following figures. Bars are SEM in this and following figures.\n\nA) The IGF-IR inhibitor PPP blocks IGF-I signalling in astrocytes. Astrocytes were treated with 120 nM PPP 1h before adding IGF-I while pAkt levels were measured 10 minutes after adding IGF-I. Ratios are shown in histograms (**p<0.01 vs. IGF-I alone). Representative blot is shown. B) Blockade of IGF-IR signalling with PPP in neurons cultured alone does not affect H2O2 toxicity after 3–4 days of exposure (F=8.124, **p<0.01 vs. respective controls). Note that PPP alone does not affect neuronal survival. C) Viability of cerebellar neurons co-cultured with forebrain astrocytes decreased significantly when treated with PPP for six days. PPP treatment in the presence of H2O2 decreased neuronal viability even further (F=8.90; *p<0.05 vs. untreated control and #p<0.05 vs. H2O2). D) Viability of forebrain neurons co-cultured with forebrain astrocytes decreased significantly when treated with PPP for five days. PPP treatment in the presence of H2O2 decreased neuronal viability even further (F=170.2, ***p<0.01 vs. untreated control and ###p<0.01 vs. H2O2). E) When co-cultured with wild type astrocytes, neuronal survival after five days of exposure to 100 µM H2O2 was moderately increased in the presence of 100 nM IGF-I (F=9.965; *p<0.05 vs. control or H2O2). I+H: IGF-I + H2O2.\n\nWe next used pharmacological blockade of the IGF-I receptor using picropodophyllin (PPP), an antagonist of IGF-IR (Figure 2A). As in this case both the neuronal and astrocyte receptors are blocked, we first determined whether neurons are affected by PPP blockade of the IGF-I receptor. In the presence of H2O2, neurons cultured alone die regardless of the presence or absence of proper IGF-I signalling since PPP did not increase neuronal death (Figure 2B). This agrees with our previous findings that IGF-I does not protect cultured neurons against oxidative stress12. Confirming the results seen with astrocytes transfected with dominant negative IGF-I receptor, a reduction in neuroprotection by astrocytes was seen when co-cultured neurons were exposed to PPP. In the presence of H2O2, significantly fewer co-cultured neurons survived with PPP (p<0.01 H2O2+PPP vs. H2O2 alone; Figure 2C). To rule out region-specific actions of astrocytes on neuroprotection we then co-cultured neurons and astrocytes from the same brain region (forebrain) and treated them with PPP. As shown in Figure 2D, forebrain neurons were similarly sensitive to blockade of IGF-IR when co-cultured with forebrain astrocytes. The observation that even supra-physiological doses of IGF-I (100 nM) added to the co-cultures only produced a modest additional effect on neuronal survival after oxidative insult confirmed the idea that endogenous IGF-I is required by astrocytes for neuroprotection (Figure 2E). Hence, endogenous production of IGF-I is necessary and sufficient to protect neurons.\n\nIGF-I-dependent neuroprotection by astrocytes appears to also involve a direct action of IGF-I on astrocytes. Because it is known that astrocytes are more resistant to oxidative damage than neurons, we explored whether IGF-I was involved in this greater resilience. Contrary to neurons (Figure 3A), IGF-I protected astrocytes against H2O2-induced death (Figure 3B). The protective effect of IGF-I involved blockade of the activation of FOXO 3, a transcription factor involved in brain responses to oxidative stress19, by H2O2 (Figure 3C). Inhibition of FOXO 3 by IGF-I was mediated by Akt; i.e.: an Akt-insensitive mutant of FOXO (M-FOXO3) abrogated IGF-I effects while wild type FOXO3 did not interfere with its protective actions (Figure 3D). Indeed, in astrocytes IGF-I activates Akt in the presence of H2O2 (Figure 3E), whereas in neurons H2O2 blocks this canonical pathway12. Underlying the protective actions of IGF-I on astrocytes was its ability to block excess ROS after exposure to H2O2 as determined by flow cytometry using MitoSOX (Figure 4A) or fluorometry with carboxy-H2DCFDA (Figure 4B).\n\nA) IGF-I does not rescue neurons from H2O2 induced death. This confirms previous observations12. Neuronal mortality was measured by counting PI+ cells 6h after treatment. H2O2 induces neuronal death in a dose-dependent manner irrespective of the presence of IGF-I (***p<0.001 vs. no H2O2). B) IGF-I treatment protects astrocytes from H2O2 induced death. Astrocyte demise was measured by counting PI+ cells 24h after H2O2 (100 µM). H2O2 exerts a dose-dependent effect that is reduced by IGF-I (*p<0.05 vs. control). C) IGF-I blocks FOXO activity induced by H2O2 (100 µM). FOXO activity was measured with a luciferase reporter in astrocytes treated with IGF-I, H2O2 or both for 24h (***p<0.001 vs. no treatment). D) Protection by IGF-I against death induced by H2O2 requires blockade of FOXO activity. Astrocyte viability was measured by counting GFP+ astrocytes after co-transfection of GFP and a FOXO wild type (wt) or an Akt-insensitive mutant of FOXO (M-FOXO); *p<0.05 vs. no IGF-I. E) IGF-I increases phosphorylation of Akt (pAkt) in the presence of H2O2 in a dose-dependent fashion. Representative blots are shown. Lower histograms indicate quantification of pAkt/Akt ratio in the presence of IGF-I as shown in the right blot. pAkt levels were measured after 15 min. (*p<0.05 and ***p<0.001 vs. no H2O2).\n\nA) H2O2 increases the number of astrocytes expressing mitochondrial O2-. This increase is prevented when cells are pre-treated with IGF-I. Mitochondrial O2- levels were detected with MitoSOX by flow cytometry. Astrocytes were treated overnight with IGF-I and for 1 hour more with 200 µM H2O2 (F=9.364; *p<0.005 H2O2 vs. control, IGF-I and IGF-I + H2O2). B) IGF-I lowers ROS levels after treatment of astrocytes with H2O2 (100 µM). Left: representative photomicrographs of astrocytes stained with carboxy-H2DCFDA to detect ROS and DAPI to stain cell nuclei. The increase in fluorescent cells elicited by H2O2 was markedly diminished by IGF-I. Right histograms: fluorimetric quantification of ROS levels with carboxy-H2DCFDA confirmed the rescuing action of IGF-I on astrocytes exposed to H2O2. (F=7.362; **p<0.01 H2O2 vs. control, IGF-I or IGF-I + H2O2.\n\nWe then determined possible mediators of the anti-oxidative actions of IGF-I on astrocytes. We examined whether modulation of SODs could be involved because these anti-oxidant enzymes constitute an important detoxifying mechanism in cases of excess ROS. We found that cytosolic Cu/ZnSOD was increased by IGF-I, H2O2, or both (Figure 5A), while mitochondrial MnSOD was increased only by H2O2 (Figure 5B). Thus, increases in SOD levels form part of the astrocyte response to H2O2, and IGF-I does not appear to interfere with these enzymes. Because FOXO participates in cellular responses to ROS, we looked for signals downstream of FOXO inactivation by IGF-I such as thioredoxin inhibitor 1 (TXNIP1), a pro-apoptotic protein dependant on FOXO activity and related to anti-oxidant responses20. We first confirmed that in astrocytes TXNIP1 is also controlled by FOXO; i.e.: in astrocytes expressing dominant negative Foxo, TXNIP1 levels were 89% reduced as compared to mock-transfected astrocytes. Accordingly, IGF-I, which inhibits FOXO, also reduced TXNIP1 levels (Figure 6A). Strikingly, H2O2, which stimulates FOXO activity in astrocytes (Figure 3D), also inhibited TXNIP1 (Figure 6A), suggesting alternative routes of TXNIP1 regulation in the presence of H2O2. When IGF-I and H2O2 were simultaneously added to astrocytes, TXNIP1 levels were markedly decreased (p<0.05 vs. IGF-I or H2O2 alone, Figure 6A). To determine the impact of downregulation of TXNIP1 on astrocyte survival we inhibited its expression with shRNA (blot in Figure 6B) and found that astrocytes became resistant to H2O2 when TXNIP1 levels were low (Figure 6B). Overexpression of TXNIP1 did not alter the response of astrocytes to H2O2 whereas co-culture of neurons with astrocytes depleted of TXNIP1 did not result in enhanced neuronal survival (data not visualized for clarity), indicating that this route is involved in the response of astrocytes to oxidative stress but not in neuroprotection. Interestingly, in neurons, TXNIP1 was downregulated only in the presence of H2O2, but not after IGF-I treatment (Figure 6C). Thus, IGF-I downregulates TXNIP1 only in astrocytes, not in neurons.\n\nA) Cu/ZnSOD levels in astrocytes are modulated by IGF-I and H2O2. B) MnSOD levels are enhanced by H2O2 but not by IGF-I (*p<0.05 and **p<0.01 vs. control).\n\nA) Levels of the pro-oxidant protein TXNIP1 are reduced by IGF-I and H2O2. Inhibition is greater when both are added together (F=156.6; ***p<0.001 vs. control and ###p<0.001 (vs. IGF-I) and #p<0.05 (vs. H2O2)). Levels of actin in each sample were measured to normalize TXNIP1 levels. B) Western blot: transfection of astrocytes with shRNA TXNIP1 results in reduced TXNIP1 levels as compared to astrocytes transfected with scrambled shRNA (SCR). TXNIP1 shRNA silencing makes astrocytes less sensitive to H2O2 toxicity. Astrocyte viability was measured by FDA in the presence of 200 µM H2O2 (F=12.09; **p<0.01 vs. control). C) In neurons, only H2O2 decreases TXNIP1 levels, whereas IGF-I does not (***p<0.001 vs. control). D) Reduction of TXNIP1 by IGF-I and H2O2 in astrocytes depends on Ca2+ as in the presence of the calcium chelator BAPTA-AM the decrease is abrogated, (F=7.226; *p<0.05 and ***p<0.001 vs. control). C=control, I=IGF-I, H=H2O2, H + I=H2O2 + IGF-I.\n\nWe then analyzed possible pathways involved in the inhibitory effect of H2O2 and IGF-I on TXNIP1. Using kinase inhibitors we ruled out the idea that the main kinases downstream of the IGF-I receptor or H2O2 were involved. In fact, inhibition of most of these kinases resulted in altered basal levels of TXNIP1 (not visualized), suggesting that basal levels of this protein are tightly regulated in astrocytes. Other inhibitory drugs of different pathways where IGF-I participates (PKC, PKA, CnA, PDK-1, NFκB among others) gave similar negative results. However, inhibition of Ca++ flux with 5 µM BAPTA abrogated TXNIP1 decreases in response to either H2O2 or IGF-I while only slightly, but not significantly affecting basal levels (Figure 6D).\n\nWe next analyzed possible neuroprotective effects of IGF-I through astrocytes. Using a commercial gene array for growth factors we screened growth factor production by IGF-I-treated astrocytes in response to H2O2. Among the several growth factors that increased, stem cell factor (SCF) showed the highest elevation (Table 3). We confirmed by qPCR that SCF is increased after H2O2 whereas IGF-I decreased it (Figure 7A). As SCF has been shown to be neuroprotective21, we determined whether it protects neurons against H2O2 and found that while SCF alone did not exert any protection, co-treatment with IGF-I resulted in significantly greater neuronal survival (p<0.05; Figure 7B). We then examined pathways underlying this cooperative action of IGF-I and SCF. Under basal conditions, the activity of extracellular signal-regulated kinase (Erk; measured as pErk/Erk ratio), a canonical kinase in IGF-I signalling, was increased by IGF-I as expected, and also by SCF (Figure 7C). Basal Erk activity was also increased by H2O2. However, Erk was no longer activated by IGF-I or SCF in the presence of H2O2. Only when both were added together to H2O2-challenged cultures Erk activity was increased (Figure 7C). No interactions were found with Akt, the other canonical kinase pathway activated by IGF-I.\n\nA battery of growth factors was screened with an RT2 ProfilerTM PCR Array. In brief, astrocytes were treated or not with IGF-I+H2O2 for 16h and total RNA was isolated. After performing the RT-PCR, total cDNA was assayed for PCR Array. PCR data was analyzed with the RT2 Profiler PCR Array Data Analysis version 3.5 software provided by the manufacturer. Significantly up- or downregulated genes are shown.\n\nA) H2O2 stimulates SCF mRNA levels in astrocytes after 16h of exposure whereas IGF-I partially counteracts this increase (F=38.67; *p<0.05 vs. control and IGF-I, #p<0.05 vs. H2O2). B) SCF and IGF-I cooperate to protect neurons from oxidative stress. Neurons were pretreated with SCF, IGF-I or both 48h before adding H2O2 (50 µM) and viability was assessed after overnight treatment (F=12.09, ***p<0.0001 vs. H2O2), H: H2O2; I: IGF-I. C) When H2O2 is present, Erk phosphorylation is significantly increased only when both SCF and IGF-I are added to the cultures but not with either alone. Neurons were treated with 100 nM IGF-I, 20 ng/ml SCF and 50 µM H2O2 for 5 minutes and pErk levels were measured by western blot and normalized for total Erk. (*p<0.05 and **p<0.01 vs. control without H2O2 and #p<0.05 vs. H2O2). D) SCF and IGF-I mRNA levels increased 16 hours after middle cerebral artery occlusion (MCAO) in the contralateral side (CONTRA) in the case of SCF (F=31.53; ***p<0.001 vs. intact control mice) and in both sides in the case of IGF-I (F=7.853; *p<0.05 and **p<0.01 vs. control). E) SCF protein levels increase after MCAO in both sides of the cortex. (F=12.38; *p<0.05 and ***p<0.001 vs. control). A representative blot is shown. Six, five and four animals were used per group, respectively. Levels of actin in each sample were measured to normalize for total protein levels.\n\nTo determine the in vivo relevance of these observations we submitted mice to brain ischemia as this brain insult is associated to oxidative stress22, and both IGF-I9 and SCF23 have been shown to be neuroprotective after ischemia. We found that IGF-I mRNA is increased after middle cerebral artery occlusion (MCAO) both in the ipsilateral and contralateral cortex, while only the contralateral side showed increased SCF mRNA levels compared to intact mice (Figure 7D). However, levels of SCF protein were elevated after MCAO in both the damaged and contralateral sides compared to normal mice (Figure 7E). This suggests that after brain ischemia the contralateral cortex produces higher amounts of SCF that eventually reach the ischemic side. Under this condition IGF-I may interact with SCF to promote neuronal survival in the ipsilateral cortex.\n\n\nDiscussion\n\nThe present results indicate that IGF-I exerts a protective action on astrocytes contributing to the resilience of these glial cells against oxidative stress. IGF-I also cooperates with astrocytes to protect neurons. These observations highlight the importance of cell-specific and cell-cooperative aspects of IGF-I protection against oxidative challenge. Thus, a better understanding of the trophic role of IGF-I in the brain requires taking into account its effects on astrocytes (and other brain cells) and the functional links of these cells with neurons. While these observations do not help settle the role of oxidative stress in brain aging they put forward an important aspect of possible mechanisms involved in aging; regulatory signals such as IGF-I may not modulate the response of the different cells and even tissues to oxidative stress in the same way.\n\nA greater resilience of astrocytes to oxidative stress provided by IGF-I will allow these cells to protect neurons that are more sensitive to oxidative challenge. Astrocytes are coupled to neurons in the response to oxidative stress and provide them with ample detoxification support11. In addition, among different anti-oxidant defences provided by astrocytes to neurons, we now find that IGF-I, which cannot protect isolated neurons against excess ROS12 cooperates with SCF secreted by astrocytes to support neurons (Figure 8). While in response to oxidative stress the production of IGF-I by cultured astrocytes and neurons is decreased, after brain ischemia IGF-I levels are actually higher due to increased synthesis and accumulation in microglia, vessels and astrocytes24. Therefore, in vivo, astrocytes and neurons will receive IGF-I input from various local sources, suggesting that the response of increased IGF-I after brain ischemia reflects an endogenous neuroprotective mechanism against oxidative injury. This conclusion apparently contradicts other evidence that IIS activity is pro-oxidant. Thus genetic ablation of ISS components in the nematode Caenorhabditis elegans25 or in higher organisms such as the fruit fly26 or mice27, increases organism resistance to oxidative stress. For example, mice with reduced IGF-I activity (hemizygous for the IGF-I receptor) have lower levels of ROS in the brain28,29. However, these mice developed greater cell damage after oxidative injury29. Conceivably, the effects of modulating IGF-I signalling prior to ROS insult (as when using genetic models) may not be the same as after insult. For example IGF-I protects nerve cells and/or the brain against diverse types of ROS-related insults30–34. In this regard, we recently reported that in a cellular model of Friedreich's ataxia (which elicits oxidative damage) neurons responded to IGF-I only when they became frataxin deficient, but not under normal conditions15. Collectively these observations emphasize the importance not only of cell type but also of context dependency of IGF-I neuroprotection in relation to oxidative stress.\n\nLeft: under basal conditions IGF-I exerts potent neuroprotective actions directly onto neurons and probably also through astrocytes. In the presence of H2O2 (right side), a number of events develop: 1) IGF-I loses its ability to directly protect neurons, 2) IGF-I secretion by astrocytes is diminished, 3) astrocytes reinforce anti-oxidant defences through IGF-I by downregulating pro-oxidant mechanisms such as TXNIP1, and 4) astrocytes potentiate neuroprotection by, among several other mediators, growth factors such as SCF that cooperate with IGF-I to protect neurons. Cytotoxic effects are depicted in red while cytoprotective actions are indicated in blue trace.\n\nA role for oxidative stress in many neurodegenerative diseases is gaining increasing acceptance35. Aberrant production of ROS in the central nervous system is linked to neurodegenerative diseases such as Alzheimer´s dementia, Parkinson’s disease or stroke, all of them associated to aging36. However, as already commented, the role of oxidative stress in brain aging is still unclear. An attempt to explain these apparently opposing observations is that moderate ROS levels may activate survival pathways37. The present findings agree with this proposal. Thus, doses of H2O2 up to 100 µM do not elicit astrocyte death probably because IGF-I helps maintain their anti-oxidant capacity and at the same time their neuroprotective action. In this regard our results show that astrocytes in response to IGF-I and/or H2O2 activate antioxidant signalling including upregulation of Cu/ZnSOD and MnSOD coupled to downregulation of pro-oxidant proteins such as Txnip1. Txnip1 inhibits thioredoxin (Trx), a protein that reduces protein disulfides as well as H2O2. The Txnip-Trx axis plays an important role in different brain diseases in which oxidative stress is implicated38.\n\nThere is ample evidence that different trophic factors, including SCF39, contribute to reduce cell damage due to oxidative stress after brain stroke40. We have found that in vitro IGF-I and SCF exert a cooperative neuroprotective effect against oxidative stress, suggesting that they may exert a similar beneficial role in vivo as after brain stroke both factors are upregulated in the lesioned area. Indeed, a cooperative neuroprotective effect of SCF with insulin has been reported41. The intracellular mechanisms mediating cooperation between these two factors involve Erk, a kinase activated by IGF-I.\n\nIn summary, cell specific and cooperative actions of IGF-I in brain responses to oxidative challenge underscores the need to design therapeutic strategies that take into account all aspects of biological organization, leading, for example, to cell-specific targeting of anti-aging drugs.\n\n\nData availability\n\nfigshare: Data on the responses of neurons and astrocytes to oxidative injury in the presence of insulin-like growth factor I, http://dx.doi.org/10.6084/m9.figshare.90490970",
"appendix": "Author contributions\n\n\n\nLaura Genis: designed, analyzed and performed experiments and wrote parts of the manuscript.\n\nDavid Dávila: designed, analyzed and performed experiments and wrote parts of the manuscript.\n\nSilvia Fernandez: designed, analyzed and performed experiments.\n\nAndrea Pozo-Rodrigálvarez: performed experiments.\n\nRicardo Martínez-Murillo: performed experiments and contributed materials.\n\nIgnacio Torres-Aleman: design the study, analyze the data and wrote the paper.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was funded by grants of the Spanish Ministry of Science (SAF2010-17036) and Centro Investigacion Biomedica en red Enfermedades Neurodegenerativas (CIBERNED) to IT-A.\n\n\nAcknowledgments\n\nWe are thankful to L Guinea, M Dominguez and M. Garcia for excellent technical support.\n\n\nReferences\n\nDemaurex N, Scorrano L: Reactive oxygen species are NOXious for neurons. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nLanfranconi S, Locatelli F, Corti S, et al.: Growth factors in ischemic stroke. J Cell Mol Med. 2011; 15(8): 1645–1687. PubMed Abstract | Publisher Full Text\n\nLi JW, Li LL, Chang LL, et al.: Stem cell factor protects against neuronal apoptosis by activating AKT/ERK in diabetic mice. Braz J Med Biol Res. 2009; 42(11): 1044–1049. PubMed Abstract | Publisher Full Text\n\nZheng M, Zhu H, Gong Y, et al.: Involvement of GMRP1, a novel mediator of Akt pathway, in brain damage after intracerebral hemorrhage. Int J Clin Exp Pathol. 2013; 6(2): 224–229. PubMed Abstract | Free Full Text\n\nKukreti H, Amuthavalli K, Harikumar A, et al.: Muscle-specific microRNA1 (miR1) targets heat shock protein 70 (HSP70) during dexamethasone-mediated atrophy. J Biol Chem. 2013; 288(9): 6663–6678. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWojtalla A, Fischer B, Kotelevets N, et al.: Targeting the phosphoinositide 3-kinase p110-alpha isoform impairs cell proliferation, survival, and tumor growth in small cell lung cancer. Clin Cancer Res. 2013; 19(1): 96–105. PubMed Abstract | Publisher Full Text\n\nCheshenko N, Trepanier JB, Stefanidou M, et al.: HSV activates Akt to trigger calcium release and promote viral entry: novel candidate target for treatment and suppression. FASEB J. 2013; 27(7): 2584–2599. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNorth AJ, Galazkiewicz B, Byers TJ, et al.: Complementary distributions of vinculin and dystrophin define two distinct sarcolemma domains in smooth muscle. J Cell Biol. 1993; 120(5): 1159–1167. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVandekerckhove J, Weber K: Actin amino-acid sequences. 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PubMed Abstract | Publisher Full Text\n\nShinder GA, Lacourse MC, Minotti S, et al.: Mutant Cu/Zn-superoxide dismutase proteins have altered solubility and interact with heat shock/stress proteins in models of amyotrophic lateral sclerosis. J Biol Chem. 2001; 276(16): 12791–12796. PubMed Abstract | Publisher Full Text\n\nMonari M, Cattani O, Serrazanetti GP, et al.: Effect of exposure to benzo[a]pyrene on SODs, CYP1A1/1A2- and CYP2E1 immunopositive proteins in the blood clam Scapharca inaequivalvis. Mar Environ Res. 2007; 63(3): 200–218. PubMed Abstract | Publisher Full Text\n\nShirasawa T, Izumizaki M, Suzuki Y, et al.: Oxygen affinity of hemoglobin regulates O2 consumption, metabolism, and physical activity. J Biol Chem. 2003; 278(7): 5035–5043. PubMed Abstract | Publisher Full Text\n\nKatsuki H, Tomita M, Takenaka C, et al.: Superoxide dismutase activity in organotypic midbrain-striatum co-cultures is associated with resistance of dopaminergic neurons to excitotoxicity. J Neurochem. 2001; 76(5): 1336–1345. PubMed Abstract | Publisher Full Text\n\nPedraza-Chaverri J, Maldonado PD, Medina-Campos ON, et al.: Garlic ameliorates gentamicin nephrotoxicity: relation to antioxidant enzymes. Free Radic Biol Med. 2000; 29(7): 602–611. PubMed Abstract | Publisher Full Text\n\nMichels S, Trautmann M, Sievers E, et al.: SRC signaling is crucial in the growth of synovial sarcoma cells. Cancer Res. 2013; 73(8): 2518–2528. PubMed Abstract | Publisher Full Text\n\nFei Z, Bera TK, Liu X, et al.: Ankrd26 gene disruption enhances adipogenesis of mouse embryonic fibroblasts. J Biol Chem. 2011; 286(31): 27761–27768. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWu M, Desai DH, Kakarla SK, et al.: Acetaminophen prevents aging-associated hyperglycemia in aged rats: effect of aging-associated hyperactivation of p38-MAPK and ERK1/2. Diabetes Metab Res Rev. 2009; 25(3): 279–286. 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Peptides. 2013; 40: 104–111. PubMed Abstract | Publisher Full Text\n\nRaucci F, Di Fiore MM: Localization of c-kit and stem cell factor (SCF) in ovarian follicular epithelium of a lizard, Podarcis s. sicula. Acta Histochem. 2011; 113(6): 647–655. PubMed Abstract | Publisher Full Text\n\nLaura Genis, David Dávila, Silvia F, et al.: Data on the responses of neurons and astrocytes to oxidative injury in the presence of insulin-like growth factor I. figshare. 2014. Data Source"
}
|
[
{
"id": "3440",
"date": "06 Feb 2014",
"name": "Marta Margeta",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this paper, the authors attempt to elucidate the role of IGF-I in the astrocyte-mediated protection of neurons against oxidative stress. While this is an important topic and the authors present a lot of interesting data, the paper is unfocused and the results do not fully support the conclusions. As such, I feel that the paper cannot be approved for indexing until substantively revised.Major comments:The paper presents three essentially independent sets of data and then tries to connect them into a single coherent story, without direct experimental evidence that it is appropriate to do so. Specifically, Figs. 1 and 2 present evidence that IGF-I plays an important role in the astrocyte-mediated neuroprotection, both at baseline conditions and under oxidative stress; this is the most interesting part of the paper (and the part that is most relevant to the paper’s current title). Figs. 3-6 show data that elucidate some aspects of IGF-I effect on astrocytes, but do not establish the importance of these effects/mechanisms for IGF-I- and astrocyte-mediated neuroprotection. (Notably, the authors have actually established that one of these mechanisms, IGF-I-induced decrease in the expression of astrocytic TXNIP1, does not play a role in the astrocyte-mediated neuroprotection. Surprisingly, these key results are not shown despite the fact that an entire figure [Fig. 6] is devoted to the IGF-I modulation of TXNIP1).Finally, Fig. 7 shows that IGF-I and SCF applied together (but not separately) have a neuroprotective effect in the absence of astrocytes and that their expression is increased in an in vivo stroke model. However, the authors again fail to show that these observations are in any way relevant for the astrocyte-mediated neuroprotection shown in Figs. 1 and 2. To connect these currently unconnected experimental threads, the authors need to use their neuron-astrocyte co-culture system to establish the link between astrocyte-mediated neuroprotection and (1) IGF-I-mediated increase in the expression of astrocyte antioxidant enzymes, (2) IGF-I-mediated decrease in the astrocyte ROS levels, and (3) astrocyte secretion of SCF; the experiments should be performed both at baseline and under oxidative stress conditions. Alternatively, the paper needs to be re-written in a way that makes it very clear (1) that the data presented in the paper represent a series of independent observations that do not add up to a coherent whole and (2) that the mechanism mediating IGF-I-induced neuroprotection in the mixed neuron-astrocyte environment currently remains unexplained. If choosing this route, the authors should also change the title of the paper to something more neutral and descriptive. The authors do not show some important experimental data, ostensibly “for clarity”; these data need to be included in the revised paper. Specifically, as already stated in point #1, the authors mention (but do not show) that depleting astrocytes of TXNIP1 does not result in increased neuronal survival (page 16). This finding, if properly established, indicates that the IGF-I-mediated decrease in TXNIP1 and the IGF-I/astrocyte-mediated neuroprotection are two entirely unrelated phenomena. Given the paper’s overall title and conclusions, this is a key experiment that needs to be shown. Similarly, it is important to show the results of IFG-IR DN experiment performed under oxidative stress conditions (page 7). In many experiments, the authors do not use appropriate statistical analyses. Specifically, given the experimental design, two-way (rather than one-way) ANOVA should be used to analyze data shown in Figs. 1B-C, 1D (after missing data are included), 2A-E, 3A-D, 4A-B, and 6B. Fig. 8 (the model) does not accurately represent the experimental results. For example, the authors state in the Fig. 8 legend that “under basal conditions IGF-I exerts potent neuroprotective actions directly onto neurons” when in fact, IGF-I has no clear neuroprotective effect when applied to neurons cultured alone (Fig. 3A, 0 µM H2O2 condition) – the IGF-I-induced decrease in neuronal cell death is small [~5% based on the graph] and does not appear statistically significant, although the authors do not comment on this one way or the other. Similarly, the figure legend mentions that IGF-1 down-regulates astrocytic TXNIP1 – a finding that is accurate but not relevant for the astrocyte-mediated neuroprotection illustrated by the figure. Thus, Fig. 8 should either be altered to more meaningfully represent the paper’s findings or, if the authors decide to re-write the paper in a more descriptive fashion, could be eliminated altogether.Minor comments:Why are neurons cultured under depolarizing conditions (25 mM KCl)? The co-culture experimental set-up should be described under a separate heading, not buried under “Cell assays”. For similar experiments, the authors should use a similar type of plot to make the paper more readable. For example, experiments in Figs. 3A and 3B have a very similar overall design – why are the results shown very differently? Neuronal viability is established by counting “all GFP-positive cells”; the authors therefore need to show that their neuronal cultures are at least 95% and preferably 99% pure (i.e., do not contain a significant population of GFP-positive glial cells). Representative flow cytometry plots should be included in Fig. 4A. To enhance readability, all figure panels should be labelled “astrocytes”, “neurons”, or “neurons + astrocytes”, as appropriate. (The information is currently largely buried in the figure legends.) In Fig. 5, authors should specify whether they are measuring mRNA or protein level (I assume the latter, but it’s difficult to be sure).",
"responses": []
},
{
"id": "3443",
"date": "07 Feb 2014",
"name": "Carlos Matute",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an excellent paper with data relevant to CNS protection against oxidative stress.",
"responses": []
},
{
"id": "3862",
"date": "06 Mar 2014",
"name": "Vince C Russo",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript by Laura Genis and co-workers is very interesting and the data presented are of significant scientific value. These studies provide further understanding of the cellular and molecular mechanisms involved in neuronal damage and rescue following oxidative stress. Of particular interest are the protective effects of IGF-I on neuronal cells mediated via the astrocytes. To these, the synergistic/additive effects of SCF on IGF-I are novel and interesting.I have only few minor suggestions - mainly to improve graphic illustrations:In figure 1B, IGF-I ELISA, the conditioned media from astrocytes and neuronal cells is analysed for IGF-I levels but the values are express in ng/ug of protein, why? In Figure 3E I asume that the graph shows the % phosphorylation for the IGF-I treatment? This should be properly label. In Figure 4 a statistical significance is shown for the 'untreated', but it should shown for the IGF-I which actually prevents MitoO2. I have a similar comment for the graph below in figure 4B.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-28
|
https://f1000research.com/articles/3-93/v1
|
22 Apr 14
|
{
"type": "Research Article",
"title": "Cytoplasmic nanojunctions between lysosomes and sarcoplasmic reticulum are required for specific calcium signaling",
"authors": [
"Nicola Fameli",
"Oluseye A. Ogunbayo",
"Cornelis van Breemen",
"A. Mark Evans",
"Oluseye A. Ogunbayo",
"Cornelis van Breemen",
"A. Mark Evans"
],
"abstract": "Herein we demonstrate how nanojunctions between lysosomes and sarcoplasmic reticulum (L-SR junctions) serve to couple lysosomal activation to regenerative, ryanodine receptor-mediated cellular Ca2+ waves. In pulmonary artery smooth muscle cells (PASMCs) it has been proposed that nicotinic acid adenine dinucleotide phosphate (NAADP) triggers increases in cytoplasmic Ca2+ via L-SR junctions, in a manner that requires initial Ca2+ release from lysosomes and subsequent Ca2+-induced Ca2+ release (CICR) via ryanodine receptor (RyR) subtype 3 on the SR membrane proximal to lysosomes. L-SR junction membrane separation has been estimated to be < 400 nm and thus beyond the resolution of light microscopy, which has restricted detailed investigations of the junctional coupling process. The present study utilizes standard and tomographic transmission electron microscopy to provide a thorough ultrastructural characterization of the L-SR junctions in PASMCs. We show that L-SR nanojunctions are prominent features within these cells and estimate that the junctional membrane separation and extension are about 15 nm and 300 nm, respectively. Furthermore, we develop a quantitative model of the L-SR junction using these measurements, prior kinetic and specific Ca2+ signal information as input data. Simulations of NAADP-dependent junctional Ca2+ transients demonstrate that the magnitude of these signals can breach the threshold for CICR via RyR3. By correlation analysis of live cell Ca2+ signals and simulated Ca2+ transients within L-SR junctions, we estimate that “trigger zones” comprising 60–100 junctions are required to confer a signal of similar magnitude. This is compatible with the 110 lysosomes/cell estimated from our ultrastructural observations. Most importantly, our model shows that increasing the L-SR junctional width above 50 nm lowers the magnitude of junctional [Ca2+] such that there is a failure to breach the threshold for CICR via RyR3. L-SR junctions are therefore a pre-requisite for efficient Ca2+signal coupling and may contribute to cellular function in health and disease.",
"keywords": [
"The importance of cytoplasmic nanojunctions to cellular signaling and thus to the modulation of cell function was recognised several decades ago1",
"2",
"henceforth the extent to which cellular nanospaces may contribute to the regulation of cell function received little attention. Nevertheless there is now a growing recognition of the widespread occurrence and functional significance of cytoplasmic nanospaces in cells from species across several kingdoms3–9."
],
"content": "Introduction\n\nThe importance of cytoplasmic nanojunctions to cellular signaling and thus to the modulation of cell function was recognised several decades ago1,2, henceforth the extent to which cellular nanospaces may contribute to the regulation of cell function received little attention. Nevertheless there is now a growing recognition of the widespread occurrence and functional significance of cytoplasmic nanospaces in cells from species across several kingdoms3–9.\n\nIn this respect, membrane-membrane junctions between lysosomes and the sarcoplasmic reticulum (L-SR junctions) are of particular interest; not least because they have been hypothesized to couple calcium signaling between these two organelles10,11.\n\nThat L-SR junctions may play an important role in cellular Ca2+ signaling was uncovered through early studies on the Ca2+ mobilizing messenger nicotinic acid adenine dinucleotide phosphate (NAADP)12, which demonstrated that NAADP released Ca2+ from a store other than the sarco/endoplasmic reticulum (S/ER)13, that could then trigger further Ca2+ release from the S/ER by Ca2+- induced Ca2+ release (CICR)14–17. A major advance in our understanding was then provided by the demonstration that the NAADP-released Ca2+ was from an acidic lysosome-related store10,18,19 in a manner that requires two pore segment channel subtype 2 (TPC2)20. However, studies on pulmonary arterial smooth muscle cells (PASMCs) had also identified a significant specialization, namely L-SR nanojunctions. It was hypothesized not only that these nanojunctions were necessary for coupling between lysosomes and the SR, but that they could both coordinate and restrict their relationship to the SR by preferentially targeting ryanodine receptors while excluding inositol 1,4,5-trisphosphate (IP3) receptors10,11. Importantly, NAADP-dependent Ca2+ bursts primarily arise from lysosomes in the perinuclear region of PASMCs and appear to promote rapid, local Ca2+ transients that are of sufficient size to activate clusters of SR resident ryanodine receptor subtype 3 (RyR3) and thus initiate, in an all-or-none manner, a propagating global Ca2+ wave10,11.\n\nThe specialization of the proposed L-SR junction is likely important in smooth muscle cell physiology, e.g., in vasomotion, and in the recycling of organelles and programmed cell death by autophagy21. However, L-SR junctions may also make as yet unforeseen contributions to vascular pathologies as highlighted by the fact that Niemann-Pick disease type C1 results, in part, from dysregulation of lysosomal Ca2+ handling22 and is known to precipitate portal hypertension23, while other lysosomal storage diseases (e.g., Pompe and Gaucher disease) accelerate pulmonary arterial hypertension24,25. Moreover, observed hypertension is often associated with dysfunction of cholesterol trafficking26, increased plasma cholesterol levels, vascular lesion formation, atherosclerosis/thrombosis and medial degradation23,27. Therefore lysosomal Ca2+ signaling is of considerable clinical interest. That L-SR junctions may be of further significance to pathology is also evident, for example, from the fact that in the pulmonary artery smooth muscle L-SR junctions underpin Ca2+ waves initiated by endothelin 1, the levels of which are elevated in pulmonary hypertension, systemic hypertension and atherosclerosis28,29. An understanding of how specific Ca2+ signals are functionally initiated therefore has important translational implications.\n\nLysosomal Ca2+ regulation has been of current interest in several recent research and review articles (e.g.,30–34), yet a quantitative elucidation of the mechanism by which Ca2+ signals are generated by the endolysosomal system is still lacking. Given the likely importance of L-SR junctions to Ca2+ signaling in health and disease, we sought to determine whether this nano-environment would indeed be able to effectively generate the previously observed NAADP-induced Ca2+ signals. In particular, we set out to verify the following hypotheses in PASMCs: (1) L-SR nanojunctions should be observable in the ultrastructure of these cells, (2) NAADP induces sufficient Ca2+ release from the lysosome to initiate activation of RyR3 embedded in the junctional SR, and (3) the combined effect of activation of L-SR nanojunctions in a cytoplasmic “trigger zone” determines the threshold of global [Ca2+]i for the biphasic release process.\n\nDue to the minute spatial scale of the nanojunctions generating the primary Ca2+ signals, accurate investigation of dynamic signaling within these spaces cannot be achieved with currently available instrumentation. To overcome this issue, we took an integrative approach by combining our own electron microscopy of L-SR nanojunctions, existing kinetic data on the Ca2+ transporters and buffers, and prior knowledge of the NAADP-induced Ca2+ signal features into a quantitative model of a typical L-SR nanojunction. This model is based on stochastic simulations of intracellular Ca2+ diffusion by Brownian motion implemented using the particle simulator MCell35–37.\n\nIn the present manuscript we have verified the existence of L-SR nanojunctions within the ultrastructure of PASMCs, and shown that lysosomes can release sufficient Ca2+ to activate CICR via RyR3 clusters embedded in the junctional SR. Perhaps most importantly, we show that L-SR coupling is determined both by the integrity of L-SR junction on the nanoscale and the quantal summation of Ca2+ release from multiple, activated junctional complexes.\n\n\nMaterials and methods\n\nAll the experiments and procedures were carried out in accordance with the guidelines of the University of British Columbia Animal Care Committee, and in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986. The Research Ethics Committees from both these institutions specifically and independently approved this study.\n\nAn adult male Wistar rat (about 12 months old, weighing 220–250 g, obtained from Charles River and housed in the institutional animal facility at the University of British Columbia Child and Family Research Institute under standard animal room conditions of 12 h light-12 h dark cycles, at 25°C, with two animals in a cage) was anesthetized with 3 ml of Thiotal (Vétoquinol, Lavaltrie, QC, Canada). For the purpose of this phase of the study, one representative healthy animal usually suffices. This choice also minimizes unnecessary animal sacrifice. The thoracic cavity was opened and flooded with warm physiological saline solution (PSS) containing 145 mM NaCl, 4 mM KCl, 1 mM MgCl2, 10 mM HEPES, 0.05 mM CaCl2, and 10 mM glucose (pH 7.4; all chemicals were purchased from Sigma-Aldrich Canada unless otherwise stated). After extraction of the heart and lungs and their placement in HEPES buffer, several rings from the primary and secondary branches of the pulmonary artery were dissected and immediately immersed in fixative solution. The primary fixative solution contained 2.5% glutaraldehyde (Ted Pella, Redding, CA, USA) in 0.1 M sodium cacodylate buffer (Canemco & Marivac, Gore, QC, Canada) at room temperature. The artery rings were then washed three times in 0.1 M sodium cacodylate (30 min in total). In the process of secondary fixation, the tissue rings were fixed with 1% OsO4 (Ted Pella, Redding, CA, USA) in 0.1 M sodium cacodylate buffer for 1 h followed by three 10-minute washes with distilled water and en bloc staining with 2% uranyl acetate (Ted Pella, Redding, CA, USA. The samples were then dehydrated in increasing concentrations of ethanol (25, 50, 75, 80, 90, and 95%). In the final process of dehydration, the samples underwent 3 washes in 100% ethanol. The artery rings were then resin-infiltrated in increasing concentrations (30, 50, and 75% in ethanol) of a 1:1 mix of Epon and Spurr’s resins (Canemco & Marivac, Gore, QC, Canada). The infiltration process was completed by three passages in 100% resin. All of the ethanol dehydration and resin infiltration steps were carried out by using a laboratory microwave oven (Pelco 3441, Ted Pella, Redding, CA, USA). The blocks were finally resin-embedded in molds and polymerized overnight in an oven at 60°C.\n\nFor standard (2D) electron microscopy imaging, 80-nm sections were cut from the embedded sample blocks on a Reichert Ultracut-E microtome (Leica Microsystems, Vienna) using a diamond knife (Diatome, Biel, Switzerland) and were collected on uncoated 100- and 200- mesh copper grids (hexagonal or square meshes; Ted Pella, Redding, CA, USA). The sections were post-stained with 1% uranyl acetate (Canemco & Marivac, Gore, QC, Canada; pH not recorded) and Reynolds lead citrate (Fisher Scientific Company, USA; pH not recorded) for 12 and 6 minutes, respectively. Electron micrographs at various magnifications were obtained with a Hitachi 7600 transmission electron microscope (Hitachi High Technologies, Japan) at 80 kV.\n\nLysosomes in these images were identified according to their well established appearance and features: they are bound by a single lipid-bilayer membrane, with a granular, more or less uniform luminal matrix that is more electron dense than the surrounding cytosol. Secondary lysosomes may also contain less granular structures within the finer matrix. Moreover, lysosomes are normally distinguishable from endosomes by their larger size, hence we set a threshold “diameter” of > 200 nm for acceptance of a lysosome, below which all vesicles were excluded.\n\nTo obtain electron microscopic tomograms, we cut 200-nm-thick sections from the same sample blocks used for standard imaging. The sections were then collected on Formvar (Canemco & Marivac, Gore, QC, Canada) coated slot copper grids (Ted Pella, Redding, CA, USA) and post-stained with 1% uranyl acetate and Reynolds lead citrate for 20 and 10 minutes, respectively. We surveyed the sample sections using a FEI Tecnai G2 200 kV transmission electron microscope (FEI Technologies, Eindhoven, The Netherlands) and identified regions of interest containing L-SR junctions. We then acquired tomograms of several of those regions by taking 2D scans through the sample every 5° of inclination as it was tilted between −60° and +60° with respect to horizontal. The scans were reconstructed with the Inspect3D (FEI Technologies, Eindhoven, The Netherlands) software tools and structures of interest, primarily lysosomal and SR membranes in the same cellular neighbourhood, were traced out using Amira (FEI Technologies, Eindhoven, The Netherlands) software.\n\nThe images of the samples were further processed using GIMP (GNU Imaging Manipulation Program, open source, available at gimp.org) to enhance membrane contrast in the interest of improving our characterization of the L-SR junctions.\n\nThe SR and lysosomal membranes were outlined, highlighted and measured in pixels using the Inkscape software program (open source, available at http://www.inkscape.org), converting the pixel measurements to nm using the scale bar in the recorded micrographs. By modifying the Inkscape script for measuring lengths, we were able to output the measurements directly into a text file, which we used to produce the histograms in Figure 3B–D. We used the Gnuplot program (open source, available at http://www.gnuplot.info) to produce the histograms and plots presented herein.\n\nTo obtain sufficient cell isolation to appropriately test significance, 1–3 adult male Wistar rats (weighing 150–300 g, obtained from Charles River and housed in the institutional animal facility at the Centre for Integrative Physiology of the University of Edinburgh under standard animal room conditions of 12 h light-12 h dark cycles, at 25°C, with 2–4 animals in a cage) were sacrificed by cervical dislocation. The heart and lungs were removed en bloc and placed in physiological salt solution (PSS) of the following composition (mM): 130 NaCl, 5.2 KCl, 1 MgCl2, 1.7 CaCl2, 10 glucose, and 10 HEPES, pH 7.45 (all chemicals were purchased from Sigma-Aldrich). Single arterial smooth muscle cells were isolated from second-order branches of the pulmonary artery. Briefly, arteries were dissected out and placed in low Ca2+ solution of the following composition (mM): 124 NaCl, 5 KCl, 1 MgCl2, 0.5 NaH2PO4, 0.5 KH2PO, 15 NaHCO3, 0.16 CaCl2, 0.5 EDTA, 10 glucose, 10 taurine and 10 HEPES, pH 7.4. After 10 min the arteries were placed in the same solution containing 0.5 mg/ml papain (Fluka) and 1 mg/ml bovine serum albumin (Sigma) and kept at 4°C overnight. The following day 0.2 mM 1,4-dithio-DL-threitol (Sigma) was added to the solution, to activate the protease, and the preparation was incubated for 1 h at room temperature (22°C). The tissue was then washed at 3°C in fresh low Ca2+ solution without enzymes, and single smooth muscle cells were isolated by gentle trituration with a fire-polished Pasteur pipette. Cells were stored in suspension at 4°C until required.\n\nPASMCs were incubated for 30 min with 5 µM Fura-2-AM in Ca2+-free PSS in an experimental chamber on a Leica DMIRBE inverted microscope and then superfused with Fura-2 free PSS for at least 30 min prior to experimentation. Intracellular Ca2+ concentration was reported by Fura-2 fluorescence ratio (F340/F380 excitation; emission 510 nm). Emitted fluorescence was recorded at 22°C with a sampling frequency of 0.5 Hz, using a Hamamatsu 4880 CCD camera via a Zeiss Fluar 40×, 1.3 n.a. oil immersion lens and Leica DMIRBE microscope. Background subtraction was performed on-line. Analysis was done via Openlab imaging software (Improvision, UK).\n\nNAADP was applied intracellularly in the whole-cell configuration of the patch-clamp technique, and in current clamp mode (I = 0) as described previously17. The pipette solution contained (in mM): 140 KCl, 10 HEPES, 1 MgCl2 and 5 µM Fura-2, pH 7.4. The seal resistance, as measured using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA), was ≥ 3 GΩ throughout each experiment. Series resistance and pipette resistance were ≤ 10 MΩ and ≤ 3 MΩ, respectively. All experiments were carried out at room temperature (≈ 22°C).\n\nThe main stages of the quantitative modeling approach are:\n\n1. The design of 3D software mesh objects (nets of interconnected triangles by which surfaces can be represented in computer graphics) representing a typical L-SR region, including a whole lysosome and a portion of neighbouring SR network. These objects are built to-scale following the ultrastructural characterization of the L-SR junctional regions as it results from our electron microscopy image analysis; this phase was carried out using the “3D content creation suite” Blender (open source, available at blender.org);\n\n2. The positioning of the relevant transporters on the reconstructed membranes (TPC2 complexes on the lysosome, SERCA2a and RyR3 on the SR) according to information gathered from the literature on their typical membrane densities and the implementation of the transporter's known kinetics and multi-state models and of the ion diffusivities (Ca2+ and mobile Ca2+ buffers);\n\n3. The simulation of molecular Brownian motion in the cytosol by random walk algorithms; this phase was performed by writing appropriate code for the stochastic particle simulator MCell (freely available at http://www.mcell.org)35–37. In a nutshell, MCell reproduces the randomness of the molecular trajectories, of the ion transporter flickering and of the relevant chemical reactions by probabilistic algorithms governed by pseudo-random number generators (iterative mathematical algorithms, which produce a pseudo-random sequence of numbers once initiated by a given number called seed). This enables the simulation of a number of microphysiological processes, all stochastically different from one another. The average outcome of the processes, to mimic the instrumental output during experimental measurements, is obtained by taking the average of a desired quantity, e.g., [Ca2+], over a large number of simulations all initiated with a different seed;\n\n4. The measurement of simulated [Ca2+] in the L-SR junctions from the process of Ca2+ release via TPC2-related signaling complex on the lysosome and SR Ca2+ uptake by the SERCA2a pumps, and static as well as dynamic visualization of the simulations; this stage is part of MCell’s output.\n\n\nResults\n\nThe work presented in this article is based on earlier Ca2+ fluorescence imaging observations by optical microscopy, of which the data in Figure 1 is a representative example, and immunofluorescence results. These studies led to the proposal that, for the lysosomal Ca2+ release to trigger CICR, L-SR nanojunctions are required and that they consist of apposing patches of lysosomal and SR membranes separated by a narrow space of nano-scale dimension10,17. The relevant background findings that stimulated the development of the work exposed here were first reported by Evans’ group10,17, and are summarized in Figure 1.\n\nUpper panel shows a series of pseudocolour images of the Fura-2 fluorescence ratio (F340/F380) recorded in an isolated pulmonary artery smooth muscle cell during intracellular dialysis of 10 nM NAADP. Note the spatially localized ‘Ca2+ burst’ (time point 1). Lower panel shows the record of the Fura-2 fluorescence ratio against time corresponding to the upper panel of pseudocolours images; note the discrete shoulder in the rising phase of the F340/F380 ratio that corresponds to the initial ‘Ca2+ burst’. Scale bar: 10 μm.\n\nThe example record in Figure 1 highlights the fact that NAADP appears to activate a two-phase Ca2+ signal, which can exhibit an identifiable “shoulder” during the initial rising phase of the signal (Figure 1, time point 1), followed by a second faster phase of signal amplification (Figure 1, time point 2). Previous studies have demonstrated that this two-phase response results from initiation by NAADP of Ca2+ bursts from lysosome-related stores10 in a manner that requires TPC238 and that Ca2+ bursts are subsequently amplified by CICR from the SR via clusters of RyR311,17. The studies cited above provided calibrated estimates of the changes in intracellular [Ca2+] input into the model below. We propose herein that L-SR junctions do indeed represent cellular nanojunctions and that they might play a role of accentuating Ca2+ gradients, akin to that of plasma membrane (PM)-SR junctions that are pivotal in the process of SR Ca2+ refilling during asynchronous [Ca2+] waves39. We furthermore hypothesize that in order for these nanojunctions to appropriately regulate Ca2+ signaling, they must be separated by a distance of approximately 20 nm and be of the order of a few hundred nm in lateral dimensions, as inferred from previous studies on PM-SR junctions10,11.\n\nTo identify lysosomes, SR regions and L-SR nanojunctions, we recorded and surveyed 74 electron micrographs of rat pulmonary arterial smooth muscle taken from samples prepared as described in the Materials and methods section. The images in Figure 2 provide a set of examples. Since we were aiming to detect L-SR junctions, namely close appositions of the lysosomal and SR membranes, immuno-gold labeling of lysosomes was prohibited, since this technique compromises membranes definition by electron microscopy to the extent that we would be unable to assess junctional architecture. Instead, in images like those in Figure 2, lysosome identification was accomplished by relying on the knowledge of lysosomal ultrastructural features, which has accumulated over the past 50 years since the discovery of the lysosomes40. In standard (2D) transmission electron microscopy (TEM) images, lysosomes typically appear as elliptical structures bound by a single lipid bilayer, a feature that distinguishes them from mitochondria. Depending on the lysosomal system stage, they also tend to have a more or less uniformly electron-dense interior as compared to the surrounding cytosol41. They can be distinguished from endosomes by their larger size and darker lumen and they differ from peroxisomes, since the latter usually display a geometrically distinct and markedly darker structure called “crystalloid” in their luminal area. Moreover, it would appear that peroxisomes are found far more frequently in liver, kidney, bronchioles and odontoblasts than in other cell types40,42 (see, for example, http://www.uni-mainz.de/FB/Medizin/Anatomie/workshop/EM/EMPeroxisomE.html).\n\nRepresentative electron micrographs of rat pulmonary artery SMC regions containing lysosomes (L), several SR cisterns, and including several examples of L-SR junctions (arrows). Also indicated are nuclei (N), Golgi apparatus (G), mitochondria (M), a multivesicular body (MVB) and extra-cellular space (ECS). Scale bars: 500 nm. Magnifications: A,C 80,000×, B, 60,000×, D, 70,000×.\n\nA, High magnification (150,000×) electron micrograph of a region of Figure 2B containing 3 L-SR junctions (arrows); coloured tracings as shown were used to measure lysosome dimensions, L-SR widths and extensions. Scale bar: 100 nm. B–D, Histograms showing distribution of several relevant lysosomal and L-SR junctional parameters, used to characterize the junctions and inform the quantitative model. B, lysosomal dimensions as major and minor axes of oval shapes in micrographs; C, L-SR junctional width; D, percentage apposition between junctional SR and lysosome perimeter as projected in 2D micrographs.\n\nOccasionally, organellar remnants are still visible inside these ovals, a characteristic that identifies them as multi-vesicular bodies (“MVB” in Figure 2A). As it is at times questionable whether MVB’s are late endosomes or endosome-lysosome hybrids40,43, we have excluded organelles (3 in total) with such characteristics from our statistical count.\n\nFrom each of the relevant smooth muscle regions surveyed, we obtained high-resolution images of areas containing lysosomes and L-SR junctions in order to quantitatively characterize them (arrows in Figure 2 and Figure 3A). Using a software graphics editor (inkscape.org) and the image scale bar as a calibration gauge, we measured the lysosome size, as the length of the major and minor axes of their elliptical 2D projections (in orange and grey, respectively, in Figure 3A), the L-SR widths, that is the distance between lysosomal and SR membranes at places where the two were about 30 nm or closer to each other (in purple in Figure 3A), and the L-SR extensions as a percentage ratio between the junctional SR and the lysosomal membranes (in turquoise in Figure 3A). From these measurements, we extrapolated the 3D junctional SR extension, both as a percentage of the lysosomal surface and as a length in nm. The histograms displayed in Figure 3B–D visually summarize the data collected from the image analysis. The mean and standard deviation values of the measured parameters are reported in Table 1.\n\nMean and standard deviation values of L-SR junction parameters, calculated from data as in Figure 3B–D.\n\nEstimates of the various parameters gathered in this phase of the study were used as a basis to build a 3D software object to represent a typical L-SR junction. This reconstruction was then used to design the simulations mimicking Ca2+ diffusion in the L-SR nanojunctions, as is described below.\n\nTo gather more direct information on the 3D morphology of L-SR junctions, we acquired a set of tomograms of those regions from the same sample blocks used to obtain the images in Figure 2. In Figure 4, we report snapshots from one of the tomograms; in these stills, we have also traced out parts of one lysosome and the closely apposed SR region that together form a L-SR junction. These tomograms are very helpful in clarifying the detailed morphology of L-SR junctions and informing on the spatial variability of the SR network. For example, while the SR segment shown in a single 2D tomographic scan (Figure 4A) would appear to be continuous and part of a large SR compartment, it actually branches out into narrower cisterns as revealed by 3D tomographic reconstruction (Figure 4B). Furthermore, it is interesting to observe the fact that one extension of the SR appears to couple with multiple organelles. Thus, the 3D views generated by tomography are paramount for demonstrating the presence of a true junctional complex and for the design of a prototypical L-SR environment as a software mesh object, on which we may simulate the NAADP-mediated localized Ca2+ release.\n\nA, Snapshot from a TEM tomogram of a L-SR region of rat pulmonary artery smooth muscle, illustrating, among other things, a single SR extension apparently forming junctions with several lysosomes. Magnification: 62,000×. B, Same snapshot shown in A, but with a lysosome (orange) and a portion of SR (turquoise) partially traced out in 3D. These pseudocolour tracings underscore how the SR can appear as a large cistern in a given plane, but can actually branch out in different directions when viewed in 3D. Scale bars ≈ 100 nm.\n\nThe model aims to verify whether NAADP-induced Ca2+ release from the lysosomal system could be responsible for the localized Ca2+ signal preceding the global Ca2+ wave (see Figure 1), which triggers a propagating wave by CICR via RyR3s localized at L-SR junctions, as predicted by earlier observations10.\n\nTo understand the generation of the signal “shoulder”, such as that observed at time point 1 in the lower panel of Figure 1, by Ca2+ bursts within L-SR nanojunctions, let us note that its magnitude corresponds to the difference in [Ca2+] between the resting level of ≈ 100 nM17 prior to NAADP stimulation (up to the point, at which NAADP enters the cytoplasm under the whole-cell configuration (WC) in Figure 1) and the value of ≈ 400 nM17, corresponding to [Ca2+]i at time point 1 in the example record shown in Figure 1, lower panel (these concentration values were obtained via a standard calibration procedure17). This leads to a ∆[Ca2+]shoulder of approximately 300 nM17. Let us now estimate the number of lysosomes required to generate a ∆[Ca2+]shoulder of such magnitude in a PASMC, given the following assumptions:\n\n1. That the luminal [Ca2+] of a lysosome, [Ca2+]lys, is in the range of 400–600 µM, as determined in mouse macrophages44 and that it is homogeneous across the lysosomal population;\n\n2. That the Ca2+ release rate during bursts is ≈ 106 ions/s (based on other experimenters’ findings45, but see next section for a detailed analysis on the rate time-variation) and that it becomes negligible for values of [Ca2+]lys below ≈ 80 µM, as suggested by the single channel kinetics of TPC2 signaling complexes in lipid bilayer studies45;\n\n3. That lysosomes are spheroids with dimensions gathered from our EM characterization (see Figure 2, Figure 3, Figure 4, and Table 1), and hence that their volume is on average V = 2.2 × 10−17 L;\n\n4. That a smooth muscle cell cytosolic volume can be calculated as Vcyt = 2.4 × 10−12 L by modeling a cell as a 130-µm-long cylinder, 6 µm in diameter46, and accounting for nuclear, SR, mitochondrial and lysosomal volumes as described in the supplementary material.\n\nWith these assumptions accepted, from points 1. and 2. above we gather that a lysosome can release a potential ∆[Ca2+]lys = (400 to 600) µM − 80 µM = 3.2 × 10-4 M to 5.2 × 10−4 M into the cytosol (as we elaborate in the next section, this should take ≈ 0.03 s, a time frame that ensures that released Ca2+ can be considered unbuffered). Each lysosome contribution to [Ca2+]i, ∆[Ca2+]i,lys, can then simply be calculated by taking the lysosome-to-cell volume ratio into account:\n\n\n\nTherefore, we can calculate the number of lysosomes that may contribute to the magnitude of ∆[Ca2+]shoulder as\n\n\n\nIn summary, between 60 and 100 lysosomes would be necessary (and possibly sufficient) to provide a ∆[Ca2+]shoulder of 300 nM, which is typically observed during the localized Ca2+ release phase of the NAADP-induced Ca2+ signal.\n\nHow many lysosomes do we actually expect to be in a PASM cell of our sample tissue? We can obtain a rough estimate of this number from the TEM imaging we performed for this study. In each 80-nm-thick TEM sample section, we see 5–10 lysosomes/cell. Lysosomes are predominantly localized to the perinuclear region of the cytoplasm, but seldom in the subplasmalemmal area, consistent with previous observations by optical microscopy10,11. If we simplify the geometry of a typical smooth muscle cell to a 130-µm-long cylinder with radius of 6 µm, and considering that lysosome radii are around 180 nm, it is reasonable to assume that separate sets of lysosomes would be observable in TEM images taken at distances into the sample of about 360 nm from each other. Neglecting the slices within one lysosomal diameter of the cylinder surface—given the near total lack of observed lysosomes in those subvolumes—then our images suggest that we can expect a total of about 110 lysosomes/cell. It is encouraging that we obtain from this count a higher number than the 60–100 lysosome range we derived in Equation (2), in that on the one hand it is plausible to think that not all of the lysosomes in a cell may be activated in synchrony, nor may they all be involved in NAADP-mediated signaling, and on the other hand experience tells us that evolution has built in some redundancy of function in order to provide a threshold and also a margin of safety for the generation of this type of Ca2+ signals. Moreover, the estimated number of junctions is based on a value for [Ca2+]lys determined in macrophages, and it is plausible that the total releasable [Ca2+]lys in a PASMC may differ from that value and that it may also vary over the lysosome population. Lastly, the uncertainty in the number of lysosomes/cell emerging from the electron micrographs as described above may also contribute to this discrepancy.\n\nIn the hypothesized model outlined previously10,11, Ca2+ bursts activate SR resident RyR3 channels within L-SR junctions and thus initiate a propagating Ca2+ wave by CICR. The stochastic simulations developed here attempt to reproduce the phenomenon of the generation of [Ca2+] transients within individual L-SR junctions, considering the Ca2+ release kinetic requirements for lysosome-resident TPC2 signaling complexes and the rate of Ca2+ capture by the SERCA2a localized on the neighbouring SR membrane47, and to link these junctional transients to the observed bursts.\n\nThe thorough quantitative image analysis described in the previous section yields critical information for our first modeling phase, in which we built a dimensionally accurate virtual lysosome and a portion of the SR system, closely apposing the lysosome (Figure 5), so as to reproduce a NAADP-triggered Ca2+ signal within the nanospace of a representative L-SR junction as faithfully as possible.\n\nA and B, 3D software reproduction of a lysosome closely apposed to a portion of SR, thereby forming an ≈ 20-nm-wide L-SR nanojunction; this rendering was inspired by a series of observations from micrographs as in Figure 2 (grey object: SR, blue object: lysosome). Included are relevant molecules traversing Brownian motion trajectories produced by the model simulations (see symbol legend below the panels). B, enlarged view of the L-SR junctional region, in which we have displayed the volume object (rust-coloured box) used to measure the [Ca2+]NJ transients like the ones reported in Figure 7. Scale bar: 100 nm. The model geometry and code files are available from the corresponding author.\n\nFrom the available literature we estimated the number of TPC2 and SERCA2a likely distributed on the lysosome and SR membranes, respectively. We obtained the former number by dividing the macroscopic whole-lysosome conductance, calculated from the current values reported in a recent study on TPC2-mediated Ca2+ current in isolated lysosomes48, by the single channel conductance45, thus estimating that a typical lysosomal membrane may contain ≈ 20 TPC2. To obtain this value, we used experimental condition data48 to extract values for the ionic potential, Eion, across the lysosomal membrane. We then employed the authors’ current-voltage (I-Vm) data to compute whole-lysosome conductance values (gWL) as a function of the applied membrane potential, Vm, from Ohm’s law: I = gWL(Vm − Eion). We report details on this calculation in the supplementary material.\n\nMoreover, we have estimated the density of SERCA2a on the SR within L-SR junctions to be equivalent to that previously predicted for PM-SR junctions as approximately 6250/μm239. Other input data for the model are the estimates of lysosomal volume and of the [Ca2+]lys, from which we calculated the actual number of ions in the lysosome prior to the beginning of Ca2+ release (see previous section). This, in turn, was used in the extrapolation of the TPC2 complex Ca2+ release rate as a function of time, as follows.\n\nA recent electrophysiological study of the Ca2+ conductance of TPC2 signaling complex provides valuable information regarding its biophysical properties and, importantly, highlights the fact that, for a given relatively low activating concentration of NAADP (10 nM, as in Figure 1), the channel open probability appears to depend on [Ca2+]lys (this is likely due to a partial neutralization of the electrochemical potential across the lysosomal membrane)45. We used the Ca2+ conductivity measured in that study and values of the lysosome membrane potential49 to calculate the maximal Ca2+ current, Imax, as 2.4 × 106 ions/s. We then applied a weighted quadratic fit to the channel’s open probability (Po) as a function of the reported [Ca2+]lys data with constraints that the Po would tend to zero at [Ca2+]lys = 80 µM, based on the observation that below [Ca2+]lys = 100 µM essentially no single channel openings were observed45. Another constraint for the fit was that the curve be within the standard deviation value of Po at the highest reported [Ca2+]lys = 1 mM. From the quadratic fit, we then obtained our own Po-vs-[Ca2+]lys table (plotted in Figure 6A) and assumed that at the beginning of Ca2+ release, the Ca2+ current would be Po,max × Imax at the maximal luminal concentration until the luminal concentration decreased to the next point in the Po-vs-[Ca2+]lys relationship. At this point, the release rate decreases to a new (lower) Po × Imax until the luminal concentration reaches the next lower point in the table, and so on until Po = 0 at [Ca2+]lys = 80 µM. The Ca2+ release rate as a function of time obtained in this manner is shown in Figure 6B.\n\nA, open probability Po of the TPC2-dependent Ca2+ conductance reproduced from a quadratic fit to the data reported in the literature45. B, Ca2+ release rate calculated as explained in the text, based on the Po in A.\n\nTo implement an approximated SERCA pump action, we used a simplified version of a multi-state kinetic model developed earlier50, which was further informed by more recent isoform-specific studies51,52. In particular, since we are interested in simulating the SERCA2a Ca2+ uptake only in terms of its influence on the shaping of the junctional [Ca2+] transient, we have opted not to implement the steps of the multistate model that deal with the Ca2+ unbinding from the SERCA on the SR luminal side of the pump. In brief, the reactions SERCA2a undergo are: (1) binding/unbinding of the first Ca2+; (2) binding/unbinding of the second Ca2+.\n\nThe Ca2+ diffusivity was obtained from studies, in which it was concluded that given the known kinetics of typical Ca2+ buffers, the range of free Ca2+ after it enters a cell can be up to 200 nm. Therefore, due to the nano-scale of our system, the trajectories of Ca2+ released by TPC2 signaling complexes in the simulations are governed by the measured diffusivity of free Ca2+ for a displacement comparable to the nanojunction extension (2.23 × 10−10 m2/s;53,54). Once Ca2+ are buffered they acquire the measured diffusivity of the buffers (8.4 × 10−11 m2/s;55).\n\nWe summarize the quantitative model input data in Table 2.\n\nSummary of input parameters used in various phases of the quantitative model, and references to the origin of their values.\n\nUsing MCell as a stochastic particle simulator, we ran a number of simulations to represent the NAADP-mediated Ca2+ release that is supposed to occur at L-SR junctions in PASM experiments. Released Ca2+ is assumed to undergo Brownian motion in the surrounding space, including the L-SR nanospace. SERCA2a placed on the neighbouring SR surface may capture Ca2+ according to our approximation of their known multistate model. To determine how this regenerated cellular environment can shape a Ca2+ transient we “measured” the junctional [Ca2+] by counting the ions within the L-SR volume at any given time and dividing the number by that volume. The snapshots in Figure 5 are part of the visual output of this phase of the work. The data to determine the simulated Ca2+ transients in the L-SR junctions were collected in a measuring volume placed between the lysosomal and SR membranes in the virtual L-SR junction (rust-coloured box in Figure 5B).\n\nPrevious experimental findings about VSMC PM-SR junctions indicate that disruption of nanojunctions can have profound consequences on Ca2+ signaling properties of the cells. For example, when calyculin A was used to separate superficial SR portions from the plasmalemma of the rabbit inferior vena cava, it was observed that [Ca2+]i oscillations would cease56. This was later corroborated by a quantitative model of the PM-SR junction’s role in the refilling of SR Ca2+ during oscillations39. In another study by the van Breemen laboratory, it was observed that the mitochondria-SR junctions of airway SMC displayed a variable average width as a function of the state of rest or activation of the cell57. It makes sense then to study the effect of changes in the L-SR junctional geometry on the transients generated by our simulations. Therefore, we ran several simulations, in which the separation between the lysosomal and SR membranes was increased from 10 nm to 100 nm in steps of 10 nm. In Figure 7A, we report three sample transients from this set of simulations obtained using three different junctional membrane separations, as indicated in the inset legend. The value of each of the points graphed in this plot is the average value of 100 simulations, in each of which the random number generator within MCell is initiated with a different seed (see Materials and methods). We report representative error bars as 3× the standard error, to convey the > 99% confidence interval of the data. As one would expect, the transient nanojunctional (NJ) Ca2+ concentration, [Ca2+]NJ, decreases in magnitude, as the junctional L-SR membrane-membrane separation increases, simply because the released Ca2+ has a larger junctional volume available over which to spread. However, it is important to make a quantitative comparison between this change in [Ca2+]NJ and the [Ca2+]i requirements to activate the putative RyR3 population of the junctional SR. This can be attained by analyzing the time scale of both the recorded and simulated Ca2+ signals.\n\nA, calculated nanojunctional [Ca2+] transient, [Ca2+]NJ, \"measured\" inside the volume of the recreated L-SR nanojunction shown in Figure 5. To show the effect of changes in the junctional geometry, we report three transients calculated using different junctional widths of 20, 50 and 100 nm. B, [Ca2+]NJ vs width of junction, concentration values are temporal averages of the transients as in panel A, calculated over an interval of 0.065 s (solid circles) and 0.046 s (empty circles); see text for explanation. The shaded area indicates the approximate threshold values for CICR at RyR3s58.\n\nIn the final step of the development of our model, we analyzed the relationship between the time scale of the [Ca2+]NJ transients resulting from our model (Figure 7) and that of the observed Ca2+ signal shoulder in Figure 1.\n\nLet us note that the typical duration of the simulated transients, ttransient, is about 0.06 s and recall that these represent ∆[Ca2+] above the resting [Ca2+]. On other hand, the build up to the maximum value of ∆[Ca2+]shoulder takes about 5 s (we refer to this time as tshoulder; Figure 1). This interval is about two orders of magnitude larger than the duration of the simulated individual transients (Figure 7A). One way to reconcile the hypothesis that L-SR junctions are at the base of the observed NAADP-induced Ca2+ signals such as the ones in Figure 1 and in particular that the signal shoulder emerges from lysosomal Ca2+ release at L-SR junctions, is to bring forward the assumption that the signal shoulder may be the result of a sequential summation effect over many L-SR junctions, each working according to an all-or-none mechanism of Ca2+ release, and that the “firing” of one junction may cause a cascading effect across the set of junctions that produces the shoulder. Then the duration of the shoulder upstroke can be expressed as:\n\n\n\nwhere NNJ is the number of L-SR nanojunctions that yield ∆[Ca2+]shoulder. Since we calculated above that Ca2+ release from NNJ ≈ 60–100 lysosomes would be necessary to produce the observed signal shoulder magnitude and have shown in the previous section that such release would need to take place at L-SR junctions, we can estimate ttransient, the duration the [Ca2+]NJ transient, by reversing Equation (3) and obtaining ttransient = 0.05–0.08 s. It is noteworthy that this range of values is obtained in a manner completely independent of our simulation results, which yielded a similar range of values.\n\nTo gain quantitative insight into the effects of varying the junctional width, we then calculated the temporal average of the [Ca2+]NJ over ttransient (using the middle value of the range calculated via Equation (3)) and graphed it as a function of the junctional width. The result of this analysis is reported in Figure 7B (solid blue circles). As we have anticipated at the end of the previous section, the decrease in magnitude of these data is to be expected, however in this plot we also indicated the range of [Ca2+]i values (shaded area) over which maximum SR Ca2+ release via RyR3 is reported to occur in skeletal muscle58. This comparison underscores the important constraint played by the width of the L-SR junctions and indicates that, unless the membrane separation is kept below about 30 nm, it is not possible for [Ca2+]NJ to breach the threshold for RyR3 Ca2+ release. Let us also point out that the [Ca2+]NJ data in Figure 7B would shift upward, toward concentration values that would make the junction more prone to promote RyR3 release, if the temporal average were taken over a shorter transient time, t′transient < ttransient around the [Ca2+]NJ peak. However, in that case Equation (3) indicates that a larger NNJ (than 80, picked as the middle of the 60–100 range) would have to contribute to the signal summation that results in a 5-second tshoulder. Interestingly, this possibility agrees with our lysosome count from TEM images (110 lysosomes/cell) and with the argument of natural redundancy we contemplated to explain the discrepancy between the calculated and observed lysosome number estimates. As an exercise we have recalculated the [Ca2+]NJ time-averaged over t′transient obtained using 110 lysosomes in Equation (3) (empty purple circles in Figure 7B), and this indeed shows that RyR3 Ca2+ release threshold would be cleared more readily. These observations suggest that activation of RyR3s in the L-SR junctions not only depends on the concentration of Ca2+ near them, but also on their exposure time to this concentration.\n\n\nDiscussion\n\nWe have recently introduced the concept of the “panjunctional SR”, which states that Ca2+ release and uptake at a family of specific nanojunctions connected by a continuous but variable SR lumen integrates cellular control over multiple functions4. The lysosome-sarco/endoplasmic reticulum (L-S/ER) junction is the most current junction to be considered in this context and exhibits perhaps the highest degree of plasticity of the family of nanojunctions of the SR; the mechanism and function of lysosomal Ca2+ signaling is currently hotly debated59.\n\nBy means of a thorough ultrastructural study in rat pulmonary artery smooth muscle, we have observed and characterized L-SR nano-junctions, which had been previously hypothesized on the basis of optical measurements of Ca2+ signals and optical immunocytochemistry10,11. Our observations corroborate the previously reported finding10,17 that lysosomes in PASMCs tend to cluster in the perinuclear region, as is evident in our micrographs (e.g., Figure 2A). We find that L-SR junctions are on average 15 nm in width (equivalent to our preliminary reports8,60 and to recent observations in cultured fibroblasts33) and extend approximately 300 nm in lateral dimensions, thereby involving about 15% of the lysosomal membrane (Table 1).\n\nIn an effort to achieve quantitative understanding of the phenomenon of NAADP-mediated Ca2+ transients and verify the proposal that these may be generated in L-SR junctions10,17, we focused on one of the prominent features of these Ca2+ signals, namely the localized Ca2+ bursts that precede the propagating Ca2+ wave (Figure 1), which we refer to as the signal shoulder (∆[Ca2+]shoulder). In the first instance, we have estimated the potential contribution of local bursts of Ca2+ release from individual lysosomes to the elevation of global [Ca2+]i observed during this shoulder in the experimental records. From this, and using the dimensions of a typical smooth muscle cell, we calculated that 60–100 lysosomes would be required to cause an elevation of comparable magnitude to the signal shoulder. This is lower than the estimate for the total number of lysosomes per cell, 110, we obtained from our ultrastructural study, but comparable in order of magnitude. We have already mentioned above a number of factors in favour of observing a greater number of lysosomes/cell than the estimated number required to generate the signal shoulder. In addition, several other elements contribute a degree of uncertainty to those estimates, such that their discrepancy may not be as large. We need to consider that published Po data for the Ca2+ conductance associated with the TPC2 signaling complex45, on which we based our TPC2 rate table, show some variability according to the standard deviation bars, which, in turn, implies an uncertainty in the interpolated Po (Figure 5A). Moreover, we cannot exclude the possibility that these data reflect a contribution from multiple channels (NPo) rather than a purely single channel Po. Lastly, the standard deviation of the simulated transient (only the standard error is shown in Figure 7A) and the variability in the experimental determination of the [Ca2+] sensitivity of RyR may allow for some uncertainty in the estimated number of nanojunctions.\n\nTo take this study further and understand whether the observed L-SR junctions could give rise to [Ca2+]i transients of appropriate magnitude to trigger Ca2+ release from RyR3 channels on the junctional SR, we developed a quantitative stochastic model of Ca2+ dynamics in the junctional nanospaces. We have previously published a simplistic version of this model, which nonetheless captured the essential features of the problem and yielded an indication that such Ca2+ transients in the L-SR junctions would be possible4. However, the simulated transients we obtained displayed unphysiological features, such as an abrupt onset and decay. We show here that this was largely due to lack of a faithful representation of the open probability for the Ca2+ conductance of the TPC2 signaling complex. We have now combined experimental information on the biophysical properties of conductance and open probability45 and on the luminal [Ca2+] of the lysosomes44 to implement a more realistic Ca2+ release rate model in the simulations. As a consequence, we are able to output a physiologically meaningful junctional transient profile (Figure 7A), and observe that the [Ca2+]NJ transients generated within our model junctions—in turn, based on those observed in our TEM images—reach peaks of about 20 µM. While we are still not able to measure these transients in individual L-SR junctions (peri-lysosomal Ca2+ probes are only recently becoming available61 and so far have not been used in smooth muscle cells, vascular or otherwise), it is worth noticing that the values we find are comparable to those recently measured in so-called Ca2+ hot spots in the mitochondria-ER junctions of neonatal ventricular cardiomyocytes (rat culture)62 and in RBL-2H3 and H9c2 cells earlier63. These results therefore suggest that the hypothesis presented for the role of L-SR junctions in cellular Ca2+ signaling is certainly plausible.\n\nAlthough our simulations were successful, comparison of the Ca2+ transients in Figure 1 and Figure 7 not surprisingly reveals a striking difference between the simulated Ca2+ kinetics of a single L-SR junction and those of the whole cell. This illustrates important aspects related to the concept of lysosome-SR “trigger zone” previously introduced by one of us (AME)10 and recently proposed as a facilitating factor in lysosomal-ER signaling in reverse, whereby Ca2+ release from ER compartments enables NAADP-mediated activation of acidic organelles32.\n\nBriefly, this concept suggests that in PSMCs clusters of lysosomes and closely apposed SR regions containing sets of RyRs may act together to form specialized intracellular compartments that transform NAADP-stimulated localized lysosomal Ca2+ release into cell-wide Ca2+ signals via RyR-supported CICR. If the firing of one junction were sufficient for subsequent initiation of the CICR across the entire SR, the experimentally observed threshold (Figure 1) would be much lower and the rate of junctional coupling by CICR faster. In other words, due to the high [Ca2+]i threshold of about 10 μM for Ca2+ activation of RyR3, CICR engendered by a single L-SR junction is likely to die out, unless reinforced by a process of quantal releases by other L-SR junctions within a cluster64. Therefore it seems more likely that multiple L-SR junctions work in concert in a process characterized by both additive and regenerative elements to provide the necessary threshold and margin of safety required to ensure the propagation by CICR of the global wave via the more widely distributed RyR2 along extra-junctional SR, once the Ca2+ release wave escapes an RyR3-enriched SR region, as previously suggested11.\n\nIn this respect, our results also allowed us to establish a time interval for the duration of the transient (ttransient) that would be compatible with the hypothesis that sequential summation of all-or-none lysosomal Ca2+ release events from a set of individual L-SR junctions is responsible for generating ∆[Ca2+]shoulder. Remarkably, this value is comparable to the average duration of the simulated transients, which was determined on the basis of ultrastructural details and completely independently of the summation effect hypothesis. The assumption of summation may imply that the role of the NAADP as a stimulus is limited to the initial lysosomal Ca2+ release, while the recruitment of subsequent L-SR junctions may be governed by further lysosomal calcium release, by SR Ca2+ release via Ca2+ activated junctional RyR3, and/or propagation and combination of calcium signals via inter-junctional clusters of RyR3. Therefore, from our model we envision the intriguing possibility of an important regulatory role of lysosomal Ca2+ content by SR Ca2+, in such a way that summation of calcium signals at multiple junctional complexes may give rise to the shoulder. While only further experiments can verify this, the quantitative corroboration provided by our findings sets on firmer ground the conclusion from earlier studies that Ca2+ bursts from lysosomes are indeed responsible for initiating the first phase of a cell wide Ca2+ wave via L-SR junctions.\n\nCarrying the signal time-scale analysis further and using the calculated ttransient to take a temporal average of the simulated [Ca2+]NJ at different junctional widths, we find that above a width of about 30 nm the transients would be unable to trigger Ca2+ release from the RyR3s (Figure 7B). Thus it is possible that heterogeneity and plasticity are controlled by a variable width of the junctional nanospace. For example, in atrial myocytes it has been proposed that NAADP evokes Ca2+ release from an acidic store, which enhances general SR Ca2+ release by increasing SR Ca2+ load and activating RyR sites34. This functional variant may be provided by either: (1) An increase in junctional distance such that [Ca2+]NJ is insufficient to breach the threshold for activation of RyR2, yet sufficient to allow for increases in luminal Ca2+ load of the SR via apposing SERCA2 clusters; or (2) L-SR junctions in cardiac muscle formed between lysosome membranes and closely apposed regions of the SR which possess dense SERCA2 clusters and are devoid of RyR2. Further ultra-structural studies on cardiac muscle and other cell types may therefore provide a greater understanding of how L-SR junctions may have evolved to provide for cell-specific modalities within the calcium signaling machinery.\n\nUnder metabolic stresses, such as hypoxia, lysosomal pathways have been proposed to provide for autophagic glycogen metabolism via acid maltase in support of energy supply, and significant levels of protein breakdown during more prolonged metabolic stress. Moreover, although controversial, in some cell types it has also been suggested that lysosomes may contribute to the energy supply by providing free fatty acids for beta-oxidation by mitochondria65. It may be significant, therefore, that TPC2 gating and thus autophagy may be modulated by mTOR31,66. This may well speak to further roles for lysosomal calcium signaling and L-SR junctional plasticity during hypoxic pulmonary vasoconstriction and even during the development of hypoxia pulmonary hypertension67, not least because AMP-activated protein kinase has been shown to modulate autophagy through the phosphorylation and inhibition of mTOR68.\n\nThe process of autophagy serves not only to regulate programmed cell death, but also to recycle organelles, such as mitochondria, through a process of degradation involving lysosomal hydrolases69. For this to occur L-SR junctions would likely be disrupted and thus select for local rather than global Ca2+ signals in order to facilitate fusion events among lysosomes, endosomes, autophagosomes and amphisomes70, which follows from the fact that Ca2+ plays a pivotal role in vesicle trafficking and fusion71. Active non-synchronous movements of TPC2-expressing vesicles have been detected in live-cell imaging experiments using GFP-tagged proteins20. Thus, lysosomal Ca2+ signaling may modulate plasticity in the manner required at all stages of multiple membrane fusion events that are dependent on Ca2+ for the effective formation of the SNARE complexes71. In short, transport of proteins between lysosomes, Golgi apparatus, and plasma membrane via lysosomes and endosomes may be coordinated at different stages by both global Ca2+ signals and spatially restricted Ca2+ release from acidic stores in a manner that is in some way determined by L-SR junctional integrity.\n\nLoss of integrity of L-SR junctions may also contribute to disease, given that lysosomal Ca2+ release both depletes luminal Ca2+ and causes intraluminal alkalinization72. Therefore, disruption of L-SR junctions may modulate the activity of pH-sensitive hydrolytic lysosomal enzymes, such as glucocerebrosidase and acid sphingomyelinase, which exhibit a marked loss of function at pH > 521,73 that could lead to accumulation of macromolecules such as glucocerebroside and sphigomylin. As mentioned previously, dysfunction of these enzyme systems consequent to L-SR junctional abnormalities could also contribute to pathologies associated with subclasses of lysosome storage disease such as Niemann-Pick disease type C122,23, Pompe and Gaucher24,25, which may include hepatic portal23, or pulmonary hypertension24,25, dysfunctions in cholesterol trafficking26 and consequent increases in plasma cholesterol levels, vascular lesion formation, atherosclerosis/thrombosis and medial degradation23,27.\n\n\nConclusion\n\nWe have determined that L-SR junctions, about 15 nm in width and extending to ≈ 300 nm in lateral dimensions are a regularly occurring feature in rat pulmonary arterial smooth muscle cells, in which L-SR junctions were first proposed. The present study provides a mechanistic basis for the observed NAADP-induced Ca2+ signals and strong support for the proposal that these Ca2+ signals are generated at L-SR junctions. Even within the variability of our recorded values of L-SR junctional widths and extension, our results suggest that localized [Ca2+] transients due to junctional Ca2+ release can without fail reach values required to breach the threshold for CICR from junctional RyR3s. Perhaps most significantly, disruption of the nanojunctions decreases [Ca2+]NJ below the value for CICR via junctional RyR3s. Therefore, consistent with previous studies on the PM-SR membrane39, we have established that L-SR junctions are required to allow such signals to be generated and that there is a 30–50 nm limit on junctional width, above which there is loss of junctional integrity and inadequate control of ion movements within the junctional space. This suggests that the observed L-SR junctions in PAMSCs are not only capable of delivering localized Ca2+ bursts of the required magnitude, but are also necessary if lysosomes are to fulfill this identified role in Ca2+ signaling. In other words, L-SR nanojunctions are a necessary and sufficient condition for generating local Ca2+ bursts essential for NAADP-induced Ca2+ waves. Most importantly, however, this study demonstrates the importance of junctional architecture on the nanoscale to the capacity for coupling across cytoplasmic nanospaces, tight regulation of ion transport and thus signal transduction. In turn, this highlights the possibility that alterations in the dimensions and architecture of intracellular nanojunctions lead to cell dysfunction and hence disease.\n\n\nData availability\n\nfigshare: L-SR nanojunction study data, doi: 10.6084/m9.figshare.988699",
"appendix": "Author contributions\n\n\n\nNF and CvB conceived the electron microscopy work, quantitative model, simulations and quantitative analysis of the results. AME and OAO obtained the background optical microscopy study. NF, OAO, CvB and AME contributed to writing the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by Grant No. CIHR MOP-84309 from the Canadian Institute of Health Research (CvB) and by the British Heart Foundation Programme Grant RG/12/14/29885 (AME).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip.t\n\n\nAcknowledgements\n\nWe are very grateful to Garnet Martens and the University of British Columbia Bioimaging Facilty for their assistance. 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PubMed Abstract\n\nMorgan AJ, Galione A: NAADP induces pHchanges in the lumen of acidic Ca2+ stores. Biochem J. 2007; 402(2): 301–10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan Weely S, van den Berg M, Barranger JA, et al.: Role of pH in determining the cell-type-specific residual activity of glucocerebrosidase in type 1 Gaucher disease. J Clin Invest. 1993; 91(3): 1167–75. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFameli N, van Breemen C: Stochastic three dimensional modelling of ionic transport in cytoplasmic nanospaces. Proc Physiol Soc. 2011; 25, SA06. Reference Source\n\nDevine CE, Somlyo AV, Somlyo AP: Sarcoplasmic reticulum and excitation-contraction coupling in mammalian smooth muscles. J Cell Biol. 1972; 52(3): 690– 718. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMackenson CA, Hikida RS: Avian smooth muscle: Morphometric analysis and comparison of different types. Zoomorphologie. 1979; 92(2): 183–189. Publisher Full Text\n\nHeng MK, Allen SG, Song MK, et al.: Mitochondrial crowding in smooth muscle cells after arterial ligation. Int J Angiol. 1996; 5: 1–7. Publisher Full Text\n\nMullock BM, Bright NA, Fearon CW, et al.: Fusion of lysosomes with late endosomes produces a hybrid organelle of intermediate density and is nsf dependent. J Cell Biol. 1998; 140(3): 591–601. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGiordano F, Saheki Y, Idevall-Hagren O, et al.: Pi(4,5)P(2)-dependent and Ca(2+)-regulated er-pm interactions mediated by the extended synaptotagmins. Cell. 2013; 153(7): 1494–509. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang C, Kotchoni SO, Samuels AL, et al.: SPIKE1 signals originate from and assemble specialized domains of the endoplasmic reticulum. Curr Biol. 2010; 20(23): 2144–9. 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Data Source\n\n\nSupplementary material\n\nThe whole-lysosome conductance was calculated based on the work reported by Schieder and collaborators [1], in which current voltage relationships of TPC2-mediated Ca2+ currents were determined for single lysosomes (see Figure 1 therein [1]). A set of these values, as well as the experimental conditions relevant for this calculation, are reported in Table S1. From these data, we first calculated the electrochemical potentials of K+ (EK) and Ca2+ (ECa) across the lysosomal membrane using the general expression\n\n\n\nwhere R is the universal gas constant, T the absolute temperature, Cz+ is a z-valent cation, F is Faraday’s constant. We then obtained the whole-lysosome, gWL, from Ohm’s law:\n\n\n\nwhere I is the measured current, Vm is the applied membrane voltage, and Eion = ECa+(PK/PCa)EK with PK/PCa the permeability ratio. The calculated conductance values are listed in Table S1.\n\nData on experimental conditions by Schieder et al. [1], used to calculate whole-lysosome TPC2-mediated Ca2+ conductance, gWL, as explained in the text. The subscripts “i” and “o” refer to the inside and outside of a lysosome, respectively.\n\nIf we assume that the whole-lysosome conductance under these conditions is due to a set of TPC2 complexes working in parallel, then gWL = NTPC2 gTPC2, NTPC2 is the number of complexes in that set and gTPC2 is the single TPC2 determined as 15 pS [2]. We can then find NTPC2 by dividing the calculated conductance, gWL, by the single channel conductance, gTPC2. For our calculation, we picked the whole-lysosome conductance value gWL = 316 pS corresponding to Vm = −20 mV, in accordance to the most recent measurements of lysosomal membrane potential [3]. Hence, our estimate of the number of TPC2 signaling complexes in a representative lysosome is:\n\n\n\nWe model a typical smooth muscle cell as a long and narrow cylinder 3 μm in radius and 130 μm long [4]. Its volume is then VSMC = π(3 μm)2 (130 μm) ≈ 3.7 × 10−12 L. To calculate an upper-limit estimate of the cell cytosolic volume, Vcyt, available to Ca2+ released from lysosomes, we need to account for the space occupied by the nucleus, the sarcoplasmic reticulum (SR), the mitochondria and the set of lysosomes we observe (Vnuc, VSR, Vmito, and Vall lys, respectively), so that\n\n\n\nThe nuclear volume, Vnuc, is estimated from a sample of 10 cells in one of our lower resolution micrographs, by measuring the minor and major axes of the oval-shaped nuclei, approximating their 3D shape as a prolate spheroid and calculating the volume of the spheroid from the measured values of the axes (Figure 2 and Table 1 in the main article). We calculate it as\n\n\n\n(about 15% of the cell volume).\n\nTo estimate Vmito, we resort once again to our own micrographs, in which we observe of the order of 10 mitochondria per sample section per cell. Using a similar procedure to the one employed to obtain the approximate number of lysosomes in a pulmonary artery smooth muscle cell (described in the main article, section ‘Generation of the signal’s “shoulder” by lysosomal Ca2+ bursts’), and pretending that mitochondria are spheres 0.8 μm in radius, we arrive at an estimate of 43 mitochondria/cell. Thus,\n\n\n\nThe volume of the SR in vascular smooth muscle has been estimated previously [5] and expressed as a percentage of the cell volume deprived of the nuclear and mitochondrial volume. These findings suggest that\n\n\n\nWhich corresponds to\n\n\n\nusing the values for VSMC, Vnuc and Vmito found above.\n\nVall lys can be obtained from the value of Vlys we found in the main article multiplied by the 110 lysosomes/cell we already estimated. However, at a value of ≈ 3 × 10−15 L it is negligible when compared to the other terms.\n\nFinally, putting Equation (5), Equation (3) and Equation (2) into Equation (1), we find\n\n\n\nClearly, the value in (6) is an upper limit, since we haven’t accounted for all of the organellar volumes in our calculations. However, the values above suggest that to a first approximation nucleus, SR, and mitochondria are by and large the main contributors to the difference between the gross cellular volume and the cytosolic volume.\n\nThe plot in the figure below was obtained from the same data, from which we produced the Ca2+ trace in Figure 1 in the manuscript. We report it again here in line-and-point form to highlight that the sampling frequency was 0.5 Hz. In a separate supplementary spread-sheet file, we also provide the raw data.\n\n[1] Michael Schieder, Katrin Rotzer, Andrea Brüggemann, Martin Biel, and Christian A Wahl-Schott. Characterization of two-pore channel 2 (tpcn2)-mediated ca2+ currents in isolated lysosomes. J Biol Chem, 285(28):21219–21222, Jul 2010.\n\n[2] Samantha J Pitt, Tim M Funnell, Mano Sitsapesan, Elisa Venturi, Katja Rietdorf, Margarida Ruas, A. Ganesan, Rajendra Gosain, Grant C Churchill, Michael X Zhu, John Parrington, Antony Galione, and Rebecca Sitsapesan. Tpc2 is a novel naadp-sensitive ca2+ release channel, operating as a dual sensor of luminal ph and ca2+. J Biol Chem, 285(45):35039–35046, Nov 2010.\n\n[3] Mirkka Koivusalo, Benjamin E Steinberg, David Mason, and Sergio Grinstein. In situ measurement of the electrical potential across the lysosomal membrane using fret. Traffic, 12(8):972–82, Aug 2011.\n\n[4] A P Somlyo. Structural characteristics, mechanisms of contraction, innervation and proliferation of smooth muscle cells. ultrastructure and function of vascular smooth muscle. Adv Exp Med Biol, 57:1–80, 1975.\n\n[5] E Devine, A V Somlyo, and A P Somlyo. Sarcoplasmic reticulum and excitation-contraction coupling in mammalian smooth muscles. J Cell Biol, 52(3):690–718, Mar 1972."
}
|
[
{
"id": "4530",
"date": "24 Apr 2014",
"name": "Anthony P Albert",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting and thought provoking study which simulates NAADP-dependent Ca2+ transients; these are produced by release of Ca2+ from lysosome stores that induce Ca2+-induced Ca2+ release from SR stores via stimulation of RyR3. It is increasingly apparent that answers to many issues concerning cellular Ca2+ signalling will be found by recording local Ca2+ events – for instance the recently identified Ca2+ sparklets representing Ca2+ influx through opening of plasma membrane ion channels. Simulation studies are always difficult to review and evaluate as they must make many assumptions. However, this work is considered and may be a useful model for how other local Ca2+ events might be modeled between discrete organelles. Moreover, unlike many simulation manuscripts, I find this one clear and well-written.\n\nDo the authors think that the use of proximity ligation assay techniques might be appropriate to measure local interactions between TPC2 and RyR3? Also, how are lysosomes refilled with Ca2+ following NAADP/TPC2 activation – a role for a lysosome store-operated Ca2+ influx?",
"responses": []
},
{
"id": "4843",
"date": "18 Jun 2014",
"name": "Alexei Tepikin",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is certainly an interesting study. The characterisation of lysosomal-SR junctions is novel and conceptually important. The data look convincing but I would recommend the authors to further expand the structural part of the study. It would perhaps be useful to further verify that the organelles participating in the formation of the contacts are indeed lysosomes and that the junctions are not simply the result of accidental collisions of the two organelles. Identifying/visualising tethers between the organelles (by analogy with tethers between mitochondria and the ER1) would be particularly advantageous.Modelling part of the paper is also useful. In this respect it would be valuable to clarify the Ca2+ buffering capacity of lysosomes (and its possible effect on the outcomes of the model). One could also perhaps consider measuring this important parameter using isolated lysosomes.",
"responses": []
},
{
"id": "5274",
"date": "27 Jun 2014",
"name": "Fan Zhang",
"expertise": [],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nNAADP mediated two-phase Ca2+ release responses were observed in smooth muscle cells, including pulmonary artery smooth muscle cells (PASMCs). Lysosomes and sarcoplasmic reticulum (L-SR) junctions had been proposed as the structural bases in facilitating local lysosomal Ca2+ bursts (first phase) followed by whole cell Ca2+ release responses from SR (second phase) through a calcium-induced calcium release (CICR) mechanism. In their studies reported here, Nicola Fameli et al. attempted to define the L-SR junctions in PASMCs with a nano-level resolution using electron microscopy (EM), and proceeded to build a quantitative L-SR junctional model. Unfortunately, all these efforts will add little to our further understanding of these L-SR junctional structures, if not at all, due to the lack of solid experimental EM results in reconstructing L-SR junctions, some dogmatic presumptions in L-SR model build up, the inadequacy of validation of the model and a failure to include information on the current disputes over the identity of NAADP-associated Ca2+ release channels in lysosomes. Some major concerns are as follows: Data are not convincing and lack quality. In defining L-SR junctions, the assurance of lysosome and SR identities is fundamental. Immune-gold staining has been widely applied in EM studies to ascertain different cell organelles as well as to conduct 3D reconstruction of cell structure 1 2. However, such a standard practice was excluded in this study as a ‘precaution’ to prevent compromising lysosome membrane definition. In line with applying empirical judgments for identifying organelles to the EM data analysis, the authors extrapolated the opposing and proximal organelles adjacent to the nucleus as lysosomes and SR, and deemed them to be worth studying (Figure 2). In addition, the authors stated that there were few lysosomes found in the subplasmalemmal region, which implied that L-SR junctions were profoundly located around the nucleus. However, the fura-2 fluorescence images in Figure 1 as well as in the two citations used as basis of this study (10,11)3 4 exclusively demonstrated that the first phase lysosomal Ca2+ release initiated from a region around the cell membrane, suggesting that L-SR junctions should have cytoplasmic locations near the plasma membrane. It is clear that more reliable methods such as immune-gold staining are needed to resolve the contradictions over their own results. The modeling is based on many assumed parameters and lacks reliable validation. To establish the L-SR nanojunctional model, the present study has introduced many assumptions to some critical parameters such as the distance between the lysosome and SR, the size of lysosome, and total number of lysosomes; many of which lack proper judgments. In the abstract section, the authors rationalized the application of EM in investigating L-SR junctions by stating “L-SR junction membrane separation has been estimated to be < 400 nm and thus beyond the resolution of light microscopy”. In defining the L-SR nanojunctions, they declared that lysosomes and SR “must be separated by a distance of approximately 20 nm and be of the order of a few hundred nm in lateral dimensions” by citing PM-SR junctions studies (10, 11)3 4. However, these two citations originated from the light microscope-based studies of L-SR junctions by the same group. Given the resolution limitation, these light microscopy results have no way to give an estimation as accurate as 20 nm. It is therefore confusing to find that these references fail to back up such claims, in my opinion. In reference 39 5, it has been proposed that the extracellular Ca2+ entered the cell through Na+/ Ca2+ exchanges, traversed PM-SR junctions to refill SR via Ca2+-ATPase and sustained cell Ca2+ oscillations. For the current L-SR model, this PM-SR modeling may be used as a comparison or reference system leading to the conclusion “that L-SR had a similar 20 nm gap measured as that in PM-SR junctions.” Given that there are no similarities of PM-SR to L-SR junctions, in terms of potency of Ca2+ movement due to Ca2+ gradient across PM and lysosomal membrane, biophysical property of Na+/ Ca2+ exchangers and TPC2 channels, and the actions of Ca2+-ATPase and RyR3 Ca2+ receptors, such a conclusion is not reliable from the modeling. In addition, a major concern is that this model is too simplistic as it excludes many other Ca2+ regulatory mechanisms in both lysosomes and SR, which may influence the accuracy of junction width or prediction in modeling. Some assumptions contradict well-established concepts due to a lack of consideration of lysosome biology. In explaining the shoulder Ca2+ signal (Figure 1), the authors introduced up to four assumptions and calculated that ~60-100 lysosomes were needed to generate this shoulder peak. They verified these lysosome numbers with their TEM images that “can expect a total of about 110 lysosomes/cell”. Considering these two calculations however, it is obviously problematic because almost all lysosomes (100 out of 110 each cell) will need to functionally contribute to the L-SR junctional structure formation. In addition, TEM measurements showed the lysosome dimensions (Table 1) to be major axis of 398 ± 91 nm and minor axis of 325 ± 65 nm. Given the L-SR junction model features “junctional membrane separation and extension are about 15 nm and 300 nm”, it could be postulated that all the lysosomes that participated in L-SR structure formation also have to align well with the SR in order to produce a 300 nm junctional extension. Therefore, this L-SR model actually describes a scenario whereby in PASMCs the majority of lysosomes act as the components of L-SR junctions and uniformly have impeccable arrangements to oppose the SR at 20 nm apart. However, this idealistic model largely contradicts the well-established concepts that lysosomes are active organelles that dynamically exert their different functions by their movements and other activities. TPCs as lysosomal Ca2+ release channels are questionable. In quantifying the L-SR model, the authors arbitrarily assumed TPC2 as the only lysosomal NADDP-activated Ca2+ channel by totally disregarding recent findings that TPC2 is not a lysosomal NAADP-associated Ca2+ channel, but a Na+ channel6, 7 . They also ignored the contribution of other lysosomal channels or transporters that may contribute to such L-SR junction activity. This prejudice in channel selection inevitably invites doubts on the reliability of this proposed L-SR model. In addition, the authors collectively adopted electrophysiological data from other research groups without taking into consideration any incompatibilities with the model. For example, in obtaining the total number of TPC2 channels on a lysosome, the authors “…dividing the macroscopic whole lysosome conductance, calculated from the current values reported in a recent study on TPC2-mediated Ca2+ current in isolated lysosomes (48) 8, by the single channel conductances(45) 9, thus estimating that a typical lysosomal membrane may contain ~20 TPC2”. Unfortunately, the lysosomes used in the whole lysosome conductance recording were not normal lysosomes, but lysosomes manipulated by overexpression of TPC2 and were undergoing size enlargement. With these changes, the modified lysosomes would have a much higher TPC2 conductance compared with normal lysosomes with a natural biogenesis. It is important to use a TPC2 conductance and other parameters of channels from lysosomes of normal cells without an artificial transgene for such modeling. Otherwise, the modeling prediction will be inaccurate and the relevance of such a model will be vague.\n\nIn conclusion; aside from the issue that the current L-SR nanojunction model fails to contribute any new insights to our understanding of the L-SR architecture, my concerns over the reliability of data and modelling parameters are rather serious – this work is therefore ‘Not Approved’ in its current version.",
"responses": [
{
"c_id": "1110",
"date": "04 Dec 2014",
"name": "Nicola Fameli",
"role": "Author Response",
"response": "Dr. Fan Zhang we respond to your concerns below, by reporting your comments in quotes first and adding our response below them in bold.\"Unfortunately, all these efforts will add little to our further understanding of these L-SR junctional structures, if not at all, due to the (1) lack of solid experimental EM results in reconstructing L-SR junctions, (2) some dogmatic presumptions in L-SR model build up, (3) the inadequacy of validation of the model and (4) a failure to include information on the current disputes over the identity of NAADP-associated Ca2+ release channels in lysosomes.\"We address points (1) to (4) in detail below (numbering ours).1. \"Data are not convincing and lack quality.\"We fully disagree with this statement. Our set of data represents the most complete survey of these structures in vascular smooth muscle currently available in the literature. The micrographs are of high quality with very well defined membranes and identifiable organelles.\"In defining L-SR junctions, the assurance of lysosome and SR identities is fundamental. Immune-gold staining has been widely applied in EM studies to ascertain different cell organelles as well as to conduct 3D reconstruction of cell structure 1 2. However, such a standard practice was excluded in this study as a ‘precaution’ to prevent compromising lysosome membrane definition.\"Immuno-gold staining was avoided not as a 'precaution', but because, while certainly enabling us to identify the desired organelles, it reduces visualization of their membranes, since the fixing procedure aimed at preserving tissue antigenicity and the staining protocol for label visualization seriously compromise the resolution of membrane structure required for accurate quantitative characterization of the L-SR junctions. The references this reviewer provides only corroborate our point. As explained in detail by first reference, SEM is less appropriate in this kind of study for a number of reasons: 1) One would need to expose lysosomes buried underneath the PM to visualize their surface membrane. This, as the article suggests, is best done by isolating the organelles. Hardly a technique that can serve the purpose of identifying L-SR junctions, whether we use immuno-gold or not; (2) As is evident from Fig 3 of the second reference, therein no membranes were visible in the micrograph with immuno-gold labeling.\"In line with applying empirical judgments for identifying organelles to the EM data analysis, the authors extrapolated the opposing and proximal organelles adjacent to the nucleus as lysosomes and SR, and deemed them to be worth studying (Figure 2).\"Figure 2, as clearly explained in the text, contains some representative examples from our set of micrographs, but even from the subset in Fig. 2 it is evident that we did not only consider regions neighbouring the nucleus in our survey. This reviewer's statement that “the authors extrapolated the opposing and proximal organelles adjacent to the nucleus as lysosomes and SR” mistakenly implies that the authors simply picked any two objects whose membrane were close enough to be deemed a junctional complex between lysosome and SR. This was most certainly not the case, as explained in detail in the article. The authors can add however that, in spite of our own laboratory's proven experience in electron microscopy (see for example references 39, 56, 57, 92, 98 in our article), we consulted with an expert in the field of electron microscopy, Prof. David Walker, and drew his immense (>30 years) experience (including smooth muscle) to our own. We not only sought and received guidance, but were enlightened with respect to common practices regarding the identification of lysosomes and their junctions with SR.\"In addition, the authors stated that there were few lysosomes found in the subplasmalemmal region, which implied that L-SR junctions were profoundly located around the nucleus. However, the fura-2 fluorescence images in Figure 1 as well as in the two citations used as basis of this study (10,11)3 4 exclusively demonstrated that the first phase lysosomal Ca2+ release initiated from a region around the cell membrane, suggesting that L-SR junctions should have cytoplasmic locations near the plasma membrane. It is clear that more reliable methods such as immune-gold staining are needed to resolve the contradictions over their own results.\"One does not exclude the other for a couple of reasons. First, the typical diameter and length of vascular smooth muscle cells are about 5 μm and 150 μm, respectively, which ensures that even so-called perinuclear lysosomes are never too far from the periphery. This fact alone would break the apparent contradiction raised. Moreover, the definition of cell periphery that one can gather from fluorescence measurements can easily cover more than 500 nm in depth. Therefore, at lower resolution fluorescence signals could appear close to the membrane yet be generated by perinuclear lysosomes.\"2. The modeling is based on many assumed parameters and lacks reliable validation. To establish the L-SR nanojunctional model, the present study has introduced many assumptions to some critical parameters such as the distance between the lysosome and SR, the size of lysosome, and total number of lysosomes; many of which lack proper judgments.\"This point raised by the reviewer verges on the ridiculous. None of the stated parameters was assumed, as should be clear upon reading the article. The L-SR distance was measured, not assumed. The size of the lysosomes observed in micrographs was measured, not assumed. The total number of lysosomes was estimated from our observations, not assumed. We find it difficult to see where proper judgment is lacking, at least with respect to our contribution. \"In the abstract section, the authors rationalized the application of EM in investigating L-SR junctions by stating “L-SR junction membrane separation has been estimated to be < 400 nm and thus beyond the resolution of light microscopy”. In defining the L-SR nanojunctions, they declared that lysosomes and SR “must be separated by a distance of approximately 20 nm and be of the order of a few hundred nm in lateral dimensions” by citing PM-SR junctions studies (10, 11)3 4. However, these two citations originated from the light microscope-based studies of L-SR junctions by the same group. Given the resolution limitation, these light microscopy results have no way to give an estimation as accurate as 20 nm. It is therefore confusing to find that these references fail to back up such claims, in my opinion.\"We thank the reviewer for drawing our attention to an unfortunate (and clearly confusing) typographical error with respect to in-text numbering of citations. Our intention was to refer to Lee CH et al 2002 and Fameli N et al 2007, or references 92 and 39, respectively, in the present version. This typographical error will be rectified in a future version of the manuscript.\"In reference 39 5, it has been proposed that the extracellular Ca2+ entered the cell through Na+/ Ca2+ exchanges, traversed PM-SR junctions to refill SR via Ca2+-ATPase and sustained cell Ca2+ oscillations. For the current L-SR model, this PM-SR modeling may be used as a comparison or reference system leading to the conclusion “that L-SR had a similar 20 nm gap measured as that in PM-SR junctions.” Given that there are no similarities of PM-SR to L-SR junctions, in terms of potency of Ca2+ movement due to Ca2+ gradient across PM and lysosomal membrane, biophysical property of Na+/ Ca2+ exchangers and TPC2 channels, and the actions of Ca2+-ATPase and RyR3 Ca2+ receptors, such a conclusion is not reliable from the modeling.\"The sentence in quotes (underlined above) does not appear in our article. Nevertheless let us provide the best rebuttal possible under these circumstances, and address the somewhat confusing comments made by this reviewer in terms of considerations of the relationship between junctional architecture and the biophysical properties of channels and transporters within different junctions. We measured the distance of observed junctional widths between lysosomes and the sarcoplasmic reticulum with closely apposed “junctional” membranes. The calculated average for the size of measured junctional width was not significantly different from the averaged size of PM-SR junctions previously published and cited in this manuscript. We draw the reader to this striking similarity, and relate this to the capacity of the “nanojunction” to hold and concentrate released calcium. In this respect the precise behaviour of each junction will of course be determined by the density and biophysical properties of local pumps and channels, much like the behaviours vary between other previously defined nanojunctions.\" In addition, a major concern is that this model is too simplistic as it excludes many other Ca2+ regulatory mechanisms in both lysosomes and SR, which may influence the accuracy of junction width or prediction in modeling.\"It is always best to start with a minimal model and to make the least number of assumptions possible while not compromising the capacity to inform by way of outcomes. This is what the authors delivered. A more complex model would require the addition of a far greater number of assumptions and, in some cases, these would be difficult to justify. Given earlier comments by this reviewer regarding the limitations of assumptions, it is surprising that such minima are not better appreciated, not least because the model in its current form is sufficient to verify a hypothesis based on a substantial body of peer reviewed work (ref. 10, 11 in the article). As the strength of supporting experimental data grows, it would of course be beneficial to the field if the present model were developed further to incorporate other “regulatory mechanisms” to assess with greater accuracy these and similar signalling mechanisms that lie beyond the reach of current technologies.\"3. Some assumptions contradict well-established concepts due to a lack of consideration of lysosome biology. In explaining the shoulder Ca2+ signal (Figure 1), the authors introduced up to four assumptions and calculated that ~60-100 lysosomes were needed to generate this shoulder peak.\"In constructing quantitative (computational) models, and particularly with respect to those that are applied to systems beyond the resolution of current technologies, one is necessarily required to make justified assumptions. All assumptions made are both appropriate and plausible, and are justified by reference to the literature.\"They verified these lysosome numbers with their TEM images that “can expect a total of about 110 lysosomes/cell”. Considering these two calculations however, it is obviously problematic because almost all lysosomes (100 out of 110 each cell) will need to functionally contribute to the L-SR junctional structure formation.\"This is a biased reading of our findings, in that we suggest that a range of 60 to 100 lysosomes would have to contribute to the shoulder magnitude, under our model’s conditions, not exactly 100.\"In addition, TEM measurements showed the lysosome dimensions (Table 1) to be major axis of 398 ± 91 nm and minor axis of 325 ± 65 nm. Given the L-SR junction model features “junctional membrane separation and extension are about 15 nm and 300 nm”, it could be postulated that all the lysosomes that participated in L-SR structure formation also have to align well with the SR in order to produce a 300 nm junctional extension.\"This reviewer’s postulate forcing the lysosomes to “align well” does not emerge naturally from our model. Would this reviewer explain what relationship between the size of the observed lysosomes (major and minor axes) and the portion of the lysosomal surface participating in the L-SR junction suggests that “all the lysosomes that participated in L-SR structure formation also have to align well with the SR in order to produce a 300 nm junctional extension”? This is most unclear from this reviewer’s comments. It is instead clear upon reading the article that nothing in our model nor in our conclusions depends on such postulate.\"Therefore, this L-SR model actually describes a scenario whereby in PASMCs the majority of lysosomes act as the components of L-SR junctions and uniformly have impeccable arrangements to oppose the SR at 20 nm apart.\"This last sentence is a conclusion that this reviewer makes and stems directly from this reviewer’s postulate—which I must re-emphasize we do not make—and not from our model.\"However, this idealistic model largely contradicts the well-established concepts that lysosomes are active organelles that dynamically exert their different functions by their movements and other activities.\"The contradiction arises from this reviewer postulated interpretation of our findings. We certainly don’t claim anything that goes against the established observations of lysosmal mobility.\"4. TPCs as lysosomal Ca2+ release channels are questionable. In quantifying the L-SR model, the authors arbitrarily assumed TPC2 as the only lysosomal NADDP-activated Ca2+ channel by totally disregarding recent findings that TPC2 is not a lysosomal NAADP-associated Ca2+ channel, but a Na+ channel6, 7 . They also ignored the contribution of other lysosomal channels or transporters that may contribute to such L-SR junction activity. This prejudice in channel selection inevitably invites doubts on the reliability of this proposed L-SR model.\"The authors used a minimal model to examine junctional communication at the L-SR nanjunction. The designation of TPC2 was supported by the fact that TPC2: (1) Is primarily targeted to lysosomes; (2) Is required to support NAAD-dependent calcium signalling; (3) Has been shown to be permeable to calcium by at least two previous studies. Controversy in the field is not ignored, as the authors cite articles that provide detailed coverage on disputes relating to TPC2 calcium permeability. That there is conflict in the field does not negate the use of data derived from papers that demonstrate calcium permeability of these channels.\"In addition, the authors collectively adopted electrophysiological data from other research groups without taking into consideration any incompatibilities with the model. For example, in obtaining the total number of TPC2 channels on a lysosome, the authors “…dividing the macroscopic whole lysosome conductance, calculated from the current values reported in a recent study on TPC2-mediated Ca2+ current in isolated lysosomes (48) 8, by the single channel conductances(45) 9, thus estimating that a typical lysosomal membrane may contain ~20 TPC2”. Unfortunately, the lysosomes used in the whole lysosome conductance recording were not normal lysosomes, but lysosomes manipulated by overexpression of TPC2 and were undergoing size enlargement. With these changes, the modified lysosomes would have a much higher TPC2 conductance compared with normal lysosomes with a natural biogenesis. It is important to use a TPC2 conductance and other parameters of channels from lysosomes of normal cells without an artificial transgene for such modeling. Otherwise, the modeling prediction will be inaccurate and the relevance of such a model will be vague.\"The authors used the best evidence available in support of the present model. At no point was it stated that values used were anything more than estimates derived by best practice from cited and peer reviewed research publications by others. Assuming something other than that which can be derived falls in to the realm of unjustifiable assumption. With present information, assuming a higher TPC2 conductance would be just that.\"In conclusion; aside from the issue that the current L-SR nanojunction model fails to contribute any new insights to our understanding of the L-SR architecture, my concerns over the reliability of data and modelling parameters are rather serious – this work is therefore ‘Not Approved’ in its current version.\"We think that, within the limits of the available techniques and knowledge, our work not only contributes \"new insights to our understanding of the L-SR architecture\", but also a novel approach to the study of nanojunctional signals."
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https://f1000research.com/articles/3-93
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https://f1000research.com/articles/3-92/v1
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17 Apr 14
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{
"type": "Research Article",
"title": "Toxoplasma gondii infection enhances the kairomonal valence of rat urine",
"authors": [
"Anand Vasudevan",
"Ajai Vyas",
"Anand Vasudevan"
],
"abstract": "Many animals use chemicals as pheromones to communicate between individuals of the same species, for example to influence mate choice or to assert dominance. Pheromonal communication is an open broadcast system that can be intercepted by unintended receivers such as predators and prey. We have recently reported that male rats infected by the protozoan parasite Toxoplasma gondii become more attractive to female rats. This suggests a facilitatory effect of infection on rat pheromone production. In view of the open nature of pheromonal communication, we postulate that Toxoplasma gondii infection collateraly enhances kairomonal valence of infected rats to their prey. We compared the strength of kairomonal interception by mice when using scent marks from rats infected with Toxoplasma gondii vs. marks from uninfected control rats. Mice exhibited greater avoidance to both fresh urine and aged rat urine marks obtained from infected animals. These results indicate that, at least in some cases, parasitism can result in opportunity costs for hosts by making prey species more averse to them.",
"keywords": [
"Behavioural manipulation",
"Mate choice",
"Scent marks",
"Pheromones"
],
"content": "Introduction\n\nThe protozoan parasite Toxoplasma gondii manipulates the behavior of its rat host in two important ways. First, it not only abolishes the rats’ innate fear of cat odors, it can also induce attraction to cat odors1–4. This plausibly increases parasite transmission to cats that serve as its definitive hosts. Second, Toxoplasma gondii infection enhances the attractiveness of infected males to females5. Interestingly, enhanced attractiveness could benefit both the parasite and its host. This plausibly increases the parasites’ transmission through sexual and vertical routes5–8 and benefits the hosts by increasing their reproductive opportunities.\n\nHost-parasite interaction is typically characterized by significant trade-offs. Behavioral manipulation itself can impose substantial direct and opportunity costs on the parasite9–11. For example, manipulation of innate fear in infected rats is optimized and not maximized12; suggesting that a dynamic balance exists for the parasite between the costs and benefits of the manipulation. From the perspective of the rat host, one of the most important trade-offs is the reproductive benefit obtained from increased attractiveness and the reproductive cost incurred by greater predation. An experimental study of this trade-off would require an ethically tenuous comparison of predation rates between control and infected animals. Another important trade-off for the host arises from the fact that pheromones produced to communicate male attractiveness are openly broadcasted, liable to be used by both the intended female audience and unintended prey or predator species. We examine this trade-off in the current study.\n\nHouse mice are predated by rats. Studies have shown that around 70% of wild rats13,14 kill mice. Mouse-killing or muricide has been shown to be influenced by rearing15, availability of food14 social16 and environmental17 conditions. In addition, mice express an innate fear of rats18,19. This is characterized by the display of defensive behavior, secretion of stress hormones and activation of brain pathways dedicated to defensive behaviors. In fact, exposure to rat urine or even a recombinant rat urinary protein is sufficient to produce aversion in mice20,21, with the presence of an actual rat not being necessary. Interestingly, in the case of rats, exposure to soiled bedding is sufficient for females to infer greater attractiveness of Toxoplasma gondii-infected males5. This suggests enhanced pheromonal production in infected males, which could result in greater kairomonal aversion in mice considering the openly broadcasted nature of urinary signals. Enhanced aversion in prey species could constitute an opportunity cost for the infected rats that need to be ‘traded-off’ with any incremental benefit of enhanced pheromone production. In light of this, we investigated whether Toxoplasma gondii infection increased the kairomonal valence of rat urine to its prey, mice.\n\n\nMaterials and methods\n\nThe Nanyang Technological University (IACUC number: ARF SBS/NIE-A-0106AZ) institutional animal care and use committee reviewed and approved all procedures. Twelve uninfected male Balb/c mice (7–8 weeks old, housed five/cage; (369 x 156 x 132mm; 1145T, Tecniplast, UK)) and four male Wistar rats (48 days old, housed two/cage (425 x 266 x 185mm; 1291H, Tecniplast, UK)) were obtained from the vivarium of National University of Singapore. Standard corn cob cage bedding was changed twice a week. Animals were placed on a 12 hours light-dark cycle, with temperature between 20–25°C and relative humidity ranging around 70–80%, respectively. Experiments were carried out during the light phase. Food and water was available ad libitum. The diet was made up of standard laboratory chow (PicoLab Rodent Diet 20, 5053) with 20% protein content. Animals from this source tested serologically negative for Toxoplasma gondii. This was done by incubating serum (1:1000) from the rats in 24 well plates that were coated with Toxoplasma gondii tachyzoites overnight at 4C. After washing the wells with PBS, polyclonal anti-rat Cy3 (1:200, Millipore, catalogue number AP189C) was added and incubated for 2 hours at room temperature. The wells were visualized under a live microscope (Nikon, 20X) under the GFP filter and Cy3 filter.\n\nWe used a Prugniaud strain of Toxoplasma gondii which has a luciferase and GFP tag (sourced from John Boothroyd, Stanford School of Medicine). Parasites were maintained as tachyzoites by passage in human foreskin fibroblast monolayers (John Boothroyd, Stanford School of Medicine). Infected fibroblasts were syringe-lysed by using a 27-gauge needle to release tachyzoites. Rats were randomly picked (a blinded person picked two rat-assigned numbers) from a population infected with tachyzoites (5 × 106, i.p in phosphate buffered saline; n = 2) or mock-infected with sterile phosphate buffered saline (0.5ml, i.p.; n = 2). The infected rats were monitored weekly for weight loss and other signs of sickness. Fresh urine and aged urine marks were collected from rats between 6 to 8 weeks post-infection, a period know to harbor chronic infection.\n\nFor testing response to fresh urine, rat urine was collected using metabolic cages (Harvard Apparatus). Rats were placed in this apparatus for at least 2 hours and food and water was provided. The urine was collected on the same day of testing. Rat urine contains both volatile and non-volatile substances. Urinary volatiles tend to dissipate quickly with passage of time, while non-volatiles can remain stable for weeks. In order to ascertain the contribution of non-volatiles (i.e. aged urine), a plastic Petri plate was placed in a rat cage for twenty-four hours on which the rats would urine mark. Three days (stored at RT) after removal of the Petri plate, it was used as the stimulus in the avoidance-avoidance test with mice.\n\nResponse of male mice to fresh urine obtained from control or infected rats (pooled from two rats) was studied using an avoidance-avoidance conflict paradigm. Avoidance was quantified by comparing time spent by mice in two opposing bisects of arena (76 × 9 cm; 15 cm high) during a 20 minute trial. Data on time spent was collected by automated behavioral tracking software (ANY-maze, version 4.3, Stoelting). Opposing bisects either had urine from control and infected rats (5 drops of 20 µl each, placed equidistant on absorbent paper of 5 × 7 cm). For testing response to aged urine, the three day old Petri plates from the control and infected rats were placed at terminal ends of opposing bisects. Again avoidance was quantified as described above with ANY-maze. The same set of mice were used for both behavioral assays. Experiments involving aged urine preceded the experiments with fresh urine.\n\nAll statistical tests were conducted using IBM SPSS software (version 20). A p value of ≤ 0.05 was considered to be significant. Student’s t-test was used to estimate statistical significance. Analysis of variance (ANOVA) was used to analyze the effect of infection status and age of urine on kairomonal communication.\n\n\nResults\n\nAvoidance of mice in response to control or infected rat scent marks was determined using a avoidance-avoidance conflict task (n = 12 uninfected control mice). Kairomonal response to fresh urine and aged urine marks was studied separately and in sequence (aged followed by fresh marks).\n\nANOVA was conducted with time spent in each bisect (control or infected) and age of urine (fresh or aged) as two sources of within subject factors. ANOVA revealed a significant main effect for the infection status of scent donors (F(1,11) = 8.049, p = 0.016). The difference between fresh and aged scent marks did not reach statistical significance (F(1,11) = 0.005, p > 0.9). The interaction between the infection status of the donor and the age of the scent marks also did not reach statistical significance (F(1,22) = 0.314, p > 0.5).\n\nOver a twenty-minute trial, mice spent more time in the bisect containing fresh urine from control rats (planned comparison; paired t-test: t22 = 2.13, p < 0.05; 674 ± 55 s in control bisect versus 510 ± 49 s in infected bisect; Figure 1A). A preference score of mice for control rat urine was computed for each trial by dividing the time spent in the control bisect by the time spent in the infected bisect. In 75% of trials, mice spent more time in the control bisect (Figure 1B; mean preference score = 1.36 ± 0.22; one outlier removed). Similar results were obtained when using aged urine marks. Mice spent more time in the bisect containing aged urine marks from control rats (planned comparison; t22 = 3.36, p < 0.01; 715 ± 52 s in the control bisect versus 469 ± 51 s in the infected rat bisect; Figure 2A). Just like the fresh urine experiment, 9 out of 12 mice spent more time near the urine marks obtained from control animals (Figure 2B; mean preference score = 1.94 ± 0.36).\n\nPreference was quantified by comparing time spent by a mouse in two opposing bisects of an arena, with each bisect containing fresh urine from either control rats or rats infected six weeks earlier (panel A; trial duration = 1200 s, n = 12 mice). Ordinate and abscissa depict time spent in infected and control bisect in seconds, respectively (p < 0.05, paired t-test). Mean and SEM of data used in scatter-plot are depicted by dot and whiskers. A preference score was computed for each mouse by dividing the time spent in the control bisect with time spent in the infected bisect (panel B; chance = 1). Each dot represents preference data from one mouse (1 outlier removed). Box plots depict median, 25th percentile and 75th percentile. Mean and SEM of data used in scatter-plot are depicted in dot and whiskers.\n\nPreference of mice for control urine marks was retained when aged (3 days old), rather than fresh, rat urine was used. Panel A depicts time spent in the control and infected bisects; Panel B depicts preference ratio (see Figure 1 for further details on what the graphs denote).\n\nThus, fresh and aged urine marks obtained from infected rats evoke a greater kairomonal response than urine obtained from uninfected rats in mice.\n\n\nDiscussion\n\nAs a result of intense predation pressure13,14, mice have developed an innate sensitivity to rat kairomones. Rat odors evoke immediate and intense defensive behaviors in laboratory mice, coupled with activation of brain pathways typical of defensive behavior and secretion of stress hormones18–20. Here we report that urine obtained from rats infected with the parasite Toxoplasma gondii generate greater avoidance in mice compared to control rats. Furthermore, we demonstrate that the active ingredient involved in parasite-induced kairomonal changes is most likely non-volatile, leading us to speculate that major urinary proteins are involved. This is in agreement with a prior report that purified recombinant rat major urinary proteins can induce kairomonal aversion in mice, precluding an essential role of urinary volatiles20.\n\nPheromones are chemical substances produced by an individual animal, which affect the behavior of conspecifics (chemical communication). Pheromones are widely used by insects to communicate danger, the presence of food or sexual receptiveness. In the mammalian world, pheromones are often used to signal dominance and to influence female mate choice22–24. Moreover, pheromones can act as kairomones when these chemical substances are received by individuals of another species25. As such, kairomones are used to the detriment of the emitter and for the benefit of the receiver26. This is because, once produced, pheromones are an openly broadcasted information system. Apart from the intended receivers of the same species, they can also be perceived by unintended receivers of a different species. These unintended receivers typically fall into two categories: predators and prey. Predators routinely use pheromones produced by their prey to locate their food27–29. Prey can also use this information to reduce their danger of predation20,30, although this possibility is relatively less well studied. Thus, pheromones of a species can be used as kairomones by both the prey and predator of that species.\n\nParasitic infections can drastically affect pheromonal production. For example, female mice typically avoid the odor of male mice infected with an array of bacteria, viruses, protozoa and nematodes31. Atypically, male rats infected with the protozoan parasite Toxoplasma gondii produce urine that is more attractive to receptive females, suggesting enhanced pheromonal production5. We have also observed that Toxoplasma gondii can be transmitted during sexual intercourse in rats5 (see6,32 for sexual transmission in other species). It is likely that increased male attractiveness is a parasitic manipulation, aiming to increase the frequency of parasite transmission between males and females (but also see33). This atypical host manipulation by Toxoplasma gondii opens a rather interesting trade-off for the infected host. An increase in pheromonal communication might lead to enhanced reproductive benefits for the infected male. At the same time, intra-species pheromonal communication can be co-opted by prey to initiate defensive behaviors, placing a probabilistic cost on the predator. Consistent with this trade-off, the data presented here suggests that male rats infected with Toxoplasma gondii suffer opportunity costs in terms of greater aversion by mice, a prey species.\n\n\nData availability\n\nfigshare: Proportion of time spent by mice near urine from rats infected with Toxoplasma gondii vs urine from uninfected rats, doi: 10.6084/m9.figshare.99387134",
"appendix": "Author contributions\n\n\n\nA.Va. designed and carried out the experiments. A. Vy. prepared the manuscript. Both authors revised and approved the final manuscript for publication.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was funded by a research grant (MOE2011-T2-2-111) to Ajai Vyas from Ministry of Education, Singapore.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe would like to thank John Boothroyd’s lab at the Stanford School of Medicine for donating us stocks of Toxoplasma gondii and human foreskin fibroblast cell line.\n\n\nReferences\n\nBerdoy M, Webster JP, Macdonald DW: Fatal attraction in rats infected with Toxoplasma gondii. Proc Biol Sci. 2000; 267(1452): 1591–1594. 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PubMed Abstract | Publisher Full Text\n\nThomas F, Guldner E, Renaud F: Differential parasite (Trematoda) encapsulation in Gammarus aequicauda (Amphipoda). J Parasitol. 2000; 86(3): 650–654. PubMed Abstract | Publisher Full Text\n\nVyas A, Kim SK, Sapolsky RM: The effects of toxoplasma infection on rodent behavior are dependent on dose of the stimulus. Neuroscience. 2007; 148(2): 342–348. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGalef BG Jr: Aggression and timidity: responses to novelty in feral Norway rats. J Comp Physiol Psychol. 1970; 70(3): 370–381. PubMed Abstract | Publisher Full Text\n\nKarli P: The Norway rat's killing response to the white mouse: an experimental analysis. Behaviour. 1956; 10(1): 81–103. Publisher Full Text\n\nGarbanati JA, Sherman GF, Rosen GD, et al.: Handling in infancy, brain laterality and muricide in rats. Behav Brain Res. 1983; 7(3): 351–359. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nVasudevan A, Vyas A: Kairomonal communication in mice is concentration-dependent with a proportional discrimination threshold. Version 2. F1000Res. 2013; 2: 195. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChamero P, Marton TF, Logan DW, et al.: Identification of protein pheromones that promote aggressive behaviour. Nature. 2007; 450(7171): 899–902. PubMed Abstract | Publisher Full Text\n\nHurst JL: Female recognition and assessment of males through scent. Behav Brain Res. 2009; 200(2): 295–303. PubMed Abstract | Publisher Full Text\n\nRamm SA, Cheetham SA, Hurst JL: Encoding choosiness: female attraction requires prior physical contact with individual male scents in mice. Proc Biol Sci. 2008; 275(1644): 1727–1735. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrown WL, Eisner T, Whittaker RH: Allomones and kairomones: Transpecific chemical messengers. BioScience. 1970; 20: 21–22. Reference Source\n\nGrasswitz TR, Jones GR: Chemical Ecology. Encyclopedia of Life Sciences, (John Wiley & Sons, Ltd). 2003. Publisher Full Text\n\nBoone CK, Six DL, Zheng Y, et al.: Parasitoids and dipteran predators exploit volatiles from microbial symbionts to locate bark beetles. Environ Entomol. 2008; 37(1): 150–161. PubMed Abstract | Publisher Full Text\n\nBranco M, van Halder I, Franco JC, et al.: Prey sex pheromone as kairomone for a new group of predators (Coleoptera: Dasytidae, Aplocnemus spp.) of pine bast scales. Bull Entomol Res. 2011; 101(6): 667–674. PubMed Abstract | Publisher Full Text\n\nStowe MK, Turlings TC, Loughrin JH, et al.: The chemistry of eavesdropping, alarm, and deceit. Proc Natl Acad Sci U S A. 1995; 92(1): 23–28. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBoersma M, Spaak P, De Meester L: Predator-mediated plasticity in morphology, life history, and behavior of Daphnia: the uncoupling of responses. Am Nat. 1998; 152(2): 237–248. PubMed Abstract | Publisher Full Text\n\nKavaliers M, Colwell DD: Discrimination by female mice between the odours of parasitized and non-parasitized males. Proc Biol Sci. 1995; 261(1360): 31–35. PubMed Abstract | Publisher Full Text\n\nGutierrez J, O'Donovan J, Williams E, et al.: Detection and quantification of Toxoplasma gondii in ovine maternal and foetal tissues from experimentally infected pregnant ewes using real-time PCR. Vet Parasitol. 2010; 172(1–2): 8–15. PubMed Abstract | Publisher Full Text\n\nBerdoy M, Webster JP, Macdonald DW: Parasite-altered behaviour: is the effect of Toxoplasma gondii on Rattus norvegicus specific? Parasitology. 1995; 111(Pt 4): 403–409. PubMed Abstract | Publisher Full Text\n\nAnand V, Ajai V: Proportion of time spent by mice near urine from rats infected with Toxoplasma gondii vs urine from uninfected rats. Figshare. 2014. Data Source"
}
|
[
{
"id": "4523",
"date": "22 Apr 2014",
"name": "Jian-Xu Zhang",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper describes an interesting result - that infected rats were more aversive to prey (mice). The authors have previously reported that infected male rats produced urine marks which were more attractive to female conspecifics. They postulated that infected male rats may produce more male pheromones to attract female conspecifics, which could also act as kairomones to repel rats' prey, e.g. house mice. Although the authors did not chemically analyze the contents of the pheromonal components, at this stage their behavioral tests reliably supported their conclusion.They should add some references to show the rat's pheromone identification.",
"responses": []
},
{
"id": "4524",
"date": "30 Apr 2014",
"name": "Jaroslav Flegr",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors clearly showed that mice avoid the smell of urine of Toxoplasma infected rats more than the smell of urine of Toxoplasma free rats. This effect was observed with both fresh and old urine samples, suggesting that non-volatile components of urine were responsible for the phenomenon.Authors used the correct experimental setup and correct techniques, including the statistical methods. I see just one potentially important problem, namely a rather common problem of pseudoreplications. Authors used urine sample mixtures originated from just two Toxoplasma infected rats and from two Toxoplasma free rats. Theoretically, existence of one abnormal urine sample among four samples could give a false positive result of a study. To avoid the problem of pseudoreplications, authors should use urine sample from different rat for each mouse.It would be interesting to test also the reaction of mice on some Toxoplasma infected and Toxoplasma free members of some non-predator species. Without doing this it could not be decided whether the mice avoided the smellier predator or the parasite. (Of course, the former explanation seems to be more probable.)",
"responses": []
}
] | 1
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https://f1000research.com/articles/3-92
|
https://f1000research.com/articles/2-289/v1
|
30 Dec 13
|
{
"type": "Research Article",
"title": "Altered functional connectivity in posttraumatic stress disorder with versus without comorbid major depressive disorder: a resting state fMRI study",
"authors": [
"Mitzy Kennis",
"Arthur R. Rademaker",
"Sanne J.H. van Rooij",
"René S. Kahn",
"Elbert Geuze",
"Arthur R. Rademaker",
"Sanne J.H. van Rooij",
"René S. Kahn",
"Elbert Geuze"
],
"abstract": "Posttraumatic stress disorder (PTSD) is an anxiety disorder that is often diagnosed with comorbid depressive disorder. Therefore, neuroimaging studies investigating PTSD typically include both patients with and without comorbid depression. Differences in activity of the anterior cingulate cortex (ACC) and insula have been shown to differentiate PTSD patients with and without major depressive disorder (MDD). Whether or not comorbid MDD affects resting state functional connectivity of PTSD patients has not been investigated to our knowledge. Here, resting state functional connectivity of PTSD patients with (PTSD+MDD; n=27) and without (PTSD-MDD; n=23) comorbid MDD was investigated. The subgenual ACC and insula were investigated as seed regions. Connectivity between the subgenual ACC and perigenual parts of the ACC was increased in PTSD+MDD versus PTSD-MDD. Reduced functional connectivity of the subgenual ACC with the thalamus was found in the PTSD+MDD group versus the PTSD-MDD group. These results remained significant after controlling for PTSD severity. In addition, the PTSD+MDD group showed reduced functional connectivity of the insula with the hippocampus compared to the PTSD-MDD group. However, this cluster was no longer significantly different when controlling for PTSD severity. Thus, resting state functional connectivity of the subgenual ACC may distinguish PTSD+MDD from PTSD-MDD. As PTSD patients with comorbid MDD are more treatment resistant, this result may be important for treatment development.",
"keywords": [
"posttraumatic stress disorder",
"PTSD",
"major depressive disorder",
"MDD",
"insula",
"ACC",
"resting state",
"functional connectivity",
"comorbidity",
"fMRI"
],
"content": "Introduction\n\nPosttraumatic stress disorder (PTSD) is an anxiety disorder that can develop after a traumatic event. It is characterized by re-experiencing the traumatic event, avoidance of trauma reminders and emotional numbing symptoms, and increased arousal1. PTSD frequently co-occurs with other Axis I psychiatric disorders, such as major depressive disorder (MDD2). Patients with both PTSD and depression were found to have more psychological distress and are also more treatment resistant than patients with PTSD or depression alone3–5. About 48% of PTSD patients were found to have comorbid MDD in a large national survey in the United States2. Therefore, studies investigating the neurobiology of PTSD often comprise patients with and without comorbid MDD. Neuroimaging studies have demonstrated dysfunction of similar brain regions in both PTSD and MDD. That is, PTSD and MDD are both associated with alterations in structure and function of the medial prefrontal cortex (mPFC), amygdala, insula, and anterior cingulate cortex (ACC6–8). To what extent comorbid MDD contributes to the reported neurobiological alterations of PTSD is yet to be determined.\n\nThus far, two neuroimaging studies have directly investigated differences in PTSD patients with and without comorbid MDD. First, reduced activity of the mPFC and amygdala was found in PTSD patients with comorbid MDD versus PTSD patients without MDD, when fearful faces were presented9. Second, during a symptom provocation paradigm PTSD patients with comorbid MDD had decreased activity in the insula, and increased ACC and posterior cingulate cortex (PCC) activation versus PTSD patients without MDD10. In addition, decreased insula activation remained significant after controlling for PTSD severity. One other study has investigated the effects of depressive symptoms in PTSD patients. A positive correlation between depressive symptoms and (para) hippocampal and ventral ACC activity during an emotional memory task was observed in PTSD patients11. A fourth fMRI study involving PTSD patients versus both controls and MDD patients found increased activity in several brain areas of PTSD patients including the insula when emotional pictures were presented12.\n\nHowever, these four studies were limited by small sample sizes (8 PTSD-MDD, 8 PTSD+MDD9, 11 PTSD-MDD and 15 PTSD+MDD10, 21 PTSD+MDD and 12 PTSD-MDD11, 16 PTSD and 16 MDD12). In addition, these studies investigated neurobiological alterations during emotional tasks, potentially inducing PTSD (and/or depressive) symptoms. It is expected that PTSD and/or MDD symptom provocation induces an altered state in PTSD with or without MDD, which is reflected by alterations in brain activity. Whether regular functioning of the brain in the absence of symptom-inducing stimuli deviates in PTSD with versus without comorbid MDD remains unclear. To our knowledge, functioning of the brain during resting state, without presenting stimuli or requiring task performance, has not been investigated in PTSD patients with and without comorbid MDD. Thus, the effect of comorbid MDD on brain functioning at baseline of PTSD patients deserves further investigation.\n\nHere, we investigate the effects of comorbid MDD on resting state functional connectivity in PTSD patients. Since the studies described above indicated that functioning of the ACC distinguishes PTSD with and without MDD during emotional tasks9–11, this brain area was chosen as a region of interest. MDD has been associated with alterations in structure13, function14, structural connectivity15, and reduced resting state functional connectivity16–18 of the subgenual ACC in particular, which is a subdivision of the ventral ACC. In addition, subgenual ACC activation and cortical thickness have been associated with symptom improvement in PTSD19,20. Therefore, the subgenual ACC was selected as a more specific region of interest. Second, alterations in activation of the insula also differed between PTSD patients with and without PTSD, even when controlling for PTSD severity10. Furthermore, insula activation distinguished PTSD patients from MDD patients12. Thus, the insula was chosen as a second region of interest. As increased ACC activity was found in PTSD with comorbid MDD, as well as a positive correlation of ACC activity with depressive symptoms, we hypothesize that functional connectivity of the subgenual ACC is increased in PTSD with versus without comorbid MDD. Since insula activity is increased in PTSD versus MDD and insula activity was reduced in PTSD with comorbid MDD versus PTSD without MDD, we expected to find lower insula functional connectivity in PTSD with MDD as compared to PTSD without MDD. In summary, in order to provide more insights into the potential effects of MDD on the neurobiology of PTSD, the present study examined the effects of comorbid MDD on subgenual ACC and insula resting state functional connectivity in PTSD patients.\n\n\nMethods\n\nIn total, 30 male veterans with PTSD with comorbid MDD (PTSD+MDD, mean age 34.2 ± 8.5), and 25 male veterans with PTSD without comorbid MDD (PTSD-MDD, mean age 37.4 ± 10.1) were included in this study. All patients were recruited from the Military Mental Health Care Center, the Netherlands. PTSD and MDD diagnoses were confirmed using the Clinician Administered PTSD scale (CAPS21) and the Structural Clinical interview for DSM-IV (SCID22). Several patients were medication naive (PTSD+MDD; n=13, PTSD-MDD; n=10), some patients were currently taking antidepressants (e.g. selective serotonin reuptake inhibitors; PTSD+MDD; n=6, PTSD-MDD; n=6), and some patients used benzodiazepines (PTSD+MDD; n=5, PTSD-MDD; n=2), or both antidepressants and benzodiazepines (PTSD+MDD; n=1, PTSD-MDD; n=3). One patient from the PTSD+MDD group used both antipsychotics and benzodiazepines. Most of the veterans had been deployed to Afghanistan (n=28) and to the former Yugoslavia (n=10). After receiving a complete written and verbal description of the study, all participants gave informed consent. Participants received financial compensation for their participation. The Medical Ethical Committee of the UMC Utrecht approved the study (protocol number NL29550.041.09), and the study was performed in accordance with the Declaration of Helsinki23.\n\nFunctional and structural images were obtained using a 3.0 Tesla magnetic resonance imaging scanner (Philips Medical System, Best, the Netherlands). Before the resting state scan, a ten minute T1-weighted high-resolution image (TR = 10 ms TE = 4.6 ms flip angle 8, 200 slices sagittal orientation, FOV 240 × 240 × 160, 304 × 299 matrix) was acquired. This image was utilized for co-registration and segmentation purposes and also allowed the participants to adapt to the scanner environment. During the nine minute resting state scan participants were asked to relax, to let their mind wander and to focus on a fixation cross. Three hundred and twenty T2* echoplanar interleaved images were collected (TR = 1600 ms, TE = 23 ms, flip angle = 72.5°, 30 transverse slices, FOV 256 × 208 × 120, 64 × 51 matrix).\n\nPre-processing was conducted with SPM5 (http://www.fil.ion.ucl.ac.uk/spm/software/spm5/), which included slice-timing correction, realignment, co-registration with the anatomical scan, normalization, and spatial smoothing (8 mm FWHM). Five participants (2 PTSD+MDD, 3 PTSD-MDD) were excluded due to excessive motion (more than 2 mm displacement in any direction (x, y or z) or 2 degrees rotation (pitch, roll or yaw)).\n\nThe Data Processing Assistant for Resting-State fMRI (DPARSF) was utilized for further analyses (restfmri.net24), which is based on MRIcroN (http://www.mricro.com), SPM5 (http://www.fil.ion.ucl.ac.uk/spm/software/spm5/), and the Resting-State fMRI Data Analysis Toolkit24. Resting state images were band-pass filtered (0.08-0.01 Hz) to reduce low-frequency drift and high-frequency noise, and detrended to correct for general signal drift. In order to correct for physiological processes and motion, the motion parameters from the realignment step, mean global signal, white matter signal, and cerebral spinal fluid signal were included as covariates in the analysis. In addition, motion scrubbing was applied to scans that surrounded a minimum of 0.5 mm frame displacement (one scan before displacement, two scans after displacement), using nearest neighbour interpolation25. A minimum of approximately 5 minutes of resting state (183 unscrubbed resting state images) was set as a required threshold for correct scrubbing. One participant was excluded due to excessive scrubbing, resulting in the following groups: 27 PTSD+MDD, and 22 PTSD-MDD.\n\nFor the subgenual ACC two spherical seeds (left and right, 3.5 mm radius) were created around two seed point coordinates, as previously described by Kelly et al. (2009)26. The anterior insula seed was created from two distinct anterior insula subdivisions that were described as the insula regions involved in emotion and cognition, as reported by Kelly et al. (2012)27. The mean time series for each of those seeds was extracted for all individuals and correlated with the time series of every voxel in the brain in order to create functional connectivity maps. These correlation maps were normalized using Fishers z-transform, resulting in a z-map for each ACC network per participant. The individual z-maps were used for second-level group analysis (full factorial design, SPM). A general effect of group (F-test) was investigated to determine group differences within the positive and negative network of the seed pairs.\n\nCluster-level multiple comparison correction was applied according to Gaussian Random Field theory28. A height threshold of p<0.001 was applied and combined with an extended cluster threshold of k>11, that corresponds to corrected p<0.05 (as determined with 1000 Monte Carlo simulations using Alphasim, implemented in the REST toolbox; FWHM 8 mm, cluster connection radius 7 mm).\n\nPost-hoc analyses were performed including the total CAPS score as a covariate, in order to assess whether the results were due to differences in PTSD severity (F-test, height threshold p<0.001 extended threshold k>11, resulting in false discovery rate (FDR) corrected p<0.05). In addition, the positive affect (PA) score from the mood and anxiety questionnaire (MASQ29), which has been reported to reflect a core feature of MDD30, was also investigated as covariate. In addition, functional connectivity values (z-values) were extracted from the peak voxels of clusters of significant differences in order to perform post-hoc correlations with PTSD and MDD symptoms. Total CAPS scores, CAPS symptom cluster B, C, and D scores, as well as MASQ PA scores were investigated. Correlations between whole brain functional connectivity and CAPS and PA scores were calculated respectively.\n\n\nResults\n\nGroups did not differ significantly in age, handedness, the number of times they were deployed, the time since their last deployment, and educational level as measured with the international standard classification of education (ISCED;31). The PTSD+MDD group differed from the PTSD-MDD group in total PTSD severity (CAPS score; p=0.008), which appeared to be largely driven by differences in avoidance and emotional numbing symptom scores (cluster C; p=0.001). In addition, the PTSD+MDD group had lower PA scores versus the PTSD-MDD group (p=0.012), while negative affect and somatic anxiety did not differ between groups. In the PTSD+MDD group 10 patients were diagnosed with a comorbid anxiety disorder (n=10), and one patient had a comorbid somatoform disorder. In the PTSD-MDD group seven patients met the current diagnostic criteria for a comorbid anxiety disorder, one patient had a somatoform disorder only, and one patient was diagnosed with both a comorbid anxiety and somatoform disorder. An overview of demographical and clinical data is presented in Table 1.\n\n*Significant differences between groups; p<0.05\n\nSpatial connectivity maps. Figure 1 shows the positive and negative networks for the bilateral insula and the bilateral subgenual ACC. Positive functional connectivity of the subgenual ACC was found with the ventromedial PFC, temporal regions (including the hippocampus) and a posterior cluster comprising the PCC/precuneus. Positive functional connectivity of the insula was found around the insular lobe, extending into the temporal and parietal lobe. A medial cluster around the dorsal ACC showed positive functional connectivity with the insula.\n\nFunctional connectivity of the subgenual ACC (a), and insula (b) seeds. Positive connectivity is represented in red-yellow and negative connectivity in blue-green. The effects were FDR corrected p<0.001 for illustrative purposes.\n\nSubgenual ACC. Reduced functional connectivity of the PTSD+MDD group versus the PTSD-MDD group was found in functional connectivity of the subgenual ACC with the bilateral thalamus (Left thalamus; 29 voxels; peak value F=25.71; peak MNI-coordinates x=-12, y=-16, z=4. Right thalamus; 16 voxels; peak value F=34.37; peak MNI-coordinates x=20, y=-12, z=4). Increased functional connectivity was found between the subgenual ACC and perigenual regions of the ACC (peak in left perigenual ACC; 100 voxels; peak value F=25.71; peak MNI-coordinates x=-12, y=40, z=-4) in the PTSD+MDD group versus the PTSD-MDD group (see Figure 2, Figure 3 and Table 2).\n\nClusters of significant different functional connectivity of the insula (a) and subgenual ACC (b) seeds. Increased functional connectivity in PTSD+MDD versus PTSD-MDD is shown in red and reduced connectivity in blue (height threshold p<0.001, extended threshold k=11, resulting in FDR corrected p<0.05).\n\nZ-values of the peak voxels for the PTSD-MDD group (red) and the PTSD+MDD (blue) group are presented.\n\nInsula. Functional connectivity of the bilateral insula with the left hippocampus (17 voxels; peak value F=19.05; peak MNI-coordinates x=-28, y=-32, z=-8) was reduced in the PTSD+MDD group as compared to the PTSD-MDD group, which showed no functional connectivity between these regions (see Figure 2, Figure 3, and Table 2).\n\nBecause of group differences in PTSD severity, we repeated the analyses after including total CAPS scores as a covariate. This did not affect significance of the subgenual ACC connectivity differences in the bilateral thalamus (cluster in left thalamus 12 voxels (p=0.022) and right thalamus 15 voxels (p=0.005)) and perigenual ACC (cluster increased to 124 voxels). However, the cluster of altered connectivity between insula and hippocampus was no longer significant after controlling for total CAPS scores (cluster size reduced to 4 voxels (p=0.813)).\n\nPost-hoc correlation analyses of the peak voxel of significant difference with CAPS total, CAPS symptom cluster, and PA scores were performed within both groups separately. CAPS cluster C scores correlated negatively with connectivity of the subgenual ACC with the peak voxel of significant difference in the left thalamus (r = -0.523, p=0.012) within the PTSD-MDD group. Within the PTSD+MDD group CAPS cluster B scores correlated negatively with connectivity of the subgenual ACC with the peak voxel of significant difference in the perigenual ACC (r = -0.396, p=0.041). No correlations were found between CAPS cluster D scores or PA scores and the peak voxels of difference in subgenual ACC and insula connectivity.\n\nExploring the relation of whole brain subgenual ACC connectivity with CAPS and PA scores revealed a negative correlation of CAPS and PA scores with subgenual ACC-PCC/precuneus connectivity, amongst other regions (see Supplementary Figure S1). In addition, a negative correlation was found between CAPS and PA scores and negative functional connectivity of the insula with the PCC/precuneus (see Supplementary Figure S1).\n\n\nDiscussion\n\nThis study showed that resting state functional connectivity of the subgenual ACC and insula differs between PTSD patients with and without comorbid depressive disorder. PTSD+MDD patients had increased functional connectivity between the subgenual ACC and the left perigenual ACC compared to PTSD-MDD patients. Reduced functional connectivity was found in PTSD+MDD between the subgenual ACC and the bilateral thalamus, and between the insula and left hippocampus. The subgenual ACC results remained significant after controlling for PTSD severity. This study complements previous task-based studies9,10 by showing that differences in the subgenual ACC/insula between PTSD patients with and without comorbid MDD are also apparent using resting state functional connectivity.\n\nIncreased subgenual ACC connectivity with the perigenual ACC was found in PTSD+MDD versus PTSD-MDD, which is in line with neuroimaging studies in MDD that demonstrated specific alterations in these regions8,13. Furthermore, increased resting state functional connectivity between the subgenual ACC and perigenual ACC has been previously reported in MDD16–18, while reduced functional connectivity of the medial PFC, including the subgenual and perigenual ACC, has been shown in PTSD patients versus controls32. Our results complement these results, by showing increased connectivity of the subgenual ACC with the perigenual ACC in PTSD+MDD versus PTSD-MDD. In addition, the perigenual ACC has been related to self-referential processing33, which underlies depressive symptoms such as helplessness, self-reproach and guilt rumination17,34. During self-referential processing tasks a reduced negative blood oxygen level dependent (BOLD) response (thus an increase in BOLD signal) has been found in medial PFC regions, including the perigenual ACC, in MDD patients versus controls34–36. On the other hand, reduced medial PFC activation, including the perigenual ACC, has been observed in PTSD versus controls during a self-referential processing task37. It can thus be suggested that the increases in subgenual-perigenual ACC connectivity in the PTSD+MDD group versus the PTSD-MDD group reflect a difference in self-referential processing. A negative correlation between re-experiencing symptoms and functional connectivity of the subgenual ACC and perigenual ACC was also found within the PTSD+MDD group. This may suggest that when more specific PTSD symptoms (re-experiencing, cluster B) are prevalent, the balance of symptoms is tilted towards a PTSD only state, and the more functional connectivity resembles the PTSD-MDD group.\n\nThe perigenual ACC is part of the default mode network, which is the network that is active during rest and deactivated during task performance. Connectivity of the perigenual ACC with default mode network regions (PCC/precuneus and medial PFC) has been negatively correlated with general symptom severity in PTSD, even when correcting for depression diagnosis38 and depression severity39. This was confirmed in our post-hoc results, as we also found a negative correlation between total CAPS and PA scores and subgenual ACC connectivity with the PCC/precuneus. In addition, a negative correlation was found between total CAPS and PA scores and negative functional connectivity between the insula and PCC/precuneus (see Supplementary Figure S1). Specific correlations between CAPS scores and subgenual ACC-PCC/precuneus connectivity were also present, whilst controlling for PA scores. Connectivity in these regions did not differ between the PTSD+MDD and PTSD-MDD groups. Thus, it seems that subgenual ACC connectivity with the perigenual ACC in particular has the capacity to distinguish PTSD with and without comorbid MDD. The importance of differences in connectivity of the perigenual ACC in particular in MDD patients has been previously described for the default mode network, the affective network, and the salience network18. Instead, functional connectivity of other regions of the default mode network may be related to general (PTSD and MDD) symptom severity.\n\nPrevious fMRI studies including resting state paradigms, have reported decreased thalamus connectivity with the ACC in both depression40 and PTSD41 versus healthy controls. The thalamus is the relay station of the brain42, and can modulate attention and arousal43. The thalamus is also implicated as a target for surgical treatment of severe MDD44. In line with these findings we found that connectivity between the thalamus and subgenual ACC is more reduced in PTSD+MDD versus PTSD-MDD, suggesting that MDD effects add up to the PTSD effects in these regions. Furthermore, the results were not influenced by general PTSD severity, which may indicate that reduced thalamus connectivity is specific for comorbid MDD. In addition, functional connectivity between the subgenual ACC and thalamus was negatively correlated with cluster C symptoms in the PTSD-MDD group only. Thus, individuals with less avoidance and reduced interest, a shared PTSD and MDD symptom, have a stronger connection between the subgenual ACC and thalamus. A possible explanation of the correlation may be that the more the clinical image shifts toward more depressive symptoms, the more the connectivity pattern resembles PTSD+MDD. However, this interpretation needs further investigation, since cluster C symptoms both include avoidance and reduced interest symptoms.\n\nInsula connectivity with the hippocampus was reduced in the PTSD+MDD group versus PTSD-MDD. However, the cluster was no longer significantly associated with comorbid MDD when PTSD severity was added as a covariate. Thus, hippocampus-insula connectivity does not seem to be specifically related to depressive symptoms, but rather to PTSD severity. The hippocampus is a brain region that is often associated with PTSD7,45,46 and is involved in memory47. Therefore, differences found in connectivity between the insula and hippocampus may be related to trauma-related memory. Thus, alterations in connectivity between the insula and hippocampus between the PTSD+MDD and PTSD-MDD group may be due to differences in PTSD severity and not to comorbid MDD diagnosis per se.\n\n\nLimitations\n\nThis study has some limitations. First, no MDD only group was included in the current study. Thus, this study does not show whether subgenual ACC and insula connectivity differs from patients with MDD only. The results only give insights in the effects of comorbid MDD in the context of PTSD, and not on general effects of PTSD or MDD. Including this group in future research can provide more insights in PTSD, MDD, and their neurobiological overlap or differences. Second, both groups included participants that were currently using antidepressants. Further studies should investigate the effect of medication on the neurobiology of PTSD with or without MDD. Third, no validated measure of the severity of all MDD symptoms was included in the study. If MDD severity was measured, it would have been possible to determine common and distinct factors of PTSD symptom severity and MDD symptom severity by including both measures in a single model (as attempted in the Supplementary Figure S1). Here, MDD diagnosis was determined with the SCID, and depressive symptom severity were approximated with the positive affect scale of the MASQ, which is only representative of a subset of symptoms (reduced positive affect). Future studies should investigate the specific effect of MDD symptom severity in the occurrence of PTSD, measured with more sensitive and comprehensive instruments.\n\n\nConclusion\n\nThis study revealed differences between PTSD+MDD and PTSD-MDD in resting state functional connectivity of the subgenual ACC, even when controlling for PTSD severity. Increased functional connectivity of the subgenual ACC with the perigenual ACC and bilateral thalamus was found in the PTSD+MDD group versus the PTSD-MDD group. Functional connectivity of the left thalamus was negatively correlated with cluster C in the PTSD-MDD group. Differences in connectivity of the insula and hippocampus were also found, but this seemed to be related to PTSD severity and not to the presence of comorbid MDD per se. Unraveling the neurobiological features of MDD and PTSD during rest can provide insights in which specific brain areas could be targeted for effective treatments. For example, tasks or therapy methods that increase functional connectivity between the regions with dysfunctional connectivity may be effective. Future studies should investigate long-term effects of training that is associated with functional connectivity alterations. This is in particular relevant for treatment of PTSD patients with comorbid MDD, since patients with this combination of psychological problems tend to be more treatment resistant.",
"appendix": "Author contributions\n\n\n\nEG and AR have made a substantial contribution to the conception and design of the study. MK and SvR have made a substantial contribution to the acquisition of data. MK performed the analyses and prepared the first draft of the manuscript. EG, AR, SvR and RK were involved in the interpretation of the data, and critically reviewing the article. All authors have agreed to the final content of the article.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was funded by the Dutch Ministry of Defence.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nIn addition, we thank Jonathan van Leeuwen for his help with data acquisition and preprocessing.\n\n\nSupplementary material\n\nCorrelations of PTSD symptom severity and reduced positive affect with subgenual ACC (a, b) and insula (c, d) functional connectivity. Violet = positive correlation with both CAPS scores and reduced PA, cyan = negative correlations with both CAPS scores and reduced PA, red = positive correlations with CAPS scores, blue = negative correlations with CAPS, yellow = positive correlations with reduced PA, and green = negative correlation with reduced PA (p<0.001, k>11, resulting in FDR-corrected p<0.05).\n\n\nReferences\n\nAmerican Psychiatric Association. Diagnostic and statistical manual of mental disorders. Washington, Author 2000. Reference Source\n\nKessler RC, Sonnega A, Bromet E, et al.: Posttraumatic stress disorder in the national comorbidity survey. Arch Gen Psychiatry. 1995; 52(12): 1048–1060. PubMed Abstract | Publisher Full Text\n\nCampbell DG, Felker BL, Liu CF, et al.: Prevalence of depression-PTSD comorbidity: implications for clinical practice guidelines and primary care-based interventions. J Gen Intern Med. 2007; 22(6): 711–718. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChan D, Cheadle AD, Reiber G, et al.: Health care utilization and its costs for depressed veterans with and without comorbid PTSD symptoms. Psychiatric Serv. 2009; 60(12): 1612–1617. PubMed Abstract | Publisher Full Text\n\nMorina N, Ajdukovic D, Bogic M, et al.: Co-occurrence of major depressive episode and posttraumatic stress disorder among survivors of war: How is it different from either condition alone? J Clin Psychiatry. 2013; 74(3): e212–e218. PubMed Abstract | Publisher Full Text\n\nShin LM, Liberzon I: The neurocircuitry of fear, stress, and anxiety disorders. Neuropsychopharmacology. 2010; 35(1): 169–191. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPitman RK, Rasmusson AM, Koenen KC, et al.: Biological studies of post-traumatic stress disorder. Nat Rev Neurosci. 2012; 13(11): 769–787. PubMed Abstract | Publisher Full Text\n\nMayberg HS: Limbic-cortical dysregulation: A proposed model of depression. J Neuropsychiatry Clin Neurosci. 1997; 9(3): 471–481. PubMed Abstract\n\nKemp AH, Felmingham K, Das P, et al.: Influence of comorbid depression on fear in posttraumatic stress disorder: An fMRI study. Psychiatry Res. 2007; 155(3): 265–269. PubMed Abstract | Publisher Full Text\n\nLanius RA, Frewen PA, Girotti M, et al.: Neural correlates of trauma script-imagery in posttraumatic stress disorder with and without comorbid major depression: A functional MRI investigation. Psychiatry Res. 2007; 155(1): 45–56. PubMed Abstract | Publisher Full Text\n\nThomaes K, Dorrepaal E, Draijer N, et al.: Increased anterior cingulate cortex and hippocampus activation in Complex PTSD during encoding of negative words. Soc Cogn Affect Neurosci. 2013; 8(2): 190–200. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWhalley MG, Rugg MD, Smith APR, et al.: Incidental retrieval of emotional contexts in post-traumatic stress disorder and depression: An fMRI study. Brain Cogn. 2009; 69(1): 98–107. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDrevets WC, Savitz J, Trimble M: The subgenual anterior cingulate cortex in mood disorders. CNS Spectrums. 2008; 13(8): 663–681. PubMed Abstract | Free Full Text\n\nGotlib IH, Sivers H, Gabrieli JD, et al.: Subgenual anterior cingulate activation to valenced emotional stimuli in major depression. Neuroreport. 2005; 16(16): 1731–1734. PubMed Abstract | Publisher Full Text\n\nCullen KR, Klimes-Dougan B, Muetzel R: Altered white matter microstructure in adolescents with major depression: a preliminary study. J Am Acad Child Adolesc Psychiatry. 2010; 49(2): 173–183.e1. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGreicius MD, Flores BH, Menon V, et al.: Resting-State Functional Connectivity in Major Depression: Abnormally Increased Contributions from Subgenual Cingulate Cortex and Thalamus. Biol Psychiatry. 2007; 62(5): 429–437. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDavey CG, Harrison BJ, Yücel M, et al.: Regionally specific alterations in functional connectivity of the anterior cingulate cortex in major depressive disorder. Psychol Med. 2012; 42(10): 2071–2081. PubMed Abstract | Publisher Full Text\n\nSheline YI, Price JL, Yan Z, et al.: Resting-state functional MRI in depression unmasks increased connectivity between networks via the dorsal nexus. Proc Natl Acad Sci U S A. 2010; 107(24): 11020–11025. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDickie EW, Brunet A, Akerib V, et al.: Neural correlates of recovery from post-traumatic stress disorder: A longitudinal fMRI investigation of memory encoding. Neuropsychologia. 2011; 49(7): 1771–1778. PubMed Abstract | Publisher Full Text\n\nDickie EW, Brunet A, Akerib V, et al.: Anterior cingulate cortical thickness is a stable predictor of recovery from post-traumatic stress disorder. Psychol Med. 2013; 43(3): 645–653. PubMed Abstract | Publisher Full Text\n\nBlake DD, Weathers FW, Nagy LM, et al.: The development of a clinician-administered PTSD scale. J Trauma Stress. 1995; 8(1): 75–90. PubMed Abstract | Publisher Full Text\n\nFirst MB, Spitzer RL, Gibbon M, et al.: Structured Clinical Interview for DSM-IV Axis I Disorders1997. Reference Source\n\nWorld Medical Association. World Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects. J Int Bioethique. 2004; 15(1): 124–129. PubMed Abstract | Publisher Full Text\n\nSong XW, Dong ZY, Long XY, et al.: REST: a toolkit for resting-state functional magnetic resonance imaging data processing. PLoS ONE. 2011; 6(1): e25031. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPower JD, Barnes KA, Snyder AZ, et al.: Spurious but systematic correlations in functional connectivity MRI networks arise from subject motion. Neuroimage. 2012; 59(3): 2142–2154. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKelly AM, Di Martino A, Uddin LQ, et al.: Development of anterior cingulate functional connectivity from late childhood to early adulthood. Cerebral Cortex. 2009; 19(3): 640–657. PubMed Abstract | Publisher Full Text\n\nKelly C, Toro R, Di Martino A, et al.: A convergent functional architecture of the insula emerges across imaging modalities. Neuroimage. 2012; 61(4): 1129–1142. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCox RW: AFNI: Software for analysis and visualization of functional magnetic resonance neuroimages. Comput Biomed Res. 1996; 29(3): 162–173. PubMed Abstract | Publisher Full Text\n\nWatson D, Clark LA: The mood and anxiety symptom questionnaire. The Mood and Anxiety Symptom Questionnaire 1991.\n\nde Beurs E, den Hollander-Gijsman ME, Helmich S, et al.: The tripartite model for assessing symptoms of anxiety and depression: Psychometrics of the Dutch version of the mood and anxiety symptoms questionnaire. Behav Res Ther. 2007; 45(7): 1609–1617. PubMed Abstract | Publisher Full Text\n\nSchneider SL: The international standard classification of education 2011. Comp Soc Res. 2013; 30: 365–379. Publisher Full Text\n\nBluhm R, Williamson P, Lanius R, et al.: Resting state default-mode network connectivity in early depression using a seed region-of-interest analysis: Decreased connectivity with caudate nucleus. Psychiatry Clin Neurosci. 2009; 63(6): 754–761. PubMed Abstract | Publisher Full Text\n\nAmodio DM, Frith CD: Meeting of minds: The medial frontal cortex and social cognition. Nat Rev Neurosci. 2006; 7(4): 268–277. PubMed Abstract | Publisher Full Text\n\nLemogne C, Delaveau P, Freton M, et al.: Medial prefrontal cortex and the self in major depression. J Affect Disord. 2012; 136(1–2): e1–e11. PubMed Abstract | Publisher Full Text\n\nGrimm S, Ernst J, Boesiger P, et al.: Reduced negative BOLD responses in the default-mode network and increased self-focus in depression. World J Biol Psychiatry. 2011; 12(8): 627–637. PubMed Abstract | Publisher Full Text\n\nJohnson MK, Nolen-Hoeksema S, Mitchell KJ, et al.: Medial cortex activity, self-reflection and depression. Soc Cogn Affect Neurosci. 2009; 4(4): 313–327. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBluhm RL, Frewen PA, Coupland NC, et al.: Neural correlates of self-reflection in post-traumatic stress disorder. Acta Psychiatr Scand. 2012; 125(3): 238–246. PubMed Abstract | Publisher Full Text\n\nLanius RA, Bluhm RL, Coupland NJ, et al.: Default mode network connectivity as a predictor of post-traumatic stress disorder symptom severity in acutely traumatized subjects. Acta Psychiatr Scand. 2010; 121(1): 33–40. PubMed Abstract | Publisher Full Text\n\nYoshimura S, Okamoto Y, Onoda K, et al.: Rostral anterior cingulate cortex activity mediates the relationship between the depressive symptoms and the medial prefrontal cortex activity. J Affect Disord. 2010; 122(1–2): 76–85. PubMed Abstract | Publisher Full Text\n\nAnand A, Li Y, Wang Y, et al.: Activity and connectivity of brain mood regulating circuit in depression: A functional magnetic resonance study. Biol Psychiatry. 2005; 57(10): 1079–1088. PubMed Abstract | Publisher Full Text\n\nYin Y, Jin C, Hu X, et al.: Altered resting-state functional connectivity of thalamus in earthquake-induced posttraumatic stress disorder: A functional magnetic resonance imaging study. Brain Res. 2011; 1411: 98–107. PubMed Abstract | Publisher Full Text\n\nSherman SM, Guillery RW: The role of the thalamus in the flow of information to the cortex. Philos Trans R Soc Lond B Biol Sci. 2002; 357(1428): 1695–1708. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPortas CM, Rees G, Howseman AM, et al.: A specific role for the thalamus in mediating the interaction of attention and arousal in humans. J Neurosci. 1998; 18(21): 8979–8989. PubMed Abstract\n\nVelasco F, Velasco M, Jiménez F, et al.: Neurobiological background for performing surgical intervention in the inferior thalamic peduncle for treatment of major depression disorders. Neurosurgery. 2005; 57(3): 439–448. PubMed Abstract | Publisher Full Text\n\nShin LM, Rauch SL, Pitman RK: Amygdala, medial prefrontal cortex, and hippocampal function in PTSD. Ann N Y Acad Sci. 2006; 1071: 67–79. PubMed Abstract | Publisher Full Text\n\nGeuze E, Vermetten E, Bremner JD: MR-based in vivo hippocampal volumetrics: 2. Findings in neuropsychiatric disorders. Mol Psychiatry. 2005; 10(2): 160–184. PubMed Abstract | Publisher Full Text\n\nSquire LR: Memory and the hippocampus: A synthesis from findings with rats, monkeys, and humans. Psychol Rev. 1992; 99(2): 195–231. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "2918",
"date": "30 Jan 2014",
"name": "Andrew Kemp",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review this interesting study on the impact of disorder comorbidity on resting state functional MRI. This is an important and under-studied area of research. I have a number of comments that may help to improve the manuscript.\n\nAbstract: The abstract is disappointing, dense and requires development. At present it is mostly comprised of statements relating to the findings obtained, rather than providing an interpretation of findings. Are all the findings reported key to the bottom-lines of the article? The authors need to add a brief interpretation after each of the KEY findings. What is the take home message; what are the bottom-lines? While the findings may indeed be “important for treatment development”, it is unclear how. Please elaborate. Introduction:The first paragraph would benefit from a little more context on why comorbidity is important. For instance, studies have demonstrated that comorbidity between the mood and anxiety disorders increases risk for CVD and mortality (Philips et al., 2009). I would recommend the authors integrate this information to help build study rationale. While the rationale for focusing on subgenual ACC as a seed region in connectivity analysis is well-founded, I thought the rationale for focusing on the insula to be much weaker. I would have thought that the amygdala region would have been a more appropriate second choice, especially given the many past studies that have focused on the amygdala as a region of interest in patients with PTSD.\n\nMethods:re Participants section: who administered the psychiatric evaluation; were they psychiatrists or research assistants? How many people conducted the evaluations? How was the reliability and validity of this measurement across assessors determined? If research assistants, how much training did they receive? What was the value of the financial compensation received by participants? re Data Acquisition: To what extent could this (incentivisation) have impacted on findings (i.e. group interaction) especially given the unrestricted participant instructions (i.e. relax and let the mind wander)? Was any questionnaire administered to assess mental state of participants during the resting state scan? Results:I recommend the authors to review the article by Miller & Chapman (2001) on the problems associated with ANCOVA when participants are not randomly allocated to a group, as in the present study. The use of total CAPS scores as a covariate in post-hoc analysis of covariance is problematic. One potential solution to this problem that the authors could consider is post-hoc correlational analysis. Although the authors ran a series of correlational analyses, these need to be conducted on all participants regardless of group, rather than by each group individually. The authors would need to determine the unique activations associated with the PTSD, with versus without depression, a contrast that is not associated with confounding variables (i.e. CAPS, positive affect). Another issue the authors need to consider is the impact of medication on their findings. While the authors note in their limitations that “future studies should investigate the effect of medication on the neurobiology of PTSD with or without MDD”, the authors have a sufficient sample of medication naive participants on which sub-analysis could be run. Error bars need to be added to Figure 3. Discussion:The authors link increases in subgenual-perigenual ACC connectivity in the PTSD+MDD group versus the PTSD-MDD group to differences in self-referential processing. This is an interesting point which I feel could be further elaborated (after of course confirming that these findings are still present after adjusting for confounds -see points I made regarding this issue under the results section). The same could be said for the discussion on the relationship between the disorders and the default mode network. I also feel the discussion section needs to be further developed. Currently, much of the discussion restates the findings without more detail on what the differences might mean (especially in the first paragraph of the discussion). This makes the discussion section a little underwhelming.",
"responses": [
{
"c_id": "760",
"date": "07 Apr 2014",
"name": "Mitzy Kennis",
"role": "Author Response",
"response": "We thank the reviewer for his valuable comments. See below our response to the comments per section of the manuscript. Abstract and introduction:We have attempted to make the abstract clearer, and added more context in both the abstract and introduction. We also acknowledge that the amygdala could have been another interesting seed to investigate PTSD with and without comorbid MDD. However, since the two previous neuroimaging studies that investigated PTSD and MDD with task paradigms reported alterations in the insula (Lanius et al., 2007; Whalley et al., 2009) while differences in the amygdala between PTSD and MDD patients were only reported by Whalley et al. (2009). Therefore, we argued that the insula is an appropriate candidate to investigate here. Methods:In order to address the concerns on the methods section, we provide more details on the administration of the interviews and financial compensation. Reliability and validity were qualitatively assessed by comparing interview scores and diagnosis between raters during interview training. With respect to the resting state design, we do not expect any effects of the financial incentive on the pattern of functional connectivity. Every participant received a similar financial compensation for participating in the study. We assessed mental states of participants during resting state after the scan session, by asking whether they felt relaxed during the rest scan. Most participants confirmed feeling relaxed during the resting state scan, and ten participants reported having some difficulties to relax (five in each group). Furthermore, we investigated co-activation patterns of the subgenual ACC and insula. Although mind states are of influence on brain activation it is not expected that different mind states (e.g. individuals that tend to think of the incentive) directly alter the spatial co-activation patterns within these functional networks. Results: We agree with the reviewer that the use of ANOVA applied here is inappropriate. However, the suggested solution, a correlation analysis with the cluster of significant differences over all participants, is also inappropriate, since groups differ on both these values (functional connectivity cluster peak and PTSD severity (total CAPS score)). Therefore misleading correlations can be induced (see Lord’s Paradox; Lord, 1967). Unfortunately, as Miller and Chapman (2001) have shown, there is no simple solution to “adjust” or “control” for differences between groups in non-randomized groups. On the other hand, we agree with the reviewer that correlations can be explored in order to provide some insight in the relation between functional connectivity and symptom severity, but then within groups rather than over all participants (to avoid misleading correlations). Thus, we have explored correlations within PTSD+MDD and PTSD-MDD groups between symptom severity scores (total CAPS and reduced PA) and the peak voxels of significant differences. There were no significant correlations between PTSD severity and functional connectivity. This suggests that functional connectivity of the clusters of significant differences was not related to differences in total CAPS or reduced PA scores. Additionally, for the correlations with the symptom clusters performed within groups (for the same reasons as described above), we plotted the data for all subjects and added a fitted line for the data for each group and for the whole group respectively (see Figure S2 and S3). This way, an objective overview of the data is presented and both whole group and subgroup correlations are visualized. Finally, as requested we performed a post-hoc analysis to investigate the effect of medication by including medication naive PTSD patients and PTSD patients that did not use benzodiazepines at least 48 hours prior to scanning (PTSD+MDD n = 20, PTSD-MDD n = 15). This revealed similar results for the subgenual ACC seed, although the hippocampal cluster of difference in connectivity with the insula disappeared. This information has been added to the paper. Figure 3 now includes error bars. Discussion:We expanded the discussion section and added more detail on the interpretation of the results. Furthermore, we have nuanced the interpretation of the correlational analyses."
}
]
},
{
"id": "2919",
"date": "26 Feb 2014",
"name": "Onno Meijer",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper shows convincing (to the technical non-specialist) differences in resting state functional connectivity between PTSD patients with or without comorbid major depression MDD.There are clear effects on connectivity of the subgenual anterior cingulate cortex with the left ACC (increased with MDD) and with the thalamus (decreased with MDD).The interpretations are hampered by the design, which ideally would be a 2 X 2, i.e. +/- MDD and +/- PTSD. Therefore sentences like 'we found that connectivity ... is more reduced in PTSD +MDD than in PTSD-MDD' cannot be made.The authors determined that effects on connectivity of insula and hippocampus are related to PTSD symptom severity - this should probably be indicated in figure 3 somehow, as this may end up being used as a graphic summary of the data.I missed whether the authors looked at the use of medication as a factor that affected connectivity, as it may interact with the condition.Second the authors, at correlation between connectivity (thalamus - sgACC) and symptom severity in the PTSD-MDD, group in a post hoc correlation. A similar effect is reported specifically for the PTSD+MDD group. It is unclear why the authors did not look at the correlation in all patients? What is the reason to now limit to a single group?Interpretation: I do not see how on p.6 the authors suggest the cluster B symptoms would tilt the balance towards a PTSD only state, based on correlation, when table 1 clearly shows no differences between the groups for cluster B symptoms.In terms of interpretation, I agree with the first reviewer that the statement in the abstract that the findings have impact on treatment development need to be substantiated. As markers? As targets? It also seems important whether or not PTSD and MDD interact in terms of connectivity, or whether the association of MDD and PTSD 'just' add up. For this a 2x2 design seems necessary, but the paper may benefit from actual meaning that is given to the data. Obviously, at present describing correlates is what is done in research like this, but an explicit sense of direction would be helpful to the non-specialist reader who is nevertheless interested in the subject matter.Minor remarks:I am not certain that (as in: I do not think that) the second, long, sentence in the discussion accurately reflects the findings, please check. The term however in the third paragraph of the intro seems needlessly negative about previous research.",
"responses": [
{
"c_id": "759",
"date": "07 Apr 2014",
"name": "Mitzy Kennis",
"role": "Author Response",
"response": "We thank the reviewer for his valuable suggestions. See below our response to the comments. Design and interpretations:We agree with the reviewer that our study is limited by design, and lacks an MDD only group and a healthy control group. We acknowledge that we can therefore not clearly interpret the effects described here, with respect to the nature of either one of these disorders or the interaction of the two. As the reviewer suggests, we have attempted to be more cautious with our interpretations and elaborated on the implications (sense of direction, specific implications for treatment development) of the results in the discussion and abstract. Analyses:As proposed, additional analyses on a subsample of medication naive PTSD patients and PTSD patients that did not use benzodiazepines at least 48 hours prior to scanning were performed to investigate the effect of medication. It was also questioned why we did not correlate the peak voxel of functional connectivity of the clusters of significant differences with all participants symptom scores, but only within groups. As also pointed out in our response to Andrew Kemp: a correlation analysis with the cluster of significant differences over all participants is inappropriate, since groups differ on both these values (functional connectivity cluster peak and PTSD severity (total CAPS score)) and therefore misleading correlations can be induced. To avoid this problem, within group correlations can be performed and seem more appropriate here. Finally, we have also added figures of the correlations with CAPS symptom clusters."
}
]
}
] | 1
|
https://f1000research.com/articles/2-289
|
https://f1000research.com/articles/3-89/v1
|
10 Apr 14
|
{
"type": "Clinical Practice Article",
"title": "Twice versus three-times weekly pulmonary rehabilitation in a real-life clinical setting",
"authors": [
"Keir Lewis",
"Lucy Thatcher",
"Carol-Anne Davies",
"Patricia Hodgson",
"Carol Llewellyn-Jones",
"Hayley Hutchings",
"Lucy Thatcher",
"Carol-Anne Davies",
"Patricia Hodgson",
"Carol Llewellyn-Jones",
"Hayley Hutchings"
],
"abstract": "Aim: Our aim was to compare a less intensive but longer pulmonary rehabilitation programme (PRP) against a more intensive but shorter PRP.Methods: We carried out an observational, cohort study in a real-life clinical setting in patients primarily with chronic obstructive pulmonary disease (COPD). We compared standard outcomes in patients who were receiving 18 sessions of PRP delivered twice weekly over 9 weeks (Group 1) against similar patients receiving an identical PRP delivered three times weekly over 6 weeks (Group 2). Outcome measures were the St. George’s Respiratory Questionnaire (SGRQ), the Incremental Shuttle Walk Test (ISWT) and the number of hospital bed-days pre- and post-PRP.Results: Both groups showed statistically significant and clinically important improvements post-PRP. The largest effects were seen immediately post-PRP and waned over the following 12 months. Group 1 showed a larger improvement in ISWT immediately post-PRP (Group 1, +92 m versus Group 2 +64 m (p=0.001), but there were no differences between groups at 6 (p=0.67) or 12 months (p=0.96). There were no differences in SGRQ between groups immediately post-PRP (p=0.09) or at 12 months (p=0.78). There were no differences between groups in the number of hospital days 12 months prior to PRP versus 12 months post-PRP (p=0.18).Conclusion: Twice weekly outpatient, multidisciplinary PRP over 9 weeks is as effective as three times weekly PRP over 6 weeks.",
"keywords": [
"Pulmonary",
"rehabilitation",
"frequency",
"duration"
],
"content": "Introduction\n\nRandomised trials have demonstrated that Pulmonary Rehabilitation Programs (PRPs) for patients with chronic obstructive pulmonary disease (COPD) can improve dyspnoea, exercise tolerance, health-related quality of life (QoL), and reduce the number of hospital days and the utilization of healthcare resources. Research has demonstrated that PRPs are cost-effective and as such they are now recommended to all patients, who remain breathless despite optimal bronchodilators, irrespective of severity and age1–4. In addition, evidence is mounting for the efficacy of PRPs in patients with non-COPD causes of pulmonary impairment5.\n\nAs evidence accumulates, there are now specific recommendations for PRPs regarding patient selection, timing (in relation to exacerbations etc.), intensity and type of exercises, educational, psychological and behavioural components, oxygen supplementation, what outcomes to measure and total duration3–7. However, there still remain unanswered questions including what are the best sites for delivery of a PRP, whether nutritional supplementation should be offered during PRP, what are optimal target populations and what post-rehabilitation maintenance strategies reduce the declines seen in nearly all settings7–9. Other important concerns relate to variable attendance and high drop-out rates in PRPs10,11. We have shown that certain factors at enrolment can predict later poor attendance at PRP10, but interventions to improve the completion rates of these more vulnerable groups are still being researched.\n\nDespite a general improvement in the provision of PRPs since 2003, there remains poor access to PRPs in some parts of the United Kingdom (UK). Those hospitals/community services that provide PRPs describe variable content and staffing and many report long waiting times12. However, there is less evidence for other chronic care programs for COPD such as Home Exercise or Hospital at Home13. In the current financial climate, reduced resources are available for developing novel strategies for COPD care so optimisation and evolution of those existing services with a good evidence base should be explored.\n\nComparable efficacy from a longer but less intensive PRPs may allow more flexible working for staff (e.g. part-time) to deliver an effective PRP or allow existing full-time staff to offer more than one PRP simultaneously, for example on different weekdays in different sites to improve local access, increase throughput and reduce waiting times.\n\nWe present immediate and one year outcomes from a real-life clinical setting comparing the delivery of identical 18-session PRPs, delivered by the same staff, simultaneously across two similar nearby hospitals. This PRP occurred three times per week over 6 weeks or twice per week over 9 weeks within the same Health Board (group of hospitals).\n\n\nMethods\n\nWe sought advice regarding whether ethical approval was required for the study. As this was a retrospective review of prospectively collected routine clinical data (service evaluation) ethics approval was not deemed necessary.\n\nPatients were referred from primary or secondary care onto the PRP. A respiratory physician first checked and optimised treatment and excluded those with dementia, unstable cardiac disease or unwillingness to commit to a program. We include data on all those who attended for pre-PRP assessment (consisting of a brief interview and description of the service) by our physiotherapist and occupational therapist.\n\nOur PRP was closely developed from our mentor’s model that has a strong evidence base14,15. It consists of 18 sessions of outpatient multidisciplinary input from occupational therapists, physiotherapists (including a teacher of the Chronic Respiratory Disease Exercise instructor course, endorsed by Loughborough College and British Lung Foundation), dietetics staff, pharmacist, physicians, specialist respiratory nurses and a smoking cessation counsellor.\n\nEach session lasted for approximately 2.5 hours starting with supervised, individual exercise prescription of lower extremity training (treadmill, step-ups) and upper extremity training (resistance bands and loose weights). This was followed by group educational activities addressing the causes and types of lung disease and psychological aspects of chronic disability. Individual goal setting, dietary interventions, physiotherapy and occupational therapy were also included with voluntary sessions of relaxation classes and breathing retraining exercises where attention was paid to emotional and social as well as physical aspects of health. Very occasional provision was made for additional sessions following non-attendance or further deterioration e.g. two people were awaiting lung transplant.\n\nCompleters (defined as attending 12 or more from 18 sessions) were referred to a follow-up component of self-exercise free classes in gyms in local leisure centres. All participants were given a pack at enrolment emphasising regular exercise with personalised goals and information on COPD.\n\nIn our health board, we split our daily PRP over 5 days per week between two similar medium-sized hospitals (25 miles apart). One hospital, which serves an urban (ex-industrial) and semi-rural area, offered 18 sessions of PRP, three times weekly over 6 weeks (Monday, Wednesday and Friday afternoons). The other hospital is based in market town serving a rural area; because of greater distances involved in travelling, it offered the identical PRP of 18 sessions but twice weekly over 9 weeks (Tuesday and Thursday afternoons). Both groups were encouraged to exercise at home and the patients receiving PRP twice weekly were asked to try an independent home exercise session on a third day each week, replicating the hospital exercises where possible. The content, staff members and time of day of the PRPs were identical. This was thus a naturalised nested, cohort study allowing a unique opportunity to compare two intensities of identical PRPs.\n\nSubjects completed the SGRQ and Medical Research Council (MRC) dyspnoea scores (under supervision) before completing a baseline ISWT16 1 week prior to PRP. They completed the same QoL survey and the ISWT immediately post-PRP and then 6 and 12 months later.\n\nInformation regarding admissions was extracted from our hospital database and all data for publication has all been anonymised and approved by our Hospital Caldicott Guardian for export.\n\nWe used the statistical package for social sciences (SPSS), version 21.0 (Chicago, Illinois). Data are expressed by means and standard deviations. Analysis of the results was by intention to treat (ITT). Between-group comparisons were carried out at each time point using unpaired samples t-tests, Mann-Whitney and Chi square. Within-group changes were assessed using paired samples t-tests and Wilcoxon rank tests. A p value of less than 0.05 was deemed statistically significant.\n\n\nResults\n\na) The overall PRP results are described below:\n\n244 patients entered PRP between March 2006 and September 2008.\n\n74% had purely COPD and 26% were disabled by lung disease primarily from other conditions including idiopathic pulmonary fibrosis (5%), bronchiectasis (11%), chronic asthma (7%), kyphoscoliosis (1%) or other respiratory conditions (2%).\n\n48% were male; overall the subjects had a mean age of 66.0±9.5 years, and the mean forced expiratory volume in 1 second (FEV1) recorded from clinics prior to PRP, was 46±19% predicted.\n\nThe median (inter-quartile range) attendance was 13 (7, 16) from a maximum 18 sessions with 56% of patients completing 12 or more sessions.\n\nComplete data on ISWT and QoL scores were available for 223 people at baseline, 161 people post-PRP, 89 people at 6 months and 53 people at 12 months.\n\nThe mean ISWT improved from 155±123 metres immediately pre-PRP to 241±152 metres immediately post-PRP (p<0.001). This had fallen back slightly to 199±146 metres at 6 months (p<0.01 from baseline) and 168±170 metres at 12 months (p<0.05 from baseline).\n\nThe total SGRQ at baseline was 63.1±15.6, immediately post-PRP was 53.6±15.5 (p<0.001), at 6 months was 56.7±15.6 (p<0.001 from baseline) at 12 months was 56.6±16.8 (p=0.07 from baseline).\n\nThe mean number of admissions 6 months prior to PRP was 0.69±1.20 and in the 6 months following PRP was 0.39±0.94 (p<0.001).\n\nThe mean number of days spent in hospital 6 months prior to PRP was 5.54±15.20 and in the 6 months following PRP was 2.70±8.11 (p<0.001).\n\nThe mean number of admissions 12 months prior to PRP was 1.05±1.60 and in the 12 months following PRP was 1.11±1.55 (p=0.66).\n\nThe mean number of days spent in hospital 12 months prior to PRP was 7.35±16.77 and in the 12 months following PRP was 8.63±19.23 (p=0.55).\n\nb) Comparison between twice weekly versus three times weekly PRPs:\n\nTable 1 describes the patients enrolled in the two separate PRPs at baseline. Apart from the ISWT, the two groups were similar in baseline characteristics.\n\n* p<0.05\n\nTable 2 compares the changes from baseline to 6 and 12 months in both groups. There was a significant difference in the ISWT post-PRP between the two groups but this was no longer apparent 6 or 12 months post-PRP. This may be due to the significantly lower mean ISWT at baseline. There was a significant difference in the SGRQ scores post-PRP, but again this was not apparent at the later time points. There were no differences in the number of hospital days pre- and post-PRP between the two PRP groups.\n\n\nDiscussion\n\nThis is the first study to report immediate and medium term outcomes from two PRPs that were identical apart from the intensity of sessions. We found statistically significant and clinically important improvements in ISWT and QoL immediately following both PRPs. These effects were waning but still apparent at 6 months. At 12 months there was an overall mean change of +13 metres in ISWT which, although statistically significant, is unlikely to be clinically important. The mean improvement in total SGRQ of around 7 units from baseline to 12 months, however, is still likely to be clinically important but did not reach statistical significance (p=0.07) and could have occurred through chance and survivor selection bias. There were reductions in the number of hospital admissions and number of days spent in hospital within both PRP groups and as a population overall, comparing the 6-month periods immediately before and after PRP, but no differences in the number of admissions and days in hospital at 12 months. In Griffiths’ landmark randomized controlled trial of a PRP with identical staffing and content to ours, occurring three times weekly, there were similar improvements in SGRQ, ISWT and days in hospital (but not number of admissions) in those receiving PRP in addition to usual care14. These differences remained statistically different from a control group of people with similar COPD, at 12 months could be at least partly due to deterioration due to progressive COPD in the usual care group. We had no such ‘usual care’ group like Griffith’s group in his randomised controlled trial as not offering PRP’s would now be considered unethical. We compared our 12-month outcomes within the same patients one year before, when they were younger and so their disease (and co-morbidity) was probably less advanced. Showing equivalent ISWT and health care utilisation over 1 year, at least shows a halting or stabilising of what has been traditionally labelled a ‘chronic progressive irreversible lung disease’ (goldcopd.org).\n\nOur patient outcomes are similar to others (that came mainly from more established academic units)6,8,14,15,18 and this helps to validate our PRP by confirming benefits within an everyday clinical setting. This indicates that PRPs should be offered in a non-teaching group of hospitals. Our outcomes are also comparable or superior to other ‘before-and-after’ studies in real-life service evaluations. For example, O'Neill et al. reported improvements in mean ISWT of around 23 metres immediately after completing a supervised, outpatient PRP over 6 weeks, but their PRP was only once weekly and only reported on 74 patients. Like us, their effects were still apparent but waning at 6 months but not reported at 12 months16.\n\nThe comparison between twice versus three times weekly PRPs revealed subtle changes. Interestingly, the less intensive, twice weekly group showed greater improvement in ISWT immediately post-PRP but the groups were not completely matched and this could be confounded by their greater exercise tolerance (higher ISWT) and better disease specific quality of life (lower SGRQ) at baseline. It is easy to suggest that the patients could exercise more at home between supervised hospital sessions so could achieve bigger overall gains in exercise performance, during their hospital attendances.\n\nWe had a mixture of people with lung disease and some with very severe airflow obstruction. Garrod et al. reported less favourable outcomes in ISWT for those with more severe disease (higher MRC dyspnoea scores), possibly because they attended PRP less frequently17. Our actual percentage change in ISWT from baseline was similar in the twice versus three times weekly PRP (6% versus 7% improvement); moreover any early differences in ISWT between the twice versus three times weekly PRP groups were no longer apparent at 6 and 12 months.\n\nBoth groups had statistically significant and clinically important improvements in SGRQ immediately following PRP. Those receiving three times weekly PRP showed a larger change and this difference could be clinically important as a difference of 4 units in the SGRQ is traditionally deemed a clinically important outcome (Meguro 2006). There was a -9.7 change in three times weekly vs -5.6 change in twice weekly so difference of 4.1 units between groups in their changes), although this clinically important change could still have occurred through chance (p=0.09). This greater early improvement in QoL may be explained by the more intensive program but is confounded by the poorer QoL at baseline and is easily explained by statistical chance. The difference between groups in SGRQ at 6 months (+0.1 from baseline in twice weekly versus -5.6 in the three times weekly) did suggest ongoing benefit only for the latter group versus an earlier return to baseline in the less intensive group. However, there were no differences between the two groups in any outcome at 12 months, and the difference in SGRQ at 6 months could be due to a statistical aberration (multiple comparisons) as this single reading went against all the other outcome trends.\n\nFew others have compared more versus less frequent sessions in PRPs. Green et al. suggested initially that a 7 week course provided greater early benefits in health status than a similarly intensive but shorter 4 week course18. The same group then later reported that a 4 week supervised PRP with similar educational content is equivalent to a 7 week supervised PRP in clinical outcomes both at 7 weeks and 6 months19.\n\nWe have previously reported that a longer PRP was associated with lower attendance10. Possible reasons could be that participants notice less incremental change and that there is more time for intercurrent illness/exacerbations or other activities to interfere with attendance. This was based on an internal study comparing three sessions over 6 weeks versus one session over 18 weeks and was entered along with other attendance predictors in a multi-regression model. The original randomised controlled trial was never completed due to the death of the lead investigator. Marciniuk et al. suggested the opposite, i.e. that longer PRPs (beyond 6 to 8 weeks) better maintain health gains7 and Rossi et al. reported that a ten session PRP provides only limited clinically significant improvements when compared with a longer 20-session course in outpatients with mild-to-moderate severity COPD20.\n\nOur study has some weaknesses; this is not a randomised controlled trial and real world observational studies may be confounded by non-randomisation of participants. Our two PRP cohorts were not exactly matched; those enrolled in the three times weekly PRP had a statistically significant higher SGRQ and lower ISWT at baseline suggesting more respiratory impairment, despite similar age, FEV1 gender and disease mix. Those in the three times weekly PRP tended to have a higher number of admissions and days in hospital before PRP although this could have occurred through chance (p>0.05). The hospital offering the three times weekly PRP serves a larger urban population with higher levels of smoking, more ex-industrial workers and deprivation and so higher COPD prevalence and likely more co-morbidities. Despite a faster turnover and a higher throughput of patients, it has longer waiting times for PRP suggesting a greater local need. Although the absolute improvement in ISWT (metres) was bigger in the hospital offering PRP twice per week, this could be influenced by their greater exercise capacity to begin with and the relative (percentage) improvement from baseline (as opposed to actual metres); immediately post-PRP was similar in both groups (p=0.39).\n\nThe study could be open to selection/reporting bias as there was incomplete data especially at 12 months with only around 20% of patients returning to hospital for completion of SGRQs and ISWTs. The lower numbers with available data at 12 months also contributed to a large data spread, especially for ISWT. These re-attenders could be those who obtained the biggest gains and wanted to support the service - or alternatively they could consist predominantly those asking for more help because of limited improvements. However, our attrition rate over 12 months is typical of others, and as a group, these long-term attenders have similar characteristics to others14. There were no differences in baseline factors between those who completed data collection and those who did not.\n\nOur study has many strengths. It provides a unique opportunity to describe a real-life service but taking advantage of a real-time ‘natural experiment’ where two PRPs are identical in staffing and process apart from the intensity. Both services are well-described and follow evidence-based guidelines. The size of our study compares well with others and our patient selection, content, delivery and staffing is typical of many UK hospitals outside of specialist centres. Our immediate and short-term outcomes of PRP are compatible with others and offer some insights into more longitudinal (12 month) data, albeit with some caveats. Real-life observational studies can sometimes yield valuable insights. Atypical staff or patient behaviour does not limit them to the same extent as a randomised controlled trial, nor are they restricted by strict age and severity inclusion criteria, or the exclusion of patients with co-morbid illnesses21. Real-life clinical studies are more generisable than randomised trials from specialist centres or those designed/funded by institutions with a financial interest in outcomes.\n\nOur data suggest that offering a longer twice weekly PRP instead of a more intensive three times-weekly PRP leads to similar attendance and has not compromised outcomes. Longer PRPs may provide more opportunities for continuing exercise. Certainly continuing exercise after PRP is now acknowledged to be a vital component on its own22. A twice weekly PRP would allow participants to have more time during the week to start exercising independently (e.g. attend leisure centres) and to identify early problems/build confidence during their hospital PRP, well before they are discharged from the scheme. This could help address some of the remaining issues with continuing exercise and slowing deterioration after PRP. Importantly, slightly less intensive PRPs allow flexibility in service provision, for example, re-allocating staff from the third PRP day-of-the-week (e.g. Wednesdays) to instead promote sustainable exercise schemes for previous completers or working in the community for those unable to attend the hospital/day care unit7,17,23. Alternatively, twice weekly PRP would allow existing staff working a typical 5 day week, to run two PRPs simultaneously per week with one day per week allocated for reviews/baseline assessments. With the increasing pressure on clinical resources, a twice weekly PRP allows less than 50% whole time equivalent staff to run a single PRP over 2 days per week without compromising standards. We now run both PRPs 2 days a week allowing part-time working and the same staff to contribute to other respiratory services within the existing team, whilst monitoring our waiting lists.\n\n\nData availability\n\nfigshare: Data sets for UK Rehabilitation Programmes in real-life clinical settings, http://dx.doi.org/10.6084/m9.figshare.98708625\n\nAll data have been anonymised and approved for publication.",
"appendix": "Author contributions\n\n\n\nKEL conceived the study idea and formulated the design of the study. LT, CD, PH, CL-J were involved in recruitment of patients and data collection. KEL and HAH carried out data analysis. KEL led on the writing of the manuscript. All authors provided input to and reviewed all drafts of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe are grateful to all staff members who have given time during the education sessions for PRP. This paper is dedicated to the fond memory of Dr T. L. Griffiths.\n\n\nReferences\n\nSundararajan L, Balami J, Packham S: Effectiveness of outpatient pulmonary rehabilitation in elderly patients with chronic obstructive pulmonary disease. J Cardiopulm Rehabil Prev. 2010; 30(2): 121–5. PubMed Abstract | Publisher Full Text\n\nNici L, Donner C, Wouters E, et al.: American Thoracic Society/European Respiratory Society statement on pulmonary rehabilitation. Am J Respir Crit Care Med. 2006; 173(12): 1390–413. PubMed Abstract | Publisher Full Text\n\nRies AL: Pulmonary rehabilitation: summary of an evidence-based guideline. Respir Care. 2008; 53(9): 1203–7. PubMed Abstract\n\nManagement of chronic obstructive pulmonary disease in adults in primary and secondary care. London: National Institute of Clinical Excellence. [cited 2011 8th August]. 2011. Reference Source\n\nHill NS: Pulmonary rehabilitation. Proc Am Thorac Soc. 2006; 3(1): 66–74. PubMed Abstract | Publisher Full Text\n\nSeymour JM, Moore L, Jolley CJ, et al.: Outpatient pulmonary rehabilitation following acute exacerbations of COPD. Thorax. 2010; 65(5): 423–8. PubMed Abstract | Publisher Full Text\n\nMarciniuk DD, Brooks D, Butcher S, et al.: Optimizing pulmonary rehabilitation in chronic obstructive pulmonary disease--practical issues: a Canadian Thoracic Society Clinical Practice Guideline. Can Respir J. 2010; 17(4): 159–68. PubMed Abstract | Free Full Text\n\nReardon J, Casaburi R, Morgan M, et al.: Pulmonary rehabilitation for COPD. Respir Med. 2005; 99(Suppl B): S19–27. PubMed Abstract | Publisher Full Text\n\nSteele BG, Belza B, Cain KC, et al.: A randomized clinical trial of an activity and exercise adherence intervention in chronic pulmonary disease. Arch Phys Med Rehabil. 2008; 89(3): 404–12. PubMed Abstract | Publisher Full Text\n\nSabit R, Griffiths TL, Watkins AJ, et al.: Predictors of poor attendance at an outpatient pulmonary rehabilitation programme. Respir Med. 2008; 102(6): 819–24. PubMed Abstract | Publisher Full Text\n\nYoung P, Dewse M, Fergusson W, et al.: Respiratory rehabilitation in chronic obstructive pulmonary disease: predictors of nonadherence. Eur Respir J. 1999; 13(4): 855–9. PubMed Abstract | Publisher Full Text\n\nStone RA, Harrison BD, Lowe D, et al.: Introducing the national COPD resources and outcomes project. BMC Health Serv Res. 2009; 9: 173. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSteuten LM, Lemmens KM, Nieboer AP, et al.: Identifying potentially cost effective chronic care programs for people with COPD. Int J Chron Obstruct Pulmon Dis. 2009; 4: 87–100. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGriffiths TL, Burr ML, Campbell IA, et al.: Results at 1 year of outpatient multidisciplinary pulmonary rehabilitation: a randomised controlled trial. Lancet. 2000; 355(9201): 362–8. PubMed Abstract | Publisher Full Text\n\nGriffiths TL, Phillips CJ, Davies S, et al.: Cost effectiveness of an outpatient multidisciplinary pulmonary rehabilitation programme. Thorax. 2001; 56(10): 779–84. PubMed Abstract | Publisher Full Text | Free Full Text\n\nO'Neill BM, Johnston D, Burrell N, et al.: Effect of once weekly pulmonary rehabilitation on exercise tolerance in patients with chronic lung disease. Ir J Med Sci. 2001; 170(4): 231–2. PubMed Abstract | Publisher Full Text\n\nGarrod R, Marshall J, Barley E, et al.: Predictors of success and failure in pulmonary rehabilitation. Eur Respir J. 2006; 27(4): 788–94. PubMed Abstract | Publisher Full Text\n\nGreen RH, Singh SJ, Williams J, et al.: A randomised controlled trial of four weeks versus seven weeks of pulmonary rehabilitation in chronic obstructive pulmonary disease. Thorax. 2001; 56(2): 143–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSewell L, Singh SJ, Williams JE, et al.: How long should outpatient pulmonary rehabilitation be? A randomised controlled trial of 4 weeks versus 7 weeks. Thorax. 2006; 61(9): 767–71. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRossi G, Florini F, Romagnoli M, et al.: Length and clinical effectiveness of pulmonary rehabilitation in outpatients with chronic airway obstruction. Chest. 2005; 127(1): 105–9. PubMed Abstract | Publisher Full Text\n\nHalpin DM: Lessons from the major studies in COPD: problems and pitfalls in translating research evidence into practice. Prim Care Respir J. 2010; 19(2): 170–9. PubMed Abstract | Publisher Full Text\n\nBourbeau J: Making pulmonary rehabilitation a success in COPD. Swiss Med Wkly. 2010; 140: w13067. PubMed Abstract | Publisher Full Text\n\nAmbrosino N, Casaburi R, Ford G, et al.: Developing concepts in the pulmonary rehabilitation of COPD. Respir Med. 2008; 102(Suppl 1): S17–26. PubMed Abstract | Publisher Full Text\n\nMeguro M, Barley EA, Spencer S, et al.: Development and Validation of an Improved COPD-Specific Version of the St. George Respiratory Questionnaire. Chest. 2006; 132(2): 456–463. PubMed Abstract | Publisher Full Text\n\nLewis KE, Thatcher L, Davies CA, et al.: Data sets for UK Pulmonary Rehabilitation Programmes in real-life clinical settings. Figshare. 2014. Data Source"
}
|
[
{
"id": "4890",
"date": "28 May 2014",
"name": "Thomas Ringbaek",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe subject (impact of the frequency of supervised training) is clinical relevant and not sufficiently evaluated. They have used a real-life study comparing two hospitals with identical rehabilitation except for the number of supervised trainings – twice or three times a week. They have discussed the limitations and strength of this design. They have included enough patients to test for relevant differences between these two programmes. The patients have been adequately characterised at baseline, and the authors have used relevant outcomes.They write that median attendance was 13 (maximum 18 sessions), but I cannot find data on difference between the two programmes. Information on home training is also welcomed.They have used proper statistical analyses. It is well written, and data are discussed sensibly in context with other studies.",
"responses": []
}
] | 1
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https://f1000research.com/articles/3-89
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https://f1000research.com/articles/3-47/v1
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13 Feb 14
|
{
"type": "Web Tool",
"title": "FeatureViewer, a BioJS component for visualization of position-based annotations in protein sequences",
"authors": [
"Leyla Garcia",
"Guy Yachdav",
"Maria-Jesus Martin",
"Guy Yachdav",
"Maria-Jesus Martin"
],
"abstract": "Summary: FeatureViewer is a BioJS component that lays out, maps, orients, and renders position-based annotations for protein sequences. This component is highly flexible and customizable, allowing the presentation of annotations by rows, all centered, or distributed in non-overlapping tracks. It uses either lines or shapes for sites and rectangles for regions. The result is a powerful visualization tool that can be easily integrated into web applications as well as documents as it provides an export-to-image functionality.Availability: https://github.com/biojs/biojs/blob/master/src/main/javascript/Biojs.FeatureViewer.js; http://dx.doi.org/10.5281/zenodo.7719",
"keywords": [
"Position-based annotation is one of the cornerstones of bioinformatics. A great number of databases",
"analysis and prediction methods are geared towards providing data that are mapped to specific sequence coordinates. Particularly in the case of proteins",
"the Pfam1 database identifies",
"marks-up",
"and characterizes different functional regions within a given protein. The coordinates of these domains are often given in terms of the start and end position within the protein. The largest pool of reviewed and automatically annotated proteins provided by the UniProt Consortium2 also contains position-based annotations for structural regions",
"modified residues",
"and functional sites among others. Finally",
"methods such as those integrated into PredictProtein3 provide positionbased feature annotations such as secondary structure states",
"buried and exposed residues",
"coiled-coil stretches",
"and disordered regions. PredictProtein also maps functional regions such as protein-protein binding sites and protein-DNA binding sites onto positions within the sequence."
],
"content": "Introduction\n\nPosition-based annotation is one of the cornerstones of bioinformatics. A great number of databases, analysis and prediction methods are geared towards providing data that are mapped to specific sequence coordinates. Particularly in the case of proteins, the Pfam1 database identifies, marks-up, and characterizes different functional regions within a given protein. The coordinates of these domains are often given in terms of the start and end position within the protein. The largest pool of reviewed and automatically annotated proteins provided by the UniProt Consortium2 also contains position-based annotations for structural regions, modified residues, and functional sites among others. Finally, methods such as those integrated into PredictProtein3 provide positionbased feature annotations such as secondary structure states, buried and exposed residues, coiled-coil stretches, and disordered regions. PredictProtein also maps functional regions such as protein-protein binding sites and protein-DNA binding sites onto positions within the sequence.\n\nBioJS4 is an open source JavaScript library of components for visualization of biological data on the web. Here, we present FeatureViewer and its current extensions: SimpleFeatureViewer that simplifies the input data format, and DasProteinFeatureViewer that retrieves the input data from a web service. The FeatureViewer is a standard, portable BioJS component designed to easily render position-based annotations, a.k.a. features. The FeatureViewer component can be easily integrated into and controlled from other applications. To our knowledge, this is the first client-side modular component to visualize position-based annotations that can be integrated into other web applications in a standard manner.\n\n\nThe FeatureViewer component\n\nThe FeatureViewer component extensively uses the Raphaël javascript library5 that renders Scalable Vector Graphics (SVG) objects in modern web browsers. The use of SVG allows the graphics to scale to any requested resolution and is portable across different computing platforms and viewing software.\n\nThe FeatureViewer component can be easily integrated into any web application by including its dependencies in the head section, e.g., jQuery6 and Raphaël, and then instantiating the component within a JavaScript section. A special dependency for some images is required as they are used for the pop-up dialogue controls. The code below shows how to instantiate the component; the corresponding visualization is shown in Figure 1. A complete example and more information can be found at http://www.ebi.ac.uk/Tools/biojs/registry/Biojs.FeatureViewer.html. The FeatureViewer component has been tested with modern browsers such as Mozilla, Chrome, and Internet Explorer (IE); however, the image export option is not available in IE.\n\nVisualization of a peptide using the FeatureViewer component.\n\n\n\nIn order to instantiate the FeatureViewer component, some options should be defined. The mandatory options correspond to (i) a place holder named target, i.e., a DIV element in the web page where the annotations will be rendered, and (ii) a JSON object, named json, with the configuration, the protein identifier, the annotations, and the legend. FeatureViewer is a dummy component in the sense that it does not make any calculations about where to render the annotations, not even when the rendering style is changed; all the rendering information is provided in the json option. A comprehensive list of the elements in the json option is available at http://www.ebi.ac.uk/Tools/biojs/registry/Biojs.FeatureViewer.html. The FeatureViewer component includes three different layouts to display the features: all features centered, features grouped by type, and features organized in non-overlapping tracks, as shown in Figure 2.\n\na. This visualization corresponds to the UniProt protein \"Amyloid beta A4 protein\" in the nonoverlapping style; interactions such as shape dragging, tooltip, and selection as well as user controls such as zooming and image exporting are illustrated in this figure; b. shows the centralized view, while c. shows the by-rows view.\n\nAdditional options can also be specified in order to customize the user controls as well as interaction with the features. User controls include the zooming slider and the export-to-image button, as shown in the Figure 2a. The zooming slider allows users to hone in on a region of interest and view it in greater detail, making it possible to move from an overview aspect into a detailed one without navigating to a different page. The export-to-image button allows users to export the rendered features into an image that can embedded into a paper or presentation.\n\nDifferent kinds of interaction are also possible. Events bound to rendered annotations include a mouse-over action that highlights and colors the \"focused\" feature. Click action is also supported. Clicking on a feature selects it so it will remain highlighted until another feature is selected; clicking an already selected feature will deselect it. Tooltips tied to each shape pop up and reveal additional information about the rendered annotations. Either shapes or lines can be used to display features covering one single amino acid; currently metal bindings can be rendered as circles, active sites as diamonds, lipidation as waves, glycosylation as hexagons, and post translational modifications as triangles. When shapes are used, it is possible to drag them making it easier to distinguish one from another when they are clustered.\n\nAs FeatureViewer requires highly detailed information in order to display the features, a simpler version, the SimpleFeatureViewer component, builds on top of it. This simplified version takes care of calculating the configuration options as well as the localization of the features; thus, developers using this version can focus on the actual data rather than on intricate details regarding styles, pixels, and coordinates. However, only the non-overlapping tracks style is supported by this component. The main advantage of this component is that its data structure is much simpler than the one required for FeatureViewer, as observed in the following code excerpt.\n\n\n\nThis component requires a place holder, a sequence identifier, a sequence length, and a features array; the width in pixels to be used to rendered the protein features can also be defined by using the option imageWidth. The features array contains information for each feature to be displayed including, for instance, identifier, start and end positions in the sequence, label, and color among others. More information is available at http://www.ebi.ac.uk/Tools/biojs/registry/Biojs.SimpleFeatureViewer.html.\n\nA second extension, the DasProteinFeatureViewer, makes use of a web service that retrieves data from Distributed Annotation System (DAS) sources. DAS defines a communication protocol used to exchange annotations on gene or protein sequences7. Multiple protein databases provide their data following the DAS principles, for instance UniProt and InterPro8. For this extension, no information about the features themselves is required as such details will be retrieved from the web service, as shown in the code below.\n\n\n\nAdditional options allow developers to specify the protein identifier, the DAS sources, the feature types – e.g., domain, chain, variant, etc., the rendering style, the image width, and some others. In order to avoid cross-domain problems, a proxy can also be specified. The feature types used by this component are those defined by UniProt, which is also used as the reference DAS source, i.e., the one providing the protein sequence. More information available at http://www.ebi.ac.uk/Tools/biojs/registry/Biojs.DasProteinFeatureViewer.html.\n\n\nUse case\n\nThe PredictProtein service3 integrates multiple algorithms that either retrieve from curated databases or automatically predict aspects of protein structure and function. Many of the predictions provided by the methods are mapped to positions within the protein. In order to easily highlight patterns, compare predictions, and cross-validate results, the PredictProtein interface lays out the predicted annotations in data tracks, i.e., in separate rows, each row presenting different predicted features. Data tracks are laid one under the other and enable the quick overview of some of the prominent features of the protein e.g., a cluster of binding sites close to the N-terminal or the count of trans-membrane regions. Figure 3 shows the implementation of the FeatureViewer component used for the PredictProtein service.\n\nFeatureViewer is used by the PredictProtein service to show a stack of predicted structure and function features.\n\n\nConclusions\n\nThe FeatureViewer component and its extensions, SimpleFeatureViewer and DasProteinFeatureViewer, provide a platform to visualize position-based biological data easily and efficiently. FeatureViewer, like any other BioJS component, can be easily integrated with other web components or extended to have greater functionality than the one shown here. We expect this component to be particularly useful to developers and users alike, requiring little technical knowledge for its full functioning.\n\n\nSoftware availability\n\nZenodo: BioJS Feature Viewer, doi: 10.5281/zenodo.77199\n\nGitHub: BioJS, https://github.com/biojs/biojs/",
"appendix": "Author contributions\n\n\n\nLG is the original author and current lead developer of the FeatureViewer, SimpleFeatureViewer, and DasProteinFeatureViewer components. GY extended FeatureViewer in order to support bridges, and provided the use case presented in this manuscript. MM conceived the original concept and contributed to the design of FeatureViewer. All authors have revised and agreed to the content in this manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nGY was supported by the Alexander von Humboldt Foundation. UniProt EMBL-EBI is funded by National Institutes of Health (NIH) [1U41HG006104-03].\n\n\nAcknowledgements\n\nGY thanks Burkhard Rost who helped fund this work and contributed ideas and feedback. GY also acknowledges Edda Kloppman and Tatyana Goldberg for helpful discussions and invaluable feedback and insight.\n\nLG thanks Pablo Moreno who spotted a bug related to multiple instantiation and contributed to fixing it, as well as Mark Bingley, Claire O’Donovan, Sangya Pundir, and Xavier Watkins in the UniProt EMBL-EBI team and Rafael Jiménez and John Gómez in the IntAct EMBL-EBI team for their comments and suggestions about the FeatureViewer component and its extensions.\n\nThe authors thank all those who funded our research as well as researchers who deposited data into publicly available datasets and programmers who provided their work under a free license: our work stands upon their shoulders and would not have been possible without them.\n\n\nReferences\n\nPunta M, Coggill PC, Eberhardt RY, et al.: The Pfam protein families database. Nucleic Acids Res. 2012; 40(Database issue): D290–301. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUniProt Consortium.Update on activities at the universal Protein Resource (uniprot) in 2013. Nucleic Acids Res. 2013; 41(Database issue): D43–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRost B, Yachdav G, Liu J: The PredictProtein server. Nucleic Acids Res. 2004; 32(Web Server issue): W321–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGómez J, García LJ, Salazar GA, et al.: BioJs: an open source Javascript framework for biological data visualization. Bioinformatics. 2013; 29(8): 1103–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRaphaël—javascript library. 2013. Reference Source\n\njquery—javascript library. 2014. Reference Source\n\nJenkinson AM, Albrecht M, Birney E, et al.: Integrating biological data--the Distributed Annotation System. BMC Bioinformatics. 2008; 9(Suppl 8): S3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHunter S, Jones P, Mitchell A, et al.: InterPro in 2011: new developments in the family and domain prediction database. Nucleic Acids Res. 2012; 40(Database issue): D306–D312. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLeyla G, Yachdav G, Moreno P: BioJS Feature Viewer. Zenodo. 2014. Reference Source"
}
|
[
{
"id": "3680",
"date": "25 Feb 2014",
"name": "Jim Procter",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummaryThis F1000Research article describes an ensemble of JavaScript components designed for bioinformatics web developers that allow the retrieval, layout and display of positional annotation on a 1D coordinate system, such as a protein or nucleotide sequence. Special support is provided for the display of protein positional annotation retrieved from the Distributed Annotation System (requires some server-side configuration), and the system provides standard glyphs and shading styles to allow active sites and common types of post-translational modifications to be effectively displayed. Importantly, the components employ the BioJS framework, which allows them to exchange messages with other BioJS biological data visualization components (as well as any jquery based module) to facilitate the creation of rich, interactive web interfaces.Using the componentThe feature viewer component example page demonstrates that it provides a clean static visualization of the positional annotation as it appears on a coordinate system (complete with annotation legend). Online documentation supports the authors’ statement that the plugin is highly configurable: attributes are provided to control practically every aspect of an annotation's appearance, and the surrounding coordinate space and additional decorators'. A few minutes spent interacting with the demonstration, however, shows that there is room for improvement:(1) Suggested improvement. Once the zoom control is adjusted to focus in on a smaller region, it doesn't allow the user to sweep across the coordinate space (i.e. by click-dragging the visible range). From a developer perspective, I would also expect the component to raise BioJS events to inform other components about the change in 'Region Of Interest' to allow them to respond in kind.(2) Suggested improvement. The authors mention that feature glyphs can be dragged to facilitate inspection in crowded regions. It is unclear how useful this capability is – since once dragged, any change in view results in the feature glyph’s shape being reset, and in crowded regions, the user must – by definition – move many features to examine the precise location of annotation. User modified glyph layout should – at the very least – be preserved on changes of scale. There may also be wisdom in including a force-directed layout algorithm to better optimize placement of nearby glyphs in response to manual adjustment of any particular one. Developing with the component(s)Apart from choosing appropriate values for the vast array of attributes and annotation display settings that FeatureViewer supports (more on that later), the most onerous aspect of deploying FeatureViewer is keeping track of the array of dependencies it requires. However, JavaScript module dependency management is a moving target, and I make the recommendation below with that in mind: (3) Suggested improvement. I strongly recommend that the authors provide examples that employ a client-side JavaScript package management system such as 'RequireJS' (see http://jquerysbestfriends.com for some other tips about more sanitary methods of deploying jquery). Dependency management is important, and demonstrating good practice will allow these tools to be more widely adopted, and more effectively maintained.One aspect of this paper that was not immediately clear on first reading was the functional relationship between FeatureViewer – which renders annotation layouts into SVG, and the helper components SimpleFeatureRenderer and DASFeatureRenderer.(4) Suggested revision/reviewer contributionA diagram such as the one I uploaded to FigShare here:http://figshare.com/articles/The_BioJS_FeatureViewer_components_for_sequence_annotation_retrieval_layout_and_display/942422may help readers of this article more effectively grasp the different functionalities provided by the components in this system. Specific revisions/corrections (5) Required revision In the abstract, the authors claim: \"To our knowledge, this is the first client-side modular component to visualize position-based annotations that can be integrated into other web applications in a standard manner.\"This is not quite correct. There have been other client-side modular web components that have been created for the visualisation of positional annotation, although some of the co-authors may have, for modesty reasons omitted mentioning the fact. In this case, Dasty3 and its forerunner, Dasty2 both provided a self-contained (ie modular) component that could be controlled through javascript and embedded in a more complex web page. Here, I recommend the authors emphasise more strongly the key novelty in this work. For example: \"because these components are built with the BioJS framework, they are the first modular visualization components for the display of position-based annotation that can be integrated with other web applications in a standard manner.\" (6) Please revise the abstract for clarity \"This component is highly flexible and customizable, allowing the presentation of annotations by rows, all centered, or distributed in non-overlapping tracks. It uses either lines or shapes for sites and rectangles for regions.\"Point 1. I'm not sure what presenting annotations \"all centered\" actually means!Point 2. (optional). The authors might consider providing a biological example. This would help the lay-reader understand what the tool does without already having deep knowledge of protein sequence annotation visualization.(7) Please revise the following sentence for clarity The authors state that DasFeatureViewer can employ different glyphs for particular types of protein sequence annotation, including post translational modifications (PTMs). Point 1. Suggested revision in bold italics“Either shapes or lines can be used to display features covering one single amino acid; currently metal bindings can be rendered as circles, active sites as diamonds, lipidation as waves, glycosylation as hexagons, and [other] post translational modifications as triangles.“The reason for this revision is that Lipidation and glycosylation are, of course, both PTMs. The authors could also mention here that triangles are used as the default for other types of ‘MOD_RES’ type PTM (glycation, hydroxylation, phosphorylation, etc). (8) Minor typos/grammar Point 1. In the Introduction section \"Particularly in the case of proteins, the Pfam database identifies, marks-up, and characterizes different functional regions within a given protein.\" THe word 'Particularly' is unnecessary.Point 2. In the following sentence (in the Introduction section): \"The largest pool of reviewed and automatically annotated proteins provided by the UniProt Consortium also contains position-based annotations for structural regions, modified residues, and functional sites among others.\" The authors probably mean to make the following statement (revision in bold italics):\"The largest pool of reviewed and automatically annotated proteins is provided by the UniProt Consortium. It also contains position-based annotations ..\"Point 3. Again, revising for clarity (in the Introduction section) (revision in bold italics):\"Finally, protein feature prediction methods such as those integrated into PredictProtein provide position-based feature..\"Point 4. In the next paragraph: \"andDasProteinFeatureViewer that retrieves the input data from a web service.\" There is a missing space between 'and' and 'DasProteinFeatureViewer' Point 5. In the second paragraph of “The FeatureViewer component\" section “The code below shows how to instantiate the component; the corresponding visualization is shown in Figure 1.” This sentence is perhaps more cleanly stated as:“The code below shows how to instantiate the component to create the visualization shown in Figure 1”. (9) Typos in Web ResourcesPlease fix the missing ‘e’ on this page: http://www.ebi.ac.uk/Tools/biojs/registry/Biojs.DasProteinFeatureViewer.html\"This component uses a DASProtein web service that builds the JSON data object used by FeatureViewer component. Version 1.0.0.\"",
"responses": [
{
"c_id": "752",
"date": "03 Apr 2014",
"name": "L. Garcia",
"role": "Author Response",
"response": "Dear James,Thanks for your review, it has have been useful to improve our work. We have tried to addressed all you comments, however those related to the component itself, i.e., JavaScript code, will be taken into account for a new version of the software, and those related to BioJS in general will be sent to BioJS core developers. Please see our responses below.Response to (1) and (2): We are currently working on an improved component to visualize protein sequence annotations. We have made notes about this suggestions and will take them into account for the new visualization. Unfortunately, such improvements are not yet ready to be integrated into the public BioJS GitHub repository. Response to (3): We agree with the reviewer on the convenience of using dependency management for BioJS components. As we think it will impact not just this component but any other currently in BioJS, we have initiated a discussion on how to improve this aspect in BioJS, but no decision has been made yet. We will take it into account for the new visualization we are working on.Response to (4): We have included a figure similar to the proposed one by the reviewer at the beginning of the 'Extensions' section. It introduces the extensions and indeed, as mentioned by the reviewer, helps the reader to understand the relation across the three components.Response to (5): We have included a new paragraph in the Introduction in order to cover other efforts on web visualization for protein sequence annotations, covering not only Dasty but others as well. We have also emphasized the novelty of this work as suggested by the reviewer.Response to (6): We have simplified the abstract.Response to (7 and 8): We have followed the reviewer’s suggestions.Response to (9): We have added the missing ‘e’ and it will be included in the next BioJS registry update.Regards"
}
]
},
{
"id": "4063",
"date": "18 Mar 2014",
"name": "Andreas Prlic",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThere are currently only a few approaches available for visualising protein features on the web. The BioJS FeatureViewer is a welcome addition to the set of tools available to web developers. The fact that it is available as a BioJS component should make it interesting as a re-usable web site element. However, I find that some of the already existing approaches provide a representation of features that I (subjectively) find graphically more refined and that go beyond simple boxes, circles, and triangles to represent sequence annotations.Minor modifications:I feel that the current version of this manuscript does not adequately summarise the already available approaches for visualising protein features. To list a few already available protein feature viewers:- Pfam (is already being mentioned in the manuscript), shows the location of Pfam domains on protein sequences. It has currently one of the graphically most appealing views and can show more than one sequence per page (e.g. http://pfam.sanger.ac.uk/family/Piwi#tabview=tab1)- PDBsum provides a graphical summary of secondary structure and pfam domains for protein sequences (e.g. https://www.ebi.ac.uk/pdbsum/1cdg and in large: https://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1cdg&template=wirpfam.html&r=wiring&l=1&chain=A&pfam=TRUE&form=TRUE&nres=150)- DASty has been developed for several years at the EBI (in fact there seems to be some shared co-authorship) and provides a feature view that is based on DAS and can also map annotations to 3D (e.g. https://www.ebi.ac.uk/dasty/client/index.html?q=P05067)- RCSB Protein Feature View: shows an SVG image for a full-length protein sequence from UniProt and how it corresponds to PDB entries. It loads annotations from external databases (e.g. http://www.rcsb.org/pdb/protein/P43379) and is available as open source from https://github.com/rcsb/proteinfeatureview- RCSB Sequence Page: can show the relationship between UniProt and PDB coordinates on a per-residue level. Also can map annotations to 3D ( e.g. http://www.rcsb.org/pdb/explore/remediatedSequence.do?structureId=1CDG).",
"responses": [
{
"c_id": "751",
"date": "03 Apr 2014",
"name": "L. Garcia",
"role": "Author Response",
"response": "Dear Andreas,Thanks for your review, it has been useful to improve our work. Please see below our responses.Response: We have included a new paragraph in the Introduction in order to cover other efforts on web visualizations for protein sequence annotations. We have included those related to protein sequence features in general rather than specialized on secondary or 3D structures."
}
]
}
] | 1
|
https://f1000research.com/articles/3-47
|
https://f1000research.com/articles/3-78/v1
|
27 Mar 14
|
{
"type": "Observation Article",
"title": "The reproductive season of scleractinian corals in Socotra, Yemen",
"authors": [
"Andrew H. Baird",
"David Abrego",
"Emily J. Howells",
"Vivian R. Cumbo",
"David Abrego",
"Emily J. Howells",
"Vivian R. Cumbo"
],
"abstract": "Determining when corals reproduce has clear management and economic implications. Here we document the reproductive condition of corals in the genus Acropora on the island of Socotra in Yemen during February 2014. Twenty percent of colonies (n = 143) contained mature gametes and 28% had immature gametes indicating that spawning will occur in both February and March in 2014, confirming previous anecdotal reports of coral spawning at this time in Socotra. Acropora typically reproduce in synchrony with many other broadcast spawning scleractinian corals, and we therefore predict that many other species are reproductively active at this time of year.",
"keywords": [
"Most hermatypic scleractinian corals have an annual gametogenic cycle that culminates in the broadcast spawning of gametes once per year1. In most reef regions",
"numerous species spawn in sychrony following full moons when sea surface temperature is either rising or falling2",
"3. Determining exactly when spawning takes place has important implications for reef management and clear economic benefits4. For example",
"activities that are likely to limit fertilization success",
"such as dredging",
"can be prohibited during these often brief spawning periods5. In addition",
"the diving industry can benefit from public interest in coral spawning."
],
"content": "Observation\n\nMost hermatypic scleractinian corals have an annual gametogenic cycle that culminates in the broadcast spawning of gametes once per year1. In most reef regions, numerous species spawn in sychrony following full moons when sea surface temperature is either rising or falling2,3. Determining exactly when spawning takes place has important implications for reef management and clear economic benefits4. For example, activities that are likely to limit fertilization success, such as dredging, can be prohibited during these often brief spawning periods5. In addition, the diving industry can benefit from public interest in coral spawning.\n\nHere, we document the reproductive condition of Acropora corals on the island of Socotra, Republic of Yemen. The island, located 240 km east of the Horn of Africa and 380 km south of the Arabian Peninsula, supports a diverse scleractinian fauna of over 250 species6, and includes sites with a high cover of Acropora (Figure 1). Acropora colonies were sampled before the full moon on 15 February 2014 to determine their reproductive condition. Three reproductive conditions were defined based on the appearance of the oocytes as observed with the naked eye in the field : 7 (1) mature - oocytes pigmented and therefore likely to spawn within a month (2) immature - oocytes pale but visible indicating that they are close to maturity and likely to spawn within two to three months (3) empty - oocytes too small to see or absent indicating either that the colony has recently spawned, or is unlikely to do so for at least three months.\n\nA total of 143 Acropora colonies from approximately 14 species were sampled at four sites on the north of Socotra (Samerhur 12°41'40.96\"N, 53°29'3.69\"E; Qaiso 12°39'58.91\"N, 53°24'33.86\"E; Dihamri 12°40'20.35\"N, 54°11'39.96\"E; Hadibo 12°40'0.77\"N, 54° 3'7.74\"E) between the 31 January and 8 February 2014 (Table 1). Twenty percent of colonies contained mature oocytes (Figure 2), 28% contained immature oocytes and no oocytes were visible in the remaining 52% of colonies (Table 1). Colonies with mature oocytes are highly likely to spawn at some time around the full moon in February 2014, whereas colonies with immature oocytes are likely to spawn in March 2014. The remaining colonies have either spawned recently, or alternatively, there could be a second reproductive season later in the year, similar to Western Australia8, Singapore9 and some locations in Indonesia1. Ten species had at least one colony with mature gametes (Table 1) suggesting a multi-species spawning event is likely in February 2014. The Acropora typically reproduce at much the same time as most other broadcast spawning scleractinian corals10,11 and therefore we predict that many other species will be spawning in February and March in Socotra. Our results confirm previous anecdotal reports of coral spawning on Socotra in February and March6. In addition, these data add to a growing body of evidence indicating that multi-specific spawning synchrony is a feature of all speciose coral assemblages1.\n\nn = number of sampled colonies.",
"appendix": "Author contributions\n\n\n\nAll authors conceived the study, collected the data and wrote the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nFunding was provided by the Australian Research Council Centre of Excellence for Coral Reef Studies COE561432 (AHB).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe thank Ahmed Eissa Ali Bin Affrar, Ali Yahai and Abdulaziz for logistical support in Socotra.\n\n\nReferences\n\nBaird AH, Guest JR, Willis BL: Systematic and biogeographical patterns in the reproductive biology of scleractinian corals. Annu Rev Ecol Evol Syst. 2009; 40: 551–571. Publisher Full Text\n\nWillis BL, Babcock RC, Harrison PL, et al.: Patterns in the mass spawning of corals on the Great Barrier Reef from 1981 to 1984. Proc 5th Int Coral Reef Symp. 1985; 4: 343–348. Reference Source\n\nGuest JR, Baird AH, Goh BPL, et al.: Seasonal reproduction in equatorial reef corals. Invertebrate Reproduction & Development. 2005; 48(1–3): 207–218. Publisher Full Text\n\nBaird AH, Kospartov MC, Purcell S: Reproductive synchrony in Acropora assemblages on reefs of New Caledonia. Pac Sci. 2010; 64(3): 405–412. Publisher Full Text\n\nBaird AH, Blakeway DR, Hurley TJ, et al.: Seasonality of coral reproduction in the Dampier Archipelago, northern Western Australia. Mar Biol. 2011; 158(2): 275–285. Publisher Full Text\n\nDeVantier L, De’Ath G, Klaus R, et al.: Reef-building corals and coral communities of the Socotra Archipelago, a zoogeographic ‘crossroads’ in the Arabian Sea. Fauna of Arabia. 2004; 20: 117–168. Reference Source\n\nBaird AH, Marshall PA, Wolstenholme J: Latitudinal variation in the reproduction of Acropora in the Coral Sea. Proc 9th Int Coral Reef Symp. 2002; 1: 385–389. Reference Source\n\nGilmour JP, Smith LD, Brinkman RM: Biannual spawning, rapid larval development and evidence of self-seeding for scleractinian corals at an isolated system of reefs. Mar Biol. 2009; 156: 1297–1309. Publisher Full Text\n\nGuest JR, Baird AH, Goh BPL, et al.: Reproductive seasonality in an equatorial assemblage of scleractinian corals. Coral Reefs. 2005; 24(1): 112–116. Publisher Full Text\n\nHarrison PL, Babcock RC, Bull GD, et al.: Mass spawning in tropical reef corals. Science. 1984; 223(4641): 1186–1189. PubMed Abstract | Publisher Full Text\n\nBabcock RC, Willis BL, Simpson CJ: Mass spawning of corals on a high latitude coral reef. Coral Reefs. 1994; 13(3): 161–169. Publisher Full Text"
}
|
[
{
"id": "4277",
"date": "28 Mar 2014",
"name": "Jean Kenyon",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nBackground information, purpose of study, data, and interpretation are all clearly and concisely presented. Documentation of coral sexual reproductive status at Socotra, Yemen in 14 species of Acropora provides a useful first contribution to improved understanding of global patterns of this reef maintenance and renewal process. Given that more than 250 species of scleractinian corals are supported at the island, it would be interesting and useful to extend these initial studies to additional species at other times of the year, and to confirm inferred spawning period and synchrony with field observations. That said, I would suggest that the article title, “The reproductive season of scleractinian corals in Socotra, Yemen” is too broad, given that only ~5% of the scleractinian fauna was sampled. A more focused title should indicate that the observations are confined to the genus Acropora. Within the article, it would also be informative to provide the number of species of Acropora reported among the scleractinian fauna, so as to give some perspective on the proportion of Acropora species sampled.",
"responses": [
{
"c_id": "763",
"date": "08 Apr 2014",
"name": "Andrew Baird",
"role": "Author Response F1000Research Advisory Board Member",
"response": "We have changed the title to “The reproductive season of Acropora in Socotra, Yemen”.We have also provided the number of Acropora species reported to occur in Socotra as listed by Devantier et al., 2004."
}
]
},
{
"id": "4321",
"date": "01 Apr 2014",
"name": "Bert W. Hoeksema",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis short publication presents clear results and is well written. I have one comment. The conclusive sentence seems redundant, and may need rephrasing or some additional explanation if the authors want to maintain it. “In addition, these data add to a growing body of evidence indicating that multi-specific spawning synchrony is a feature of all speciose coral assemblages.” The present results concern a selection of Acropora species. I wonder whether this would be sufficiently representative for a speciose coral assemblage. In theory any assemblage of two coral species can be considered multi-specific. With many Acropora species in a speciose coral assemblage, some degree of spawning synchrony is not surprising. So, in the present context I wonder if there is a minimum number of species for what counts as speciose and if the authors have a minimum number or percentage in mind for what exactly counts as multi-species spawning. If the authors cannot clarify their remark or cannot give more supportive information to this statement, I suggest that this sentence should be removed.",
"responses": [
{
"c_id": "762",
"date": "08 Apr 2014",
"name": "Andrew Baird",
"role": "Author Response F1000Research Advisory Board Member",
"response": "We have removed the sentence as suggested."
}
]
}
] | 1
|
https://f1000research.com/articles/3-78
|
https://f1000research.com/articles/3-8/v1
|
13 Jan 14
|
{
"type": "Web Tool",
"title": "Validation of predicted mRNA splicing mutations using high-throughput transcriptome data",
"authors": [
"Coby Viner",
"Stephanie N. Dorman",
"Ben C. Shirley",
"Peter K. Rogan",
"Coby Viner",
"Stephanie N. Dorman",
"Ben C. Shirley"
],
"abstract": "Interpretation of variants present in complete genomes or exomes reveals numerous sequence changes, only a fraction of which are likely to be pathogenic. Mutations have been traditionally inferred from allele frequencies and inheritance patterns in such data. Variants predicted to alter mRNA splicing can be validated by manual inspection of transcriptome sequencing data, however this approach is intractable for large datasets. These abnormal mRNA splicing patterns are characterized by reads demonstrating either exon skipping, cryptic splice site use, and high levels of intron inclusion, or combinations of these properties. We present, Veridical, an in silico method for the automatic validation of DNA sequencing variants that alter mRNA splicing. Veridical performs statistically valid comparisons of the normalized read counts of abnormal RNA species in mutant versus non-mutant tissues. This leverages large numbers of control samples to corroborate the consequences of predicted splicing variants in complete genomes and exomes.",
"keywords": [
"DNA variant analysis of complete genome or exome data has typically relied on filtering of alleles according to population frequency and alterations in coding of amino acids. Numerous variants of unknown significance (VUS) in both coding and non-coding gene regions cannot be categorized with these approaches. To address these limitations",
"in silico methods that predict biological impact of individual sequence variants on protein coding and gene expression have been developed",
"which exhibit varying degrees of sensitivity and specificity1. These approaches have generally not been capable of objective",
"efficient variant analysis on a genome-scale."
],
"content": "Introduction\n\nDNA variant analysis of complete genome or exome data has typically relied on filtering of alleles according to population frequency and alterations in coding of amino acids. Numerous variants of unknown significance (VUS) in both coding and non-coding gene regions cannot be categorized with these approaches. To address these limitations, in silico methods that predict biological impact of individual sequence variants on protein coding and gene expression have been developed, which exhibit varying degrees of sensitivity and specificity1. These approaches have generally not been capable of objective, efficient variant analysis on a genome-scale.\n\nSplicing variants, in particular, are known to be a significant cause of human disease2–5 and indeed have even been hypothesized to be the most frequent cause of hereditary disease6. Computational identification of mRNA splicing mutations within DNA sequencing (DNA-Seq) data has been implemented to varying degrees of sensitivity, with most software only evaluating conservation solely at the intronic dinucleotides adjacent to the junction (i.e.7). Other approaches are capable of detecting significant mutations at other positions with constitutive, and in certain instances, cryptic, splice sites5,8,9 which can result in aberrations in mRNA splicing. Presently, only information theory-based mRNA splicing mutation analysis has been implemented on a genome scale10. Splicing mutations can abrogate recognition of natural, constitutive splice sites (inactivating mutation), weaken their binding affinity (leaky mutation), or alter splicing regulatory protein binding sites that participate in exon definition. The abnormal molecular phenotypes of these mutations comprise: (a) complete exon skipping, (b) reduced efficiency of splicing, (c) failure to remove introns (also termed intron retention or intron inclusion), or (d) cryptic splice site activation, which may define abnormal exon boundaries in transcripts using non-constitutive, proximate sequences, extending or truncating the exon. Some mutations may result in combinations of these molecular phenotypes. Nevertheless, novel or strengthened cryptic sites can be activated independently of any direct effect on the corresponding natural splice site. The prevalence of these splicing events has been determined by ourselves and others5,11–13. The diversity of possible molecular phenotypes makes such aberrant splicing challenging to corroborate at the scale required for complete genome (or exome) analyses. This has motivated the development of statistically robust algorithms and software to comprehensively validate the predicted outcomes of splicing mutation analysis.\n\nPutative splicing variants require empirical confirmation based on expression studies from appropriate tissues carrying the mutation, compared with control samples lacking the mutation. In mutations identified from complete genome or exome sequences, corresponding transcriptome analysis based on RNA sequencing (RNA-Seq) is performed to corroborate variants predicted to alter splicing. Manually inspecting a large set of splicing variants of interest with reference to the experimental samples’ RNA-Seq data in a program like the Integrative Genomics Viewer (IGV)14, or simply performing database searches to find existing evidence would be time-consuming for large-scale analyses. Checking control samples would be required to ensure that the variant is not a result of alternative splicing, but is actually causally linked to the variant of interest. Manual inspection of the number of control samples required for statistical power to verify that each displays normal splicing would be laborious and does not easily lend itself to statistical analyses. This may lead to either missing contradictory evidence or to discarding a variant due to the perceived observation of statistically insignificant altered splicing within control samples. In addition, a list of putative splicing variants returned by variant prediction software can often be extremely large. The validation of such a significant quantity of variants may not be feasible, for example, in certain types of cancer, in instances where the genomic mutational load is high and only manual annotation is performed. We have therefore developed Veridical, a software program that automatically searches all given experimental and control RNA-Seq data to validate DNA-derived splicing variants. When adequate expression data are available at the locus carrying the mutation, this approach reveals a comprehensive set of genes exhibiting mRNA splicing defects in complete genomes and exomes. Veridical and its associated software programs are available at: www.veridical.org.\n\n\nMethods\n\nThe program Veridical was developed to allow high-throughput validation of predicted splicing mutations using RNA sequencing data. Veridical requires at least three files to operate: a DNA variant file containing putative mRNA splicing mutations, a file listing of corresponding transcriptome (RNA-Seq) BAM files, and a file annotating exome structure. A separate file listing RNA-Seq BAM files for control samples (i.e. normal tissue) can also be provided. Here, we predict mutations in a set of breast tumours which are validated with RNA-Seq data from the same individuals, with RNA-Seq data from normal breast tissues as controls. However, in principle, potential splicing mutations for any disease state with available RNASeq data can be investigated. In each tumour, every variant is analyzed by checking the informative sequencing reads from the corresponding RNA-Seq experiment for non-constitutive splice isoforms, and comparing these results with the same type of data from all other tumour and normal samples that do not carry the variant in their exomes.\n\nVeridical concomitantly evaluates control samples, providing for an unbiased assessment of splicing variants of potentially diverse phenotypic consequences. Note that control samples include all non-variant containing files, as well any normal samples provided. Maximizing the set of control samples, while computationally more expensive, increases the statistical robustness of the results obtained.\n\nFor each variant, Veridical directly analyzes sequence reads aligned to the exons and introns that are predicted to be affected by the genomic variant. We elected to avoid indirect measures of exon skipping, such as loss of heterozygosity in the transcript, because of the possibility of confusion with other molecular etiologies (i.e. deletion or gene conversion), unrelated to the splicing mutations. The nearest natural site is found using the exome annotation file provided, based upon the directionality of the variant, as defined within Table 1. The genomic coordinates of the neighboring exon boundaries are then found and the program proceeds, iterating over all known transcript variants for the given gene. A diagram of this procedure is provided in Figure 1. The variant location, C, is specifically referring to the, variant-induced, location of the predicted mRNA splice site, which is often proximate to, but distinct from the coordinate of the actual genomic mutation itself.\n\nα – 5′ splice site β – 3′ splice site\n\nA diagram portraying the definitions used within Veridical to specify genic variant position and read coordinates. We employ the same conventions as IGV14. Blue lines denote genes, wherein thick lines represent exons and thin lines represent introns. Grey lines denote reads, wherein thick lines denote a read mapping to some particular location in the genome and thin lines represent connecting segments of reads that are split across spliced-in regions (i.e. exons or included introns).\n\nThe program uses the BamTools API15 to iterate over all of the reads within a given genomic region across experimental and control samples. Individual reads are then assessed for their corroborating value towards the analysis of the variant being processed, as outlined in the flowchart in Figure 2. Validating reads are based on whether they alter either the location of the splice junction (i.e. junction-spanning) or the abundance of the transcript, particularly in intronic regions (i.e. read-abundance). Junction-spanning reads contain DNA sequences from two adjacent exons or are reads that extend into the intron. These reads directly show whether the intronic sequence is removed or retained by the spliceosome, respectively. Read-abundance validated reads are based upon sequences predicted to be found in the mutated transcript in comparison with sequences that are expected to be excised from the mature transcript in the absence of a mutation. Both types of reads can be used to validate cryptic splicing, exon skipping, or intron inclusion. A read is only said to corroborate cryptic splicing if and only if the variant under consideration is expected to activate cryptic splicing. Junction-spanning, cryptic splicing reads are those in which a read is exactly split from the cryptic splice site to the adjacent exon junction. For read-abundance cryptic splicing, we define the concept of a read fraction, which is the ratio of the number of reads corroborating the cryptically spliced isoform and the number of reads that do not support the use of the cryptic splice site (i.e. non-cryptic corroborating) in the same genomic region of a sample. Cryptic corroborating reads are those which occur within the expected region where cryptic splicing occurs (i.e. spliced-in regions). This region is bounded by the variant splice site location and the adjacent (direction dependent) splice junction. Non-cryptic corroborating reads, which we also term \"anti-cryptic\" reads, are those that do not lie within this region, but would still be retained within the portion that would be excised, had cryptic splicing occurred. To identify instances of exon skipping, Veridical only employs junction-spanning reads. A read is considered to corroborate exon skipping if the connecting read segments are split such that it connects two exon boundaries, skipping an exon in between. A read is considered to corroborate intron inclusion when the read is continuous and either overlaps with the intron-exon boundary (and is then said to be junction-spanning) or if the read is within an intron (and is then said to be based upon read-abundance).\n\nThe algorithm employed by Veridical to validate variants. Refer to Table 1 for definitions concerning direction and Figure 1 for variable definitions.\n\nWe proceed to formalize the above descriptions as follows. A given read is denoted by r, with start and end coordinates (rs, re), if the read is continuous, as diagrammed within Figure 1(c), or otherwise, with start and end coordinate pairs, (rs1, re1) and (rs2, re2), as diagrammed within Figure 1(d). Let ℓ be the length of the read. The set ζ denotes the totality of validating reads. The criterion for r ∈ ζ is detailed below. It is important to note that validating reads are necessary but not sufficient to validate a variant. Sufficiency is achieved only if the number of validating reads is statistically significant relative to those present in control samples. ζ itself is partitioned into three sets: ζc, ζe, and ζi for evidence of cryptic splicing, exon skipping, and intron inclusion, respectively. We allow partitions to be empty. Let S denote the relevant splice junctions as defined by Figure 1 and Table 1. Without loss of generality, we consider only the red (i.e. direction is right) set of labels within Figure 1(b). Then the (splice consequence) partitions of ζ are given by:\n\nr ∈ ζc ⇔ variant is cryptic ∧ rs2 – re1 = C – S ∨ (rs > A ∧ re < S)\n\nr ∉ ζc ∧ variant is cryptic ∧ ¬ ( rs2 – re1 = C – S) ⇒ r ∈ anticryptic\n\nr ∈ ζe ⇔ (re1=A∧ rs2=B)\n\nr ∈ ζi ⇔ (A∈ [rs, re]) ∨ B ∈ [rs, re]) ∨ [(A ∉ [rs, re] ∧ B ∉ [rs, re]) ∧ (rs < A – ℓ ∧ re > B ∧ r ∉ (A, B))]\n\nWe separately partition ζ by its evidence type, the set of junction-spanning reads, δ and read-abundance reads, α, as follows:\n\nr ∈ δ ⇔ (A ∈ [rs, re] ∨ B ∈ [rs, re]) ∨ [r ∈ ζc ∧ rs2 – re1 = C – S]. r ∈ α ⇔ r ∉ δ\n\nOnce all validating reads are tallied for both the experimental and control samples, a p-value is computed. This is determined by computing a z-score upon Yeo-Johnson (YJ)16 transformed data. This transformation, shown in Equation 1, ensures that the data is sufficiently normally distributed to be amenable to parametric testing.\n\n\n\nThe transform is similar to the Box-Cox power transformation, but obviates the requirement of inputting strictly positive values and has more desirable statistical properties. Furthermore, this transformation allowed us to avoid the use of non-parametric testing, which has its own pitfalls regarding assumptions of the underlying data distribution17. We selected λ = 12, because Veridical’s untransformed output is skewed left, due to their being, in general, less validating reads in control samples and the fact that there are, by design, vastly more control samples than experimental samples. We found that this value for λ generally made the distribution much more normal.\n\nA comparison of the distributions of untransformed and transformed data is provided in Figure S1. We were not concerned about small departures from normality as a z-test with a large number of samples is robust to such deviations18. It is important to realize, therefore, that the p-values given by Veridical are much more robust when the program is provided with a large number of samples.\n\nThus, we can compute the p-value of the pairwise unions of the two sets of partitions of ζ, except the irrelevant ζe ∪ α = ∅. We only provide p-values for these pairwise unions and do not attempt to provide p-values for the partitions for the different consequences of the mutations on splicing. While such values would be useful, we do not currently have a robust means to compute them. Our previous work provides guidance on interpretation of splicing mutation outcomes3–5,10. Thus for ζx ∈ {ζc, ζe, ζi}, let ΦZ(z) represent the cumulative distribution function of the one-sided (right-tailed — i.e. P[X > x]) standard normal distribution. Let N represent the total number of samples and let V represent the set of all ζx validations, across all samples. Then:\n\n\n\nThe program outputs two tables, along with summaries thereof. The first table lists all validated read counts across all categories for experimental samples, while the second table does the same for the control samples. P-values are shown in parentheses within the experimental table, which refer to the column-dependent (i.e. the category is given in the column header) p-value for that category with respect to that same category in control samples. The program produces three files: a log file containing all details regarding validated variants, an output file with the programs progress reports and summaries, and a filtered validated variant file. The filtered file contains all validated variants of statistical significance (set as p < 0.05, by default), defined as variants with one or more validating read categories which achieve statistical significance in a relevant category (i.e. a cryptic variant for which p = 0.04 in the junction-spanning cryptic column would meet this criteria).\n\nWe elected to use RefSeq19 genes for the exome annotation, as opposed to, the more permissive exome annotation sets, UCSC Known Genes20 or Ensembl21. The large number of transcript variants within Ensembl, in particular, caused many spurious intron inclusion validation events. This occurred because reads were found to be intronic in many cases, when in actuality they were exonic with respect to the more common transcript variant. In addition, the inclusion of the large number of rare transcripts in Ensembl significantly increased program runtime and made validation events much more challenging to interpret unequivocally. The use of RefSeq, which is a conservative annotation of the human exome, resolves these issues.\n\nWe also provide an R program22 which produces publication quality histograms displaying embedded Q-Q plots and p-values, to evaluate for normality of the read distribution and statistical significance, respectively. The R program performs the YJ transformation as implemented in the car package23. The histograms generated by the program use the Freedman-Draconis24 rule for break determination, and the Q-Q plots use algorithm Type 8 for their quantile function, as recommended by Hyndman and Fan25. Lastly, a Perl program was implemented to automatically retrieve and correctly format an exome annotation file from the UCSC database20 for use in Veridical. All data uses hg19/GRCh37, however when new versions of the genome become available, this program can be used to update the annotation file.\n\n\nResults\n\nVeridical validates predicted mRNA splicing mutations using high-throughput RNA sequencing data. The performance of the software is affected by the number of predicted splicing mutations, the number of abnormal samples containing mutations and control samples and the corresponding RNA-Seq data for each type of sample. Veridical has the ability to analyze approximately 3000 variants in approximately 4 hours assuming an input of 100 BAM files of RNA-Seq data. The relationship between time and numbers of BAM files and variants are plotted in Figure 3 for a 2.27 GHz processor. Veridical uses memory in linear proportion to the number and size of the input BAM files. In our tests, using RNA-Seq BAM files with an average size of approximately 6 GB, Veridical used approximately 0.7 GB for ten files to 1 GB for 100 files. Currently, splicing consequences that are reported include intron inclusion, exon skipping, and cryptic splicing, which are validated through junction-spanning reads, or based on read-abundance in the region circumscribing the variant (see Methods for details). For example, a cryptic splicing junction-spanning read will show that the mRNA contains a truncated or extended exon at the predicted location, which is directly attached to the sequence of the corresponding adjacent exon. For mutations that alter read-abundance, each read within the genomic location assessed (i.e. intron for intron inclusion) is counted for the variant-containing samples and then compared with the number of reads in the control files. For each input variant, Veridical outputs the number of validating reads (i.e. RNA-Seq reads which corroborate the predicted splicing consequence) for a given splice consequence within the variant-containing tumour samples and within control samples (i.e. non-variant containing tumour samples and normal samples). The program provides read counts for the different categories for all experimental and control samples as tab-delimited tables, along with the relevant p-values, indicating the statistical probability that the predicted mutation exhibits a normal expression pattern.\n\nProfiling data for Veridical runtime. Tests were conducted upon an Intel Xeon @2.27 GHz. Visualizations were generated with R22 using Lattice26 and Effects27. A surface plot of time vs. numbers of BAM files and variants is provided in (A). Effect plots are given in (B) and demonstrate the effects of the numbers of BAM files and variants upon runtime. The effect plots were generated using a linear regression model (R2 = 0.7525).\n\nWe demonstrate how Veridical and its associated R program are used to validate predicted splicing mutations in somatic breast cancer. Each example depicts a particular variant-induced splicing consequence, analyzed by Veridical, with its corresponding significance level. The relevant primary RNA-Seq data are displayed in IGV, along with histograms and Q-Q plots showing the read distributions for each example. The source data are obtained from controlled-access breast carcinoma data from The Cancer Genome Atlas (TCGA)28. Tumour-normal matched DNA sequencing data from the TCGA consortium was used to predict a set of splicing mutations, and a subset of corresponding RNA sequencing data was analyzed to confirm these predictions with Veridical. The following examples demonstrate the utility of Veridical to identify potentially pathogenic mutations from a much larger subset of predicted variants.\n\nLeaky mutations are those variants that reduce, but not abolish, the spliceosome’s ability to recognize the intron/exon boundary3. This can lead to the mis-splicing (intron inclusion and/or exon skipping) of many but not all transcripts. An example of a predicted leaky mutation (chr5:162905690 G>T) in the HMMR gene in which both junction-spanning exon skipping (p < 0.01) and read-abundance-based intron inclusion (p = 0.04) are observed is provided within Figure 4. We predict this mutation to be leaky because its final Ri exceeds 1.6 bits — the minimal individual information required to recognize a splice site and produce correctly spliced mRNA4. Indeed, the natural site, while weakened by 2.16 bits, remains strong — 10.67 bits. This prediction is validated by the variant-containing sample’s RNA-Seq data (Figure 4), in which both exon skipping (5 reads) and intron inclusion (14 reads) are observed, along with 70 reads portraying wild-type splicing. Only a single normally spliced read contains the G→T mutation. These results are consistent with an imbalance of expression of the two alleles, as expected for a leaky variant. Figure 5 shows that for the distribution of read-abundance-based intron inclusion is statistically significant (p = 0.04).\n\nIGV images depicting a predicted leaky mutation (chr5:162905690 G>T) within the natural acceptor site of exon 12 (162905689–162905806) of HMMR. This gene has four transcript variants and the given exon number pertains to isoforms a and b (reference sequences NM_001142556 and NM_012484). RNA-Seq reads are shown in the centre panel. The bottom blue track depicts RefSeq genes, wherein each blue rectangle denotes an exon and blue connecting lines denote introns. In the middle panel, each rectangle (grey by default) denotes an aligned read, while thin lines are segments of reads split across exons. Red and blue coloured rectangles in the middle panel denote aligned reads of inserts that are larger or smaller than expected, respectively. (A) depicts a genomic region of chromosome 5: 162902054–162909787. The variant occurs in the middle exon. Intron inclusion can be seen in this image, represented by the reads between the first and middle exon (since the direction is right, as described within Table 1). These 14 reads are read-abundance-based, since they do not span the intron-exon junction. (B) depicts a closer view of the region shown in (A) — 162905660–162905719. The dotted vertical black lines are centred upon the first base of the variant-containing exon. The thin lines in the middle panel that span the entire exon fragment are evidence of exon skipping. These 5 reads are split across the exon before and after the variant-containing exon, as seen in (A).\n\nHistogram of read-abundance-based intron inclusion with embedded Q-Q plots of the predicted leaky mutation (chr5:162905690 G>T) within HMMR, as shown in Figure 4. The arrowhead denotes the number of reads (14 in this case) in the variant-containing file which is more than observed in the control samples (p = 0.04).\n\nVariants that inactivate splice sites have negative final Ri values3 with only rare exceptions4, indicating that splice site recognition is essentially abolished in these cases. We present the analysis of two inactivating mutations within the PTEN and TMTC2 genes from different tumour exomes, namely: chr10:89711873 A>G and chr12:83359523 G>A, respectively. The PTEN variant displays junction-spanning exon skipping events (p < 0.01), while the TMTC2 gene portrays both junction-spanning and read-abundance-based intron inclusion (both splicing consequences with p < 0.01). In addition, all intron inclusion reads in the experimental sample contain the mutation itself, while only one such read exists across all control samples analyzed (p < 0.01). The PTEN variant contains numerous exon skipping reads (32 versus an average of 2.466 such reads per control sample). The TMTC2 variant contains many junction-spanning intron inclusion reads with the G→A mutation (all of its junction-spanning intron inclusion reads: 22 versus an average of 0.002 such reads per control sample). IGV screenshots for these variants are provided within Figure 6. This figure also shows an example of junction-spanning cryptic splice site activated by the mutation (chr1:985377 C>T) within the AGRN gene. The concordance between the splicing outcomes generated by these mutations and the Veridical results indicates that the proposed method detects both mutations that inactivate splice sites and cryptic splice site activation.\n\n(A) depicts an inactivating mutation (chr10:89711873 A>G) within the natural acceptor site of exon 6 (89711874–89712016) of PTEN. The dotted vertical black line denotes the location of the relevant splice site. The region displayed is 89711004–89712744 on chromosome 10. Many of the 32 exon skipping reads are evident, typified by the thin lines in the middle panel that span the entire exon. There is also a significant amount of read-abundance-based intron inclusion, shown by the reads to the left of the dotted vertical line. Exon skipping was statistically significant (p < 0.01), while read-abundance-based intron inclusion was not (p = 0.53). Panels (B) and (C) depict an inactivating mutation (chr12:83359523 G>A) within the natural donor site of exon 6 (83359338–83359523) of TMTC2. (B) depicts a closer view (83359501–83359544) of the region shown in (C) and only shows exon 6. Some of the 22 junction-spanning intron inclusion reads can be seen. In this case, all of these reads contain the mutation, shown by the green adenine base in each read, between the two vertical dotted lines. (C) depicts a genomic region of chromosome 12: 83359221–83360885, TMTC2 exons 6–7. The variant occurs in the left exon. 65 read-abundance-based intron inclusion can be seen in this image, represented by the reads between the two exons. Panel (D) depicts a mutation (chr1:985377 C>T) causing a cryptic donor to be activated within exon 27 (the second from left, 985282–985417) of AGRN. The region displayed is 984876–985876 on chromosome 1 (exons 26–29 are visible). Some of the 34 cryptic (junction-spanning) reads are portrayed. The dotted black vertical line denotes the cryptic splice site, at which cryptic reads end. Refer to the caption of Figure 4 for IGV graphical element descriptions.\n\nRecurrent genetic mutations in some oncogenes have been reported among tumours within the same, or different, tissues of origin. Common recurrent mutations present in multiple abnormal samples are recognized by Veridical. This avoids including a variant-containing sample among the control group, and outputs the results of all of the variant-containing samples. A relevant example is shown in Figure 7. The mutation (chr1:46726876 G>T) causes activation of a cryptic splice site within RAD54L in multiple tumours. Upon computation of the p-values for each of the variant-containing tumours, relative to all non-variant containing tumours and normal controls, not all variant-containing tumours displayed splicing abnormalities at statistically significant levels. Of the six variant-containing tumours, two had significant levels of junction-spanning intron inclusion, and one showed statistically significant read-abundance-based intron inclusion. Details for all of the aforementioned variants, including a summary of read counts pertaining to each relevant splicing consequence, for experimental versus control samples, are provided in Table 2.\n\nIGV images and their corresponding histograms with embedded Q-Q plots depicting all six variant containing files with a mutation (chr1:46726876 G>T) which, in some cases, causes a cryptic donor to be activated within the intron between exons 7 and 8 of RAD54L. This results in the extension of the downstream natural donor (the 5´ end of exon 8). This gene has two transcript variants and the given exon numbers pertain to isoform a (reference sequence NM_003579). Only samples IV and V have statistically significant intron inclusion relative to controls. read-abundance-based intron inclusion can be seen in (A), between the two exons. The region displayed is on chromosome 1: 46726639–46726976. (B) depicts the corresponding histogram for the 15 read-abundance-based intron inclusion reads (p = 0.05) that are present in sample IV. The intron-exon boundary on the right is the downstream natural donor. (C) typifies some of the 13 junction-spanning intron inclusion reads that are a direct result of the intronic cryptic site’s activation. In these instances, reads extending past the intron-exon boundary are being spliced at the cryptic site, instead of the natural donor. In particular, samples IV and V both have a statistically significant numbers of such reads, 7 (p = 0.01) and 5 (p = 0.04), respectively. This is further typified by the corresponding histogram in (D). (C) focuses upon exon 8 from (A) and displays the genomic positions 46726908–46726957. Refer to the caption of Figure 4 for IGV graphical element descriptions. In the histograms, arrowheads denote numbers of reads in the variant-containing files. The bottom of the plots provide p-values for each respective arrowhead. Statistically significant p-values and their corresponding arrowheads are denoted in red.\n\nHeaders containing R with some subscript denote numbers of validated reads for the specified variant’s splicing consequence(s) and evidence type(s). RE denotes reads within variant-containing tumour samples. RT and RN denote control samples, for tumours and normal cells, respectively. Rμ is the per sample mean of RT and RN. Splicing consequences: CS denotes cryptic splicing, ES denotes exon skipping, and II denotes intron inclusion. Evidence types: JS denotes junction-spanning and RA denotes read-abundance.\n\n\nDiscussion\n\nWe have implemented Veridical, a software program that automates confirmation of mRNA splicing mutations by comparing sequence read-mapped expression data from samples containing variants that are predicted to cause defective splicing with control samples lacking these mutations. The program objectively evaluates each mutation with statistical tests that determine the likelihood of and exclude normal splicing. To our knowledge, no other software currently validates splicing mutations with RNA-Seq data on a genome-wide scale, although many applications can accurately detect conventional alternative splice isoforms (i.e.29). Veridical is intended for use with large data sets derived from many samples, each containing several hundred variants that have been previously prioritized as likely splicing mutations, regardless of how the candidate mutations are selected. It is not practical to analyze all variants present in an exome or genome, rather only a filtered subset, due to the extensive computations required for statistical validation. As such, Veridical is a key component of an end-to-end, hypothesis-based, splicing mutation analysis framework that also includes the Shannon splicing mutation pipeline10 and the Automated Splice Site Analysis and Exon Definition server5.\n\nThere is a trade-off between lengthy run-times and statistical robustness of Veridical, especially when there are either a large number of variants or a large number of RNA-Seq files. As with most statistical methods, those employed here are not amenable to small sample sets, but become quite powerful when a large number of controls are employed. In order to ensure that mutations can be validated, we recommend an excess of control transcriptome data relative to those from samples containing mutations (> 5 : 1), regardless of the computational expense. We do not recommend the use of a single control to corroborate a sample containing a putative mutation. Not surprisingly, we have found that junction-spanning reads have the greatest value for corroborating cryptic splicing and exon skipping. Even a single such read is almost always sufficient to merit the validation a variant, provided that sufficient control samples are used. For intron inclusion, both junction-spanning and read-abundance-based reads are useful and a variant can readily be validated with either, provided that the variant-containing experimental sample(s) show a statistically significant increase in the presence of either form of intron inclusion corroborating reads.\n\nVeridical is able to automatically process variants from multiple different experimental samples, and can group the variant information if any given mutation is present in more than one sample. The use of a large sample size allows for robust statistical analyses to be performed, which aid significantly in the interpretation of results. The main utility of Veridical is to filter through large data sets of predicted splicing mutations to prioritize the variants. This helps to predict which variants will have a deleterious effect upon the protein product. Veridical is able to avoid reporting splicing changes that are naturally occurring through checking all variant-containing and non-containing control samples for the predicted splicing consequence. In addition, running multiple tumour samples at once allows for manual inspection to discover samples that contained the alternative splicing pattern, and consequently, permits the identification of DNA mutations in the same location which went undetected during genome sequencing.\n\nThe statistical power of Veridical is dependent upon the quality of the RNA-Seq data used to validate putative variants. In particular, a lack of sufficient coverage at a particular locus will cause Veridical to be unable to report any significant results. A coverage of at least 20 reads should be sufficient. This estimate is based upon alternative splicing analyses in which this threshold was found to imply concordance with microarray and RT-PCR measurements30–33. There are many potential legitimate reasons why a mutation may not be validated: (a) nonsense-mediated decay may result in a loss of expression of the entire transcript, (b) the gene itself may have multiple paralogs and reads may not be unambiguously mapped, (c) other non-splicing mutations could account for a loss of expression, and (d) confounding natural alternative splicing isoforms may result in a loss of statistical significance during read mapping of the control samples. The prevalence of loci with insufficient data is dependent upon the coverage of the sequencing technology used. As sequencing technologies improve, the proportion of validated mutations is expected to increase. Such an increase would mirror that observed for the prevalence of alternative splicing events34. It is important to note that acceptance of the null hypothesis, due to an absence of evidence required to disprove it, does not imply that the underlying prediction of a mutation at a particular locus is incorrect, but merely that the current empirical methods employed were insufficient to corroborate it.\n\nWhile there is considerable prior evidence for splicing mutations that alter natural and cryptic splice site recognition, we were somewhat surprised at the apparent high frequency of statistically significant intron inclusion revealed by Veridical. In fact, evidence indicates that a significant portion of the genome is transcribed34, and it is estimated that 95% of known genes are alternatively spliced30. Defective mRNA splicing can lead to multiple alternative transcripts including those with retained introns, cassette exons, alternate promoters/terminators, extended or truncated exons, and reduced exons35. In breast cancer, exon skipping and intron retention were observed to be the most common form of alternative splicing in triple negative, non-triple negative, and HER2 positive breast cancer36. In normal tissue, intron retention and exon skipping has been predicted to affect 2572 exons in 2127 genes and 50 633 exons in 12 797 genes, respectively37. In addition, previous studies suggest that the order of intron removal can influence the final mRNA transcript composition of exons and introns38. Intron inclusion observed in normal tissue may result from those introns that are removed from the transcript at the end of mRNA splicing. Given that these splicing events are relatively common in normal tissues, it becomes all the more important to distinguish expression patterns that are clearly due to the effects of splicing mutations — one of the guiding principles of the Veridical method.\n\nVeridical is an important analytical resource for unsupervised, thorough validation of splicing mutations through the use of companion RNA-Seq data from the same samples. The approach will be broadly applicable for many types of genetic abnormalities, and should reveal numerous, previously unrecognized, mRNA splicing mutations in exome and complete genome sequences.\n\n\nData availability\n\nfigshare: Input, output, and explanatory files for Veridical, http://dx.doi.org/10.6084/m9.figshare.89497139.",
"appendix": "Author contributions\n\n\n\nPKR elaborated the problem and conceived of the analytic solution. CV implemented the algorithm, wrote, and tested the Veridical software and its accompanying Perl and R scripts. CV and SND designed the methods used, generated, and interpreted mutation results. BCS wrote and tested the Shannon pipeline, which provided the predictions evaluated by Veridical. BCS and CV implemented procedures and code for software distribution. CV, SND, and PKR wrote the manuscript, which has been approved by all of the authors.\n\n\nCompeting interests\n\n\n\nPKR is the inventor of US Patent 5,867,402 and other patents pending, which underlie the prediction and validation of mutations. He is one of the founders of Cytognomix, Inc. which is developing software based on this technology for complete genome or exome splicing mutation analysis. BCS is an employee of Cytognomix, Inc. CV and SND declare that they have no competing interests. Both trial and licensed versions of Veridical are fully functional, however the length of the trial period is limited.\n\n\nGrant information\n\nPKR is supported by the Canadian Breast Cancer Foundation, Canadian Foundation for Innovation, Canada Research Chairs Secretariat and the Natural Sciences and Engineering Research Council of Canada (NSERC Discovery Grant 371758-2009). SND received fellowships from the Ontario Graduate Scholarship Program, the Pamela Greenaway-Kohlmeier Translational Breast Cancer Research Unit, and the CIHR Strategic Training Program in Cancer Research and Technology Transfer, and the University of Western Ontario.\n\n\nAcknowledgments\n\nWe acknowledge the TCGA consortium for providing the data used in this article. The data used in this article is part of TCGA project #988: \"Predicting common genetic variants that alter the splicing of human gene transcripts\". This work was made possible by the facilities of the Shared Hierarchical Academic Research Computing Network (SHARCNET) and Compute/Calcul Canada.\n\n\nSupplementary materials\n\nThis input format most easily accepts formatted output from the Shannon Pipeline. In particular, all variants of interest should be concatenated into a single file. Once a, tab-delimited, concatenated file has been generated, it can easily be formatted correctly by using FilterShannonPipelineResults.pl. One can also manually ensure the following: the header line has no quotation marks or special characters, empty columns have been replaced by a period (.) and each variant line contains only a single gene (comma-delimited gene lists must be split such that there is only one gene per line). If one wishes Veridical to consider variants pertaining to more than one experimental sample, a comma-delimited list of experimental samples, in the form of BAM file names, must be provided as the key column. The key column must always contain at least one file name that is present as the base name of one of the files listed in the BAM file list that must be passed to Veridical.\n\nAlternatively, one can prepare the input format as follows. The header must contain at least the following, case-insensitive, values to which the file’s columns must adhere to: chromosome, splice&coordinate, strand, type, gene, location, location_type, heterozygosity, variant, input, key. The column headers need only contain the given text (i.e. a column labeled gene_name would be sufficient to satisfy the above requirement for a “gene” column). Column headers with ampersands (&) denote that all words joined by this symbol must be present for that column (i.e. Splice_site_coordinate satisfies the “splice&coordinate” requirement). The order of the columns is immaterial. The input column can contain any identifier for the variant and need not be unique. The location column specifies if the site is natural or cryptic. For Veridical, all that matters is that cryptic variants contain the word “cryptic” as part of their value in this column and that non-cryptic variants do not. The location_type column is only used for cryptic variants and specifies if the variant is intronic or exonic. It is not currently used by the program. This column must be present but can always be set to null (i.e. .).\n\nA few rows from a sample variant file is provided below (text wrapped for readability):\n\n\n\nThis input format can be generated via Convert To Exome Annotation.pl. The file must be tab-delimited, excepting its header, which must be comma-delimited. It must have the following, case-insensitive, header columns, to which its data must adhere: transcript, chromosome, exon chr start, exon chr end, exon rank, gene. The column headers need only contain the given text (i.e. a column labeled gene_name would be sufficient to satisfy the above requirement for a “gene” column). The order of the columns is immaterial.\n\nA few rows from a sample exome annotation file is provided below (text wrapped for readability):\n\n\n\nIf a variant contains any validating reads, Veridical outputs the variant in question, along with some summary information and a table specifying the numbers of each validating read type detected for both the experimental and control samples. Within the output of Veridical, the phrase: “Validated (x ) variant n times” means that the variant was validated mainly for splicing consequence x and has n validating reads. The variant will only appear within the *.filtered output file if the p-value for either junction-spanning or read-abundance-based reads for splicing consequence x was statistically significant (defined, by default, as: p < 0.05). After the variant being validated is provided, along with its primary predicted splicing consequence, the output is divided into two sections with identical contents: one for the experimental sample(s) and another for control samples. The summary enumerates the number of reads of each splicing consequence, partitioned by evidence type (junction-spanning or read-abundance-based), and by sample type (tumour or normal for control samples, and only tumour for experimental samples). A table describing the number of each read type for every file follows this summary. An example of this output, for the variant within RAD54L, as shown by Figure 7 and the last portion of Table 2, is provided. While Veridical outputs this as plain text, with the table in a tab-delimited format, we provide this output as an Excel document with descriptions of the meaning of each table heading, to clarify the presentation of the data. All input and output files for the five variants presented are provided. VeridicalOutExample.xls contains the output for the variant within RAD54L, along with descriptions of the terms used and the output format. all.vin contains the input variant file. allTumoursBAMFileList.txt and allNormalsBAMFileList.txt are the BAM file lists for tumour and normal samples, respectively. all.vout contains the Veridical output. The exome file can be retrieved using ConvertToExomeAnnotation.pl, available with the other programs at: www.veridical.org. The BAM file lists contain the TCGA file UUID, followed by a slash, followed by the file name. The RNA-Seq data itself can be downloaded from TCGA at: https://tcga-data.nci.nih.gov/tcga/.\n\n\nReferences\n\nRogan PK, Zou GY: Best practices for evaluating mutation prediction methods. Hum Mutat. 2013; 34(11): 1581–1582. PubMed Abstract | Publisher Full Text\n\nKrawczak M, Reiss J, Cooper DN: The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: Causes and consequences. Hum Genet. 1992; 90(1–2): 41–54. PubMed Abstract | Publisher Full Text\n\nRogan PK, Faux BM, Schneider TD: Information analysis of human splice site mutations. Hum Mutat. 1998; 12(3): 153–171. PubMed Abstract | Publisher Full Text\n\nRogan PK, Svojanovsky S, Leeder JS: Information theory-based analysis of CYP2C19, CYP2D6 and CYP3A5 splicing mutations. Pharmacogenetics. 2003; 13(4): 207–218. PubMed Abstract | Publisher Full Text\n\nMucaki EJ, Shirley BC, Rogan PK: Prediction of mutant mRNA splice isoforms by information theory-based exon definition. Hum Mutat. 2013; 34(4): 557–565. PubMed Abstract | Publisher Full Text\n\nLópez-Bigas N, Audit B, Ouzounis C, et al.: Are splicing mutations the most frequent cause of hereditary disease? FEBS Lett. 2005; 579(9): 1900–1903. PubMed Abstract | Publisher Full Text\n\nWang K, Li M, Hakonarson H: ANNOVAR: Functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 2010; 38(16): e164. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChurbanov A, Vorechovský I, Hicks C: A method of predicting changes in human gene splicing induced by genetic variants in context of cis-acting elements. BMC Bioinformatics. 2010; 11(1): 22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPertea M, Lin X, Salzberg SL: GeneSplicer: A new computational method for splice site prediction. Nucleic Acids Res. 2001; 29(5): 1185–1190. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShirley BC, Mucaki EJ, Whitehead T, et al.: Interpretation, stratification and evidence for sequence variants affecting mRNA splicing in complete human genome sequences. Genomics Proteomics Bioinformatics. 2013; 11(2): 77–85. PubMed Abstract | Publisher Full Text\n\nEswaran J, Cyanam D, Mudvari P, et al.: Transcriptomic landscape of breast cancers through mRNA sequencing. Sci Rep. 2012; 2: 264. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEswaran J, Horvath A, Godbole S, et al.: RNA sequencing of cancer reveals novel splicing alterations. Sci Rep. 2013; 3: 1689. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKwan T, Benovoy D, Dias C, et al.: Genome-wide analysis of transcript isoform variation in humans. Nat Genet. 2008; 40(2): 225–231. PubMed Abstract | Publisher Full Text\n\nThorvaldsdóttir H, Robinson JT, Mesirov JP: Integrative genomics viewer (IGV): High-performance genomics data visualization and exploration. Brief Bioinform. 2013; 14(2): 178–192. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBarnett DW, Garrison EK, Quinlan AR, et al.: BamTools: A C++ API and toolkit for analyzing and managing BAM files. Bioinformatics. 2011; 27(12): 1691–1692. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYeo IK, Johnson RA: A new family of power transformations to improve normality or symmetry. Biometrika. 2000; 87(4): 954–959. Publisher Full Text\n\nJohnson DH: Statistical sirens: the allure of nonparametrics. Ecology. 1995; 76: 1998–2000. Reference Source\n\nHubbard R: The probable consequences of violating the normality assumption in parametric statistical analysis. Area. 1978; 10(5): 393–398. Reference Source\n\nPruitt KD, Tatusova T, Maglott DR: NCBI reference sequence (RefSeq): A curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res. 2005; 33(Database Issue): D501–D504. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHsu F, Kent JW, Clawson H, et al.: The UCSC known genes. Bioinformatics. 2006; 22(9): 1036–1046. PubMed Abstract | Publisher Full Text\n\nHubbard T, Barker D, Birney E, et al.: The Ensembl genome database project. Nucleic Acids Res. 2002; 30(1): 38–41. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRDC Team R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing, 2008. Reference Source\n\nFox J, Weisberg S: An R Companion to Applied Regression, 2nd ed. Thousand Oaks CA: Sage, 2011. Reference Source\n\nFreedman D, Diaconis P: On the histogram as a density estimator: L2 theory. Zeitschrift für Wahrscheinlichkeitstheorie und Verwandte Gebiete. 1981; 57(4): 453–476. Publisher Full Text\n\nHyndman RJ, Fan Y: Sample quantiles in statistical packages. American Statistician. 1996; 50(4): 361–365. Publisher Full Text\n\nSarkar D: Lattice: Multivariate Data Visualization with R. New York: Springer, 2008. Reference Source\n\nFox J: Effect displays in R for generalised linear models. J Stat Softw. 2003; 8(15): 1–27. Reference Source\n\nKoboldt DC, Fulton RS, McLellan MD, et al.: Comprehensive molecular portraits of human breast tumours. Nature. 2012; 490(7418): 61–70. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShen S, Park JW, Huang J, et al.: MATS: A bayesian framework for flexible detection of differential alternative splicing from RNA-Seq data. Nucleic Acids Res. 2012; 40(8): e61. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPan Q, Shai O, Lee LJ, et al.: Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat Genet. 2008; 40(12): 1413–1415. PubMed Abstract | Publisher Full Text\n\nGriffith M, Griffith OL, Mwenifumbo J, et al.: Alternative expression analysis by RNA sequencing. Nat Methods. 2010; 7(10): 843–847. PubMed Abstract | Publisher Full Text\n\nKatz Y, Wang ET, Airoldi EM, et al.: Analysis and design of RNA sequencing experiments for identifying isoform regulation. Nat Methods. 2010; 7(12): 1009–1015. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShen S, Lin L, Cai JJ, et al.: Widespread establishment and regulatory impact of Alu exons in human genes. Proc Natl Acad Sci U S A. 2011; 108(7): 2837–2842. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKapranov P, Willingham AT, Gingeras TR: Genome-wide transcription and the implications for genomic organization. Nat Rev Genet. 2007; 8(6): 413–423. PubMed Abstract | Publisher Full Text\n\nFeng H, Qin Z, Zhang X: Opportunities and methods for studying alternative splicing in cancer with RNA-Seq. Cancer Lett. 2013; 340(2): 179–191. PubMed Abstract | Publisher Full Text\n\nEswaran J, Horvath A, Godbole S, et al.: RNA sequencing of cancer reveals novel splicing alterations. Sci Rep. 2013; 3: 1689. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPal S, Gupta R, Davuluri RV: Alternative transcription and alternative splicing in cancer. Pharmacol Ther. 2012; 136(3): 283–294. PubMed Abstract | Publisher Full Text\n\nTakahara K, Schwarze U, Imamura Y, et al.: Order of intron removal influences multiple splice outcomes, including a two-exon skip, in a COL5A1 acceptor-site mutation that results in abnormal pro-a1 (V) N-propeptides and Ehlers-Danlos syndrome type I. Am J Hum Genet. 2002; 71(3): 451–465. PubMed Abstract | Publisher Full Text | Free Full Text\n\nViner C, Dorman SN, Shirley BC, et al.: Input, output, and explanatory files for Veridical. figshare, 2013. Data Source"
}
|
[
{
"id": "3120",
"date": "27 Jan 2014",
"name": "Stefania Bortoluzzi",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper “Validation of predicted mRNA splicing mutations using high-throughput transcriptome data” by Viner et al. presents Veridical, a new software for the interpretation and validation of genetic variants identified by DNA sequencing that alter mRNA splicing, leveraging RNA-seq data. The method is based on statistical comparisons of the normalized read counts of abnormally-spliced RNA species in mutant versus non-mutant tissues. Actually, the interpretation of genetic variants is a difficult and key issue in current research.The integration of genomic and transcriptomic data, namely the use of RNA-seq-based transcriptome characterization as a “molecular phenotype” of cells is useful and meaningful. The software is standalone (not a web-tool) and it is completed by perl scripts, facilitating data management.The manuscript declare that “Veridical and its associated software programs are available at: www.veridical.org”.Actually, Veridical is commercially available to the scientific community. A trial version lasting 30 days can be downloaded by the website, but in order to obtain binaries, the website requests a registration with an institutional email address - they reserve the right to deny access to users who register with third-party mail servers (Gmail, Yahoo, Hotmail, etc.).No pricing information is included in the manuscript and, more importantly, in the webpages accessible to download the software, either before or after registration.After downloading the software, I was not able to find R scripts that can be useful to generate some plots, as indicated in the manuscript. Saying that, the paper is written in a clear language and it is quite complete.I propose a few revisions that in my opinion can improve the manuscript readability and clarity.Introduction Line 13 (minor): indicate which hereditary disease (colon cancer?). Methods 2nd Par, Line 5 (minor): “Maximising” is used, but probably the meaning is “increasing” (the number of). Figure 1 (major): I feel that the info provided by points C and D is trivial, whilst point A's images, sentences and legends can be improved. Figure 1 C and D shows simply examples of reads that are mapped continuously and discontinuously to the reference genome. I think that every potential user of this type of software known well this concept. On the other hand, regarding A and B (upper part of the figure) there is not clear correspondence between the text in the legend and the image, and between the image and the text below (the arch overlap the point A in the figure B, whereas the text says \"reads between A and B\"). In general, in many cases in the manuscript, the correspondence between legend and figure can be improved, by indicating more clearly the points specific sentences in the legend refer to. Regarding this issue, for instance in Figure 4 I can see indicated neither the “exon 12” nor the “14 reads” mentioned in the legend. Please indicate (using colors, boxes, arrows or overlapping text) key elements in the figure, and revise all figures using the same criterion. Page 5 (minor): Consider revising the sentence “Furthermore, this transformation allowed us to avoid the use of non-parametric testing, which has its own pitfalls regarding assumptions of the underlying data distribution”, since normally it is assumed that parametric tests ground on assumptions on data distributions, but non-parametric tests by definition can be used without information about data distribution. End of the next paragraph (major): “It is important to realize, therefore, that the p-values given by Veridical are much more robust when the program is provided with a large number of samples.” This is a pretty clear concept. Please, indicate a general rule to the user/reader: How many samples are required? Setting a reasonable minimum can be more useful for experimental design than saying the larger the sample size the most robust the result. Results I have two important criticisms about the Results section:The section is not organized in paragraphs, and mixes performance info (run time using different number of samples and variants) with example results. Not clearly saying how these results were obtained. This is important to guarantee repeatability. (Major) I propose to reorganize the results (considering skipping less important examples; retain surely Fig. 4 and 6) and insert a first paragraph providing information about the dataset used for variants validation (how many samples, how many controls) and about the variant calling (BAM files can be obtained with different settings and criteria and the same apply to calling and filtering of variants). Moreover, please explain how RNA-seq data are treated, and particularly how they are normalized to guarantee cross-samples comparability. (Major) Also, a brief discussion about the impact of disease samples not carrying the given mutation can be useful, as well as regarding the possibility that a tumour sample not carrying the considered variant can present altered transcriptome since other variants (or factors) impact on the “molecular phenotype”. Figure 4 (minor): Please comment about the possible existence of intronic transcripts (totally unknown or also annotated in Ensemble, but not displayed in the more conservative RefSeq annotations). Figure 5 (minor): Please define better the measure “Read−Abundance Total Intron Inclusion”.",
"responses": [
{
"c_id": "739",
"date": "27 Mar 2014",
"name": "Peter K Rogan",
"role": "Author Response",
"response": "Thank you for your constructive comments. Regarding the R program, it is embedded within a Perl script to facilitate expeditious parameter and data parsing. All of our figures were generated using the Perl script, which, in-turn, invokes the R script. The program used to generate the histograms is called: VeridicalHist.pl. The invocations of this program are provided in the Perl program documentation on www.veridical.org. We have clarified this in the revision of the manuscript. We will update this web page to include pricing information and details about trial access to the software. The requirement to provide an institutional email address is quite ubiquitous across a number of academic disciplines, including many fields of informatics. In bioinformatics, for example, ANNOVAR requires non-profit users to register an address and HGMD does as well. ChemAxon, software for cheminformatics requires this. In astroinformatics, Sloan Digital Sky Survey III (SDSSIII) also requires an institutional email. These examples are represent a fraction of the numerous scientific websites that request such emails. We consider it a professional courtesy to provide legitimate email information, and reserve the right to disapprove addresses of individuals who seek to mask their identity, which may enable them to avoid acknowledging the provenance of the software. We specifically address each of your other comments below. All references to figures pertain to the first version of the manuscript. \"Line 13 (minor): indicate which hereditary disease (colon cancer?).\"López-Bigas et al. (2005) conducted a general analysis using the complete sets of SwissProt genes and OMIM known disease genes. Their mathematical model does not depend upon any specific hereditary disease. Nevertheless, certain genes have been demonstrated to exhibit very high frequencies of splicing mutations (ATM, NF1). \"2nd Par, Line 5 (minor): “Maximising” is used, but probably the meaning is “increasing” (the number of).\"This has been corrected. \"Figure 1 (major): I feel that the info provided by points C and D is trivial, whilst point A's images, sentences and legends can be improved. Figure 1 C and D shows simply examples of reads that are mapped continuously and discontinuously to the reference genome. I think that every potential user of this type of software known well this concept. On the other hand, regarding A and B (upper part of the figure) there is not clear correspondence between the text in the legend and the image, and between the image and the text below (the arch overlap the point A in the figure B, whereas the text says \"reads between A and B\").\"This has been revised accordingly. \"In general, in many cases in the manuscript, the correspondence between legend and figure can be improved, by indicating more clearly the points specific sentences in the legend refer to. Regarding this issue, for instance in Figure 4 I can see indicated neither the “exon 12” nor the “14 reads” mentioned in the legend. Please indicate (using colors, boxes, arrows or overlapping text) key elements in the figure, and revise all figures using the same criterion.\"In general, we provide exon numbers for reference and to allow for future exon identification. While we specifically describe in the captions which exon contains the variant and relate this to the exon number, we agree that graphical indications of key figure elements within IGV screenshots could improve the clarity. These figures have been revised accordingly. \"Page 5 (minor): Consider revising the sentence “Furthermore, this transformation allowed us to avoid the use of non-parametric testing, which has its own pitfalls regarding assumptions of the underlying data distribution”, since normally it is assumed that parametric tests ground on assumptions on data distributions, but non-parametric tests by definition can be used without information about data distribution.\"In this sentence, we are not referring to the ubiquitous assumption of an underlying normal distribution, which is indeed not required for non-parametric tests. Instead, the reference is to, other, lesser known, assumptions that are tacit in most non-parametric methods. The citation we provide (Johnson, 1995) refutes the commonly-held notion that non-parametric tests make no assumptions about the underlying data distribution, and describes numerous pitfalls that can occur when using non-parametric methods. For example, the author articulates a particular assumption implicit in a comparison of means via the Mann-Whitney test which actually requires that, “the two distributions are identical, in shape and scale, differing only in their means. This assumption can be harder to justify than the asymptotic normality demanded by the t test, and is rarely evaluated.” \"End of the next paragraph (major): “It is important to realize, therefore, that the p-values given by Veridical are much more robust when the program is provided with a large number of samples.” This is a pretty clear concept. Please, indicate a general rule to the user/reader: How many samples are required? Setting a reasonable minimum can be more useful for experimental design than saying the larger the sample size the most robust the result.\"The revised manuscript describes the procedure and criteria for determining the number of control samples needed. \"The section is not organized in paragraphs, and mixes performance info (run time using different number of samples and variants) with example results.\"The profiling information and example results are more clearly demarcated in the revision. \"(Major) I propose to reorganize the results (considering skipping less important examples; retain surely Fig. 4 and 6) and insert a first paragraph providing information about the dataset used for variants validation (how many samples, how many controls) and about the variant calling (BAM files can be obtained with different settings and criteria and the same apply to calling and filtering of variants). Moreover, please explain how RNA-seq data are treated, and particularly how they are normalized to guarantee cross-samples comparability.\"We now elaborate upon experimental protocols, processing, and data providence. The details of the RNA-Seq data and BAM file generation can obtained from the TCGA consortia, which generated them, specifically in the supplementary methods of Koboldt et al. (2012). All of our data input directly into Veridical are available, and even the program used to generate the histograms is provided. This ensures that all of our results are reproducible.The input data were obtained by analyzing the original TCGA dataset with the Shannon pipeline, as the paper describes. The pipeline has been published in a peer-reviewed context and is also available on a trial basis. The details of the TCGA analytical results obtained with the Shannon pipeline are considerable in length, putting them beyond the scope of this paper and will be described elsewhere.The journal guidelines concerning “web tools” state: “The article should provide examples of suitable input data sets and include an example of the output that can be expected from the tool and how this output should be interpreted.” The examples shown are representative of the splicing analysis outcomes present in the full TCGA analysis. The examples provided in this paper were designed to illustrate the capabilities of Veridical and are not an attempt to forge biological conclusions of this large dataset. \"(Major) Also, a brief discussion about the impact of disease samples not carrying the given mutation can be useful, as well as regarding the possibility that a tumour sample not carrying the considered variant can present altered transcriptome since other variants (or factors) impact on the “molecular phenotype”.\"We provide in Table 2 the read counts for non-variant containing tumour samples, and for the variant containing samples. We acknowledge that cancer gene expression results in a gross dysregulation of mRNA splicing thereby causing the presentation of the altered transcriptome. This was a motivating factor behind our choice of statistical method. \"Figure 4 (minor): Please comment about the possible existence of intronic transcripts (totally unknown or also annotated in Ensemble, but not displayed in the more conservative RefSeq annotations).\"If such exons exist, which are annotated as introns by RefSeq, and are actively transcribed in normal or breast cancer tissues, the large number of control samples will reflect this and such events will accordingly not trigger statistically significant intron inclusion events. We have added a comment to the Discussion reflecting this. \"Figure 5 (minor): Please define better the measure “Read−Abundance Total Intron Inclusion”.\"This has been addressed in the revision."
}
]
},
{
"id": "3119",
"date": "28 Jan 2014",
"name": "Francesc Xavier Roca",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript describes a new computational tool named Veridical, which detects mutant-allele specific splicing changes from large RNAseq datasets. This outstanding tool appears very useful to screen the wealth of transcriptomic data for effects in splicing due to mutations in disease samples, and I think that it will potentially be of interest for many if not all such RNAseq-based studies. In addition, this could spur further efforts to derive similar tools with improved efficiencies. Use of this method should help establish the importance of aberrant splicing in disease as well as the effects of genomic mutations at the RNA level. I only have two comments, that do not diminish my overall rating of this work as of high value: I personally disagree with the widespread use of the word “validation” in the title, abstract and text. Authors describe Veridical as a tool to “validate” DNA sequence variants that alter splicing. Indeed, I think that this tool provides an “association” between the variants and splicing, but not a formal proof of their connection. As the genomic and RNA samples usually come from different individuals with many confounding variables, the possibility that the splicing changes arise from factors other than the individual DNA mutations cannot be ruled out. In other words, changes in the levels of trans-acting splicing factors could account in part or totally for the splicing changes across samples. The statistical tests properly conducted in Veridical are designed to minimize such possibility but do not rule it out. In addition, the inherent noisy nature of RNAseq datasets also prompts for caution in the conclusions. To me, the direct proof that a DNA mutation changes splicing of its pre-mRNA can only be provided using minigenes and cell transfection (or in vitro splicing), in which the substrate sequences and cellular context are under almost absolute control. Indeed, the Veridical method is reminiscent of GWAS (Genome-Wide Association Studies), in which the genotype in the DNA, wild-type or mutant, is associated to its phenotype, such as normal versus disease (or other traits) in GWAS, or normal versus aberrant splicing in this study. Thus, for me Veridical provides strong associations – but not validations – between DNA mutations and their effects on splicing. As mentioned briefly at the beginning of Discussion, Veridical has built-in prediction tools to prioritize the mutations that are more likely to affect splicing, such as those mapping to splice sites. Even if other sources and tools are cited, a more extensive explanation of these components of Veridical would help the reader/user.",
"responses": [
{
"c_id": "740",
"date": "27 Mar 2014",
"name": "Peter K Rogan",
"role": "Author Response",
"response": "We greatly appreciate your review of this paper. Regarding our use of the term “validation”, we understand that in silico validation is not comparable to in vitro or in vivo validation assays. That said, we would like to elaborate. The DNA and RNA samples were matched from the same individual, which is not the case for many in vitro assays, i.e. mini-gene expression analysis by reverse transcription followed by PCR. The very large set of controls used in our analysis is also atypical in experimental validation of proposed mutations. When results are obtained that are statistically significant, it is conventional to refer to them as “validating”. Even if DNA-Seq and RNA-Seq data were not matched from the same individual, Veridical would still determine if known splicing mutations were expressed in a known subset of tumors, however we would instead refer to this as \"corroborating\".The comparison with GWAS is inappropriate: we are not comparing means of distinct case and control distributions; rather we are computing the read distribution from the control samples and determining the probability that the mutation bearing sample falls within that distribution. Additionally, the initial hypothesis in a GWAS is quite vague. Thus, the resultant associations are not of the same category as those provided by Veridical. While Veridical does indeed validate the splicing consequence observed, when we say that it validates the mutation we do only mean that it strongly corroborates the mutation as a causative agent of the splicing consequence. The responsibility to decide if the p-value reported by the program is sufficient is left up to the user, who should avoid incorrect post hoc ergo propter hoc arguments. Our reference to prioritization of variants for subsequent verification is based upon the result of Veridical's statistical tests. We explicitly mention that the software is not well suited for the analysis of raw output from genome-scale analyses, and that filtering should be performed a priori, as we conducted, with a separate Perl script, which is available with Veridical."
}
]
},
{
"id": "3524",
"date": "07 Mar 2014",
"name": "Liliana D. Florea",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe a method and the associated software, Veridical, for assessing the effects on pre-mRNA splicing of predicted splicing-affecting mutations. To do so the program compares splicing effects, measured by the supporting read counts, in variant-containing (disease) samples against a distribution derived from very large numbers of 'normals', either normal tissue from the same individual or samples from healthy individuals.The idea is ingenious and novel as applied to mutations affecting splicing, although not in general (see VAAST [Yandell et al. 2011], which exploits the availability of large numbers of samples to identify likely deleterious variants; it is also the premise for the HapMap and 1000 Genomes projects). The software is fast and practical, being able to test thousands of variants in hundreds of samples within hours. This is the first software of its kind, and if accurate it will be a very valuable resource for clinical genomics.That being said, while the article provides proof-of-concept and clearly demonstrates the potential of the tool with specific examples, there are several missing pieces that are needed to provide the readers with a view of its overall performance and limitations and to help them use it effectively.Major comments:The article shows numerous positive examples, however there is no indication of the tool's performance in general. The authors should include the results from running the tool on a full data set, to give potential users an idea of the expected outcome.Also, several other tools (e.g., MATS, Miso, SpliceTrap) have been developed for the related problem of discovering alternative splicing events and comparing them among samples. MATS in particular, allows differential splicing analyses with multiple replicates. Ideally the paper would include a comparison with MATS on the data set analyzed; this comparison is informative even if MATS is used with only a subset of the samples. The method uses the YJ-transformed distribution of supporting read counts across the 'normals' to determine a p-value for the variant, and thus judge its significance and impact on splicing. This is an interesting concept that assumes that with large numbers of 'normals' sample and batch effects will even out; hence, large numbers of samples are required to ensure accuracy. Since these are absolute (non-normalized) counts, however, the method may not work if the variant sample is obtained with a different method, e.g. by rRNA depletion of total RNA whereas most normal samples would come from polyA+ libraries. The authors should clearly discuss this and other possible limitations of their approach. Related to the above, the authors mention on several occasions the difficulty in identifying intron inclusion (II) events, in particular the large number of false positives. Indeed, IIs are generally difficult to predict due to the presence of intronic reads ('noise') from unspliced RNA. The levels can vary from sample to sample and across the genome, depending on the sample preparation, gene expression level, splicing efficiency, etc. By comparing read counts exclusively among samples and without taking into account the gene- or genome-level background, Veridical is likely to produce many false positives.In particular, the 14 supporting reads in the left intron on Figure 4 seem hardly sufficient to indicate an II event, all the more as there is a larger number of reads in the neighboring intron (not predicted to be II). The authors should provide other type of evidence for this event. The mathematical formulas for the various classes of supporting reads and their locations (page 4, continued on page 6) are hard to understand. It would greatly help the readers to include a figure showing schematically the event and read location with respect to the introns and exons.Minor comments:As another reviewer pointed out, the software requires a registration to obtain a temporary license for 30 days, after which the availability and terms of use are unclear. This mode of distribution is not a problem, but the terms should be clearly stated in the manuscript. Also, this is a stand-alone software and not a web tool as implied by the article. The authors use the term 'cryptic' splice sites throughout the manuscript (I assume meaning 'aberrantly activated'), but some of the events discovered could be alternative exon ends. It would be helpful to clarify in the context.This is a potentially very powerful and useful tool. I gave the article an 'Approved with reservation' because it is critical to include results in the aggregate to complement the showcased examples, as well as to discuss its limitations. I will gladly change once these few issues are addressed.",
"responses": [
{
"c_id": "741",
"date": "27 Mar 2014",
"name": "Peter K Rogan",
"role": "Author Response",
"response": "We greatly appreciate your remarks and useful suggestions. All references to figures pertain to the first version of the manuscript. First, we address your comments regarding VAAST and de novo exome annotation software packages. VAAST uses a feature-based, maximal likelihood, approach to identify variants suspected to be pathogenic. While Yandell et al. present a general, and indeed very useful method, it is very different from Veridical in two key respects. Most importantly, Veridical does not aim to identify putatively pathogenic variants, but rather, to confirm existing in silico predictions thereof. Although it is not designed for this purpose, one could envision using the likelihood approach used by VAAST to extract solely splicing variants, annotating them, and then confirming these variants using Veridical with corresponding RNA-Seq data (assuming such data exists). Veridical is the only software available for statistical validation of splicing mutations. While both programs prioritize variants, their goals are rather dichotomous. Similarly, Veridical depends upon the use of an (ideally conservative) exome annotation. We do infer exon skipping events (i.e. alternative splicing) de novo, but only to catalog dysregulated splicing “phenotypes” due to genomic sequence variants. Since we rely upon existing annotations, improvements in this area will serve to benefit Veridical and complement it well. In many ways, our parsimonious approach was possible precisely because the problem we address is quite a lot easier to address than de novo alternative splicing inference. We are not suggesting that Veridical be used to analyze all variants, resulting from exome or whole-genome sequencing experiments. The Shannon Pipeline, which predicts candidate variants, contains several default or modifiable filters which can limit the number of candidates input to Veridical. We are suggesting that our approach be coupled to existing approaches to act as an a posteriori, hypothesis-driven, check on the veridicality of specific variants. We consider alternative splicing to be a different problem, for the following reasons. The authors state: “MATS calculates a P-value for each exon isoform by comparing the observed posterior probability with a set of simulated posterior probabilities from the null hypothesis.” We do not simulate posterior probabilities; we calculate them directly from the distribution of control samples, which represent the null hypothesis. Further, MATS requires the user to set uniform thresholds for all alternative splicing events, which in our case, may vary between variants (i.e. inactivating versus leaky mutations). Finally, neither MATS, nor Miso or SpliceTrap provide any means of analyzing novel isoforms created by the activation of cryptic splice sites. We appreciate your insightful summary of our underlying statistical methodology and appreciate the limitations of our statistical inference method. It is certainly the case that the use of different RNA-Seq protocols could prove problematic. Veridical is intended to be used with RNA-Seq data from the same individual as matched DNA-Seq data. We assume that the control RNA-Seq data are generated with comparable sequencing protocols. This has been articulated in the Discussion. We agree that levels of intron inclusion and alternative splicing can be highly variable across samples, due to factors extrinsic to the mutation itself. Such variability is a significant contributory factor to the production of transcriptomic “noise”. Given that statistically significant levels of intron inclusion were observed across all control samples, the prevalence of adjacent intron inclusions (which is not uncommon, per se), does not weaken the inference that the predicted splicing mutation increases the levels of intron inclusion. However, in such cases, the statistical threshold to achieve significance would be higher. A recent analysis indicates that p-value thresholds of 0.005 to 0.001 are appropriate (Johnson, 2013). Our statistical approach tacitly accounts for this and indeed greater levels of such “noise” could actually increase the statistical utility of our computation. Because of a wide degree of variation of intron inclusion in both normal and tumor transcriptomes, we deliberately elected against normalizing gene level expression in the program. Regarding the specific example cited, the 14 intronic reads in the sample containing the variant in the read-abundance category (Figure 4 (A)) are contrasted with the per sample mean of 4.051 reads across all non-variant-containing tumours and normal samples in this intron (i.e. the totality of control samples, which results in a p-value of 0.04. We agree that this is not a strong result, and would not permit us to reject the null hypothesis had the threshold significance level been reduced to ≤ 0.01 (which the user is free to stipulate).In this particular case, the variant is still strongly validated, due to the presence of junction-spanning, exon skipping reads (that are absent from all of the control samples). We do not stipulate that the predicted mutation have only one abnormal read type, but rather, that the test assesses each consequence separately. We suggest that the corroborating evidence be taken together in support of variant-induced missplicing. Furthermore, junction-spanning exon skipping is, in general, rarer than intron inclusion in these data. Nevertheless, we do show examples of read-abundance tests for intron inclusion that achieve a significance level of < 0.01, such as within TMTC2 in Figure 6(C). As you point out, intron inclusion patterns also vary by intron and there are a large number of such intron inclusion reads in the downstream intron in Figure 4(A). However, in our entire set of predicted splicing mutations, we did not find any pertaining to the intron between exons 12 and 13. The observed increased intron inclusion between exons 12 and 13 may be related to the weak (5.2 bit) donor (5’) splice site in exon 12. In fact, substantial intron inclusion was expected in intron 11, due to the minimal strength of the exon 11 donor of 2.1 bits. Regarding the comment concerning cryptic sites, we always mean non-canonical splice sites activated by sequence-level variation, which are distinctly different from tissue-related alternatively spliced isoforms lacking one or more constitutive exons. We and others have reported numerous examples of common SNPs inducing cryptic splice site activation, which could result in the annotation of accordingly altered transcripts. It is unfortunate that Veridical had to be categorized as a “web tool”. We would have preferred to indicate that the program is a standalone-tool. F1000Research does not provide this category, even though other standalone programs have been published by the journal with the Web Tool designation. In the Web Tools category, the journal guidelines require that we provide a set of examples (complete with input and output files) representing the range of results that are obtained with the software. Therefore, providing a full analysis of the complete TCGA breast cancer dataset is beyond the scope of this paper. It will be presented elsewhere. We believe that the statements in the manuscript's conflict of interests section are sufficient to describe the terms of use. We have added the requested details on Veridical.org, both indicating its duration and instructions for obtaining longer-term access to this software."
}
]
},
{
"id": "3121",
"date": "18 Mar 2014",
"name": "Peter N. Robinson",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper from the Rogan group presents a methodology for validation of DNA sequencing variants that alter mRNA splicing. While variants of the most conserved splice site nucleotides at the intron-exon boundary can be predicted to cause splice defects with high reliability, it remains difficult to predict whether variants deeper in the intron or those that potentially affect exonic splicing enhancers actually cause splice defects. RNA-seq data, when coupled with variant data, potentially provide a means of correlating variation data with observations of (mis-)splicing patterns. The program fulfils an important need in the community, the results appear promising and will be of special interest to groups performing RNA-seq analysis in medical settings. I have only some minor suggestions that the authors may like to consider. Suggestions:The explanation of the methodology is relatively difficult to follow, and I wonder if it might not be better to simplify Figures 1 and 2 for didactic sake. For instance, in Figure 1A, it is unclear where the location of variant C is. Does the curved line mean that it could be anywhere in the middle exon? Also, I assume that exons are being shown in blue and reads shown in gray? Also, the legend text is overly complicated: D > E swap D and E. While aficionados of first order logic will follow without problems, I would suggest that it would be better for didactic purposes to delete this and to implicitly assume that D<E for the sake of this figure. Figure 1B is confusing at this point in the manuscript because the motivation for switching the variable A,B, D, and E is not yet clear. On the other hand, panel C and panel D are trivial and do not add anything. I would suggest using Figure 1 to provide one concrete example one a simple level, and stating in the text that the variables are to be switched if the candidate mutation is located on the other side of the exon. Also, the explanations of the method that are couched in first order logic-like notation are difficult to follow, because it is not stated whether the variant C can precede the start of the read (in which case C-S would be negative). The subscripts for r in turn have the subscript s1 but the variable S in the formula does not. Although in the end, I think I follow the overall method, the reader is forced to make arbitrary assumptions in order to interpret the formulae being used to explain the method. A similar comment pertains to the flow chart in Figure 2.Therefore, I would suggest the authors take some pains to improve the clarity of the explanation of the method. I would suggest that they show one of two concrete examples and provide English language specifications of the FOL-like formulae that describe the partitioning of reads. I am a little unclear on the use of control samples vs experimental samples. Assuming the experimental samples come from different individuals, what is the reason to assume that they will have the same distribution of splice mutations? And given that one finds dozens of splice variants in normal individuals, what exactly is meant by a control sample? Will control samples not also have lots of splice mutations? How does the method deal with this? And if we are dealing with cancer samples, why not user a paired control to detect cancer-specific mutations? In light of this, the statement \"Maximizing the set of control samples, while computationally more expensive, increases the statistical robustness of the results obtained.\", does not appear to be supported by evidence presented in the manuscript. It would be interesting to see a comparison of the distribution of Ri values and results of Veridical analysis? How does Veridical decide which sequence variant is causative if there are multiple variations located in the vicinity of a given mis-spliced exon? The mutation nomenclature chr1:985377 C>T should not have a space between the position and the nucleotides. It is unclear to me why a linear regression model was used to show the performance of the method. The authors could provide timings from real runs. It would be interesting to see a plot on the relationship of the p-values called by Veridical and the sequencing depth covered. The authors state \"In particular, a lack of sufficient coverage at a particular locus will cause Veridical to be unable to report any significant results. A coverage of at least 20 reads should be sufficient.\", but they do not provide evidence for this assertion. This is an important question given that low-expressed genes are thus likely to be systematically under-represented in the results of Veridcal, and this should be commented on somewhere in the paper. It would be good if the authors provided Sanger validation of at least some of the mis-splicing events reported in the paper. The input format for Veridical is described as \"This input format most easily accepts formatted output from the Shannon Pipeline.\" Why not allow VCF files and filter them for potential splice variants informatically prior to Veridcal analysis? It was unclear to me how the variants are to be selected and whether Veridical can be easily used outside of the Shannon pipeline?",
"responses": [
{
"c_id": "742",
"date": "27 Mar 2014",
"name": "Peter K Rogan",
"role": "Author Response",
"response": "We really appreciate your constructive comments. In particular, we have revised figure 1 as you suggested. Furthermore, we have endeavored to clarify the methods by referencing every corresponding equation pertaining to each informal description of a methodological principle. All references to figures pertain to the first version of the manuscript. \"It is unclear to me why a linear regression model was used to show the performance of the method. The authors could provide timings from real runs.\"All data points in Figure 3 (a) consist of data from actual runs. We did not conduct a sufficient number of large-scale runs to accurately determine performance and believe that the regression model provides information for users who wish to use the software in that context. \"The input format for veridical is described as “This input format most easily accepts formatted output from the Shannon Pipeline.” Why not allow VCF files and filter them for potential splice variants informatically prior to veridcal analysis? It was unclear to me how the variants are to be selected and whether veridical can be easily used outside of the Shannon pipeline?\" It would be prohibitively difficult for a user and cumbersome to attempt to add such support. The Shannon Pipeline and most other splicing analysis software accept variant call format (VCF) input and outputs their own custom format. VCF is very poorly suited for the annotation of detailed splicing information. The position of the variant is not necessarily the same as the affect splice junction coordinate. While it is possible to label the junction using custom fields in the VCF “INFO” column, the format is not standardized; it would be specific to the particular software used, and thus provides no real advantage over the Shannon pipeline output.This would become feasible if the community were to agree upon a standard definition of custom fields; this concept could be generalized to other scenarios in which both a variant and one (or more) other associated coordinates (in this case, a splice site, but elsewhere, perhaps the site of some specific interaction). This process would ultimately require an explicit definition of the content and schema for such additional fields, and would need to solicit views from the community to ensure widespread adoption. We would be interested in contributing to this endeavor.\"It would be interesting to see a plot on the relationship of the p-values called by Veridical and the sequencing depth covered.\" We have conducted a preliminary comparison of p-values for specific evidence types and splicing consequences vs. coverage per exonic base (the coverage of the gene divided by the length of the pertinent exon) and found that the results were quite difficult to interpret. We suspect that this could be explained by the fact that, due to significant intron inclusion, normalizing by exonic length is not really appropriate. Cryptic sites and alternative splicing make direct comparison of these values difficult to compute. \"How does Veridical decide which sequence variant is causative if there are multiple variations located in the vicinity of a given mis-spliced exon?\"Veridical does not currently address cases in which multiple independent variants pertain to the same splice site. In such a case, each variant is analyzed independently. Based on Mendelian disorders, we have rarely observed multiple independent variants alter the strength of the same splice site, and in any case, the phase of these variants is unknown, so they could reside on different chromosomes. \"I am a little unclear on the use of control samples vs experimental samples. Assuming the experimental samples come from different individuals, what is the reason to assume that they will have the same distribution of splice mutations? And given that one finds dozens of splice variants in normal individuals, what exactly is meant by a control sample? Will not control samples also have lots of splice mutations? How does the method deal with this? And if we are dealing with cancer samples, why not user a paired control to detect cancer-specific mutations? In light of this, the statement “Maximizing the set of control samples, while computationally more expensive, increases the statistical robustness of the results obtained.“ Does not appear to be supported by evidence presented in the manuscript.\"Control samples consist of all non-variant containing (NVC) tumour samples (relative to the variant being analyzed) and normal samples. In the examples shown in the paper, we maximized the number control samples used (106 normal samples and, in general, over 400 NVC samples). Control samples do indeed contain splicing mutations and this is a strength of our statistical paradigm. We term them control samples, because we consider their read distribution to correspond to that expected from the null hypothesis, as they do not contain the specific variant in question. The “noise” introduced by these splicing variants in control samples ensures that we are testing the variant containing samples against samples with many, distinct, splicing mutations. Thus, the greater the number of samples, the smaller the batch effects, which makes the method more reliable. While the normal and tumor DNA sequencing data was paired to call somatic variants, we do not require paired controls for the RNA-Seq data. In fact, TCGA does not provide such data. Since we initially designed the software for the task of analyzing their dataset, we did not wish to assume that paired RNA-Seq data was available. \"It would be good if the authors provided Sanger validation of at least some of the mis-splicing events reported in the paper.\"We do not have access to the TCGA tumour samples used. While TCGA performed Sanger validation of a small subset of their variants, none of the variants validated by Veridical had any associated Sanger sequencing data. \"It would be interesting to see a comparison of the distribution of Ri values and results of Veridical analysis?\"The variants predicted by the Shannon splicing mutation analysis pipeline exhibit changes in information content (ΔRi). The change in information content of a splice site is directly related to the thermodynamics of the binding event (Schneider, 1997) and therefore the strength of the splice site (Rogan et al., 1998; Rogan et al., 2003). Therefore, one would expect ΔRi to be highly predictive of valid splicing variants, where cryptic sites are expected to result in increased splice site strength, and natural sites would be expected to be weakened.The median ΔRi for cryptic variants in the set of pre-Veridical variants (those variants called as somatic mutations and predicted by the Shannon Pipeline to affect mRNA splicing) was 2.6 bits, while the median for validated variants was 2.38 bits. For non-cryptic variants the pre- and post-validation median ΔRi values were -2.52 and -3.18 bits, respectively.The information content of cryptic variants actually decreased slightly in the validated set. This likely is related to other factors, such as the initial strength of the natural site and the exon length, which are not accounted for by this analysis. However, the average information content of natural sites did decrease, as expected. The reason for the decrease not being more substantial, again, is likely due to the involvement of other factors, such as the distance of the variant to the natural site – which impacts its Ri value. We have refrained from including this discussion in the manuscript, since there is no requirement per se for Veridical to be used with the Shannon Pipeline. An explicit definition of the required input format is provided in the paper."
},
{
"c_id": "1522",
"date": "14 Aug 2015",
"name": "Peter Robinson",
"role": "Reviewer Response F1000Research Advisory Board Member",
"response": "Thanks for the detailed response to my comments."
}
]
}
] | 1
|
https://f1000research.com/articles/3-8
|
https://f1000research.com/articles/3-88/v1
|
07 Apr 14
|
{
"type": "Case Report",
"title": "A 13 year-old boy with post-transplantation lymphoproliferative disorder presenting with obscure gastrointestinal bleeding: a case report",
"authors": [
"Edith Y. Ho",
"Vijay George",
"Marjorie McCracken",
"James W. Ostroff",
"Vijay George",
"Marjorie McCracken",
"James W. Ostroff"
],
"abstract": "One well recognized and potentially serious complication of chronic immunosuppression in organ transplant recipients is post-transplantation lymphoproliferative disorders (PTLD). This accounts for 20% of all malignancies in transplant recipients, which is four times higher than the general population1,2. The diagnosis of PTLD is often difficult, due to various manifestations resulting in late diagnosis. We report an unusual presentation of PTLD in a pediatric patient where the diagnosis was achieved only after extensive investigation.",
"keywords": [
"This case involves a 13 year-old Caucasian boy with a history of cystic fibrosis status post deceased donor EnBloc combined liver and pancreas transplantation",
"presenting with gastrointestinal bleeding. His immune suppression regimen after the transplant included tacrolimus 1.5 mg twice daily."
],
"content": "Case presentation\n\nThis case involves a 13 year-old Caucasian boy with a history of cystic fibrosis status post deceased donor EnBloc combined liver and pancreas transplantation, presenting with gastrointestinal bleeding. His immune suppression regimen after the transplant included tacrolimus 1.5 mg twice daily.\n\nTwo years after his transplant, the patient presented to the hospital with “a few days of dark tarry stool”. He did not complain of any nausea, vomiting, or abdominal pain. He denied taking any anti-platelet agents or non-steroidal inflammatory drugs. On admission, the patient was hypotensive with a blood pressure of 70/50 mmHg and tachycardic with a heart rate of 112 beats per minute. His hemoglobin was 7.4 g/dL, which represented a drop from a baseline hemoglobin of 10 g/dL. After fluid resuscitation, the patient was started on pantoprazole continuous infusion at 5 mg/hour and sulcrafate suspension 300 mg four times daily. He then underwent an urgent upper endoscopy, which revealed multiple small prepyloric and duodenal ulcerations without signs of recent hemorrhage. Biopsies of these ulcers showed acute inflammation. A capsule endoscopy was then performed, which showed scattered duodenal erosions and two adjacent erosions in the distal duodenum without stigmata of recent bleeding. During the work-up, the patient continued to have melena, necessitating transfusions every three days. This prompted a second upper endoscopy, which showed complete healing of the prior ulcers. Interestingly, portal hypertensive gastropathy and a gastric varix were also noted. To target bleeding lesions potentially related to portal hypertension, octreotide continuous infusion at 2 mcg/kg/hour was initiated. Unfortunately, his melena persisted. The patient underwent a technetium-labeled red blood cell bleeding scan, where a potential bleeding source “near the anastomosis of the native to transplanted duodenum or proximal jejunum” was identified. A visceral arteriogram was obtained but it failed to detect a bleeding source. Following these radiological tests, a deep enteroscopy was performed. No signs of recent bleed within the suspected proximal hepatic limb of the Roux were detected. In the jejunum, however, there was a 10 mm clean-base, friable ulcer with significant oozing when it was biopsied. The pathological diagnosis of the ulcer was reported as follows: “the morphologic and immunophenotypic features indicate involvement of this patient’s jejunum of a large B-cell lymphoproliferative disorder consistent with diffuse large B-cell lymphoma (DLBCL). These findings thus support the diagnosis of a post-transplantation lymphoid disorder, involving small intestinal mucosa”. Epstein-Barr virus (EBV) was positive in the biopsied sample (See Figure 1).\n\n1A: Haematoxylin & Eosin stain, 1000x. Large, centroblastic lymphoid cells with hyperchromatic, pleomorphic nuclei and moderate cytoplasm. These tumor cells almost completely efface the jejunal mucosa. Scattered mitotic activity and apopotic debris are present as well. 1B: Immunohistochemical stain for CD20, 1000x. Diffusely positive membranous staining in the tumor cells is indicative of lymphoid differentiation. In the context of the morphology seen in Figure 1A, the findings are compatible with a Diffuse Large B-Cell Lymphoma (DLBCL). Per the 2008 World Health Organization (WHO) classification, a lymphoid proliferation that meets the criteria for a high grade malignancy (in this case DLBCL) that occurs in a recipient of a solid organ, bone marrow, or stem cell transplant is diagnostic of a post-transplant lymphoproliferative disorder (PTLD). 1C: In situ hybridization for Epstein-Barr virus encoded RNA (EBER), 1000x. Strong nuclear positivity is seen in the majority of the tumor cells, a finding seen in many monomorphic PTLD cases.\n\n\nFollow-up and outcome\n\nAn abdominal MRI performed a few days later demonstrated a mild thickening of the segments of small bowel in the left upper quadrant and portal hypertension. No lymphadenopathy was found in the abdomen and pelvis. The patient was then evaluated by the pediatric oncology service. A bone marrow biopsy reassuringly showed a normocellular marrow. The patient was started on cyclophosphamide, rituximab, and prednisone. His gastrointestinal bleeding eventually stopped a few weeks later.\n\n\nDiscussion\n\nPTLD are rare but potentially fatal complications of solid organ transplantation3. The overall incidence of lymphoproliferative disorders in the transplant population is approximately 1% at 10 years, which is significantly higher than the general population3. The risk increases with the intensity of induction or rescue immunosuppression, and particularly following monoclonal or polyclonal anti-lymphocyte therapy4–6. The EBV serostatus of the recipient is another risk factor7, although PTLD can occur in EBV-negative diseases as well. Time post-transplant is also a key factor as the majority of PTLD occurs within the first year post-transplant8. Over half of the patients with PTLD present with extranodal masses9. PTLD can affect many organs, including the gastrointestinal tract, lungs, liver, central nervous system, and the allograft itself. Clinical manifestations vary, ranging from benign polyclonal lymphoproliferation (infectious mononucleosis-type acute illness) to aggressive and disseminated malignant disease. Gastrointestinal features typically include abdominal pain, fever, and bowel perforation in serious cases. There have been case reports describing gastrointestinal bleeding as the initial presentation of PTLD in the pediatric population10–13, but this is less common. This case highlights the importance of considering the diagnosis of post-transplantation lymphoproliferative disorders in transplant receipts presenting with unexplained gastrointestinal hemorrhage.\n\n\nConsent\n\nInformed consent for publication of clinical details and clinical images was obtained from the next of kin.",
"appendix": "Author contributions\n\n\n\nEH: acquisition of data, interpretation of data, drafting of manuscript, assistance in clinical care.\n\nVG: preparation and processing of pathology slides, interpretation of data.\n\nMM, JO: acquisition of data, interpretation of data, guidance of clinical management plan, revision of manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nPenn I: Cancers complicating organ transplantation. N Engl J Med. 1990; 323(25): 1767–1769. PubMed Abstract | Publisher Full Text\n\nAdami J, Gäbel H, Lindelöf B, et al.: Cancer risk following organ transplantation: a nationwide cohort study in Sweden. Br J Cancer. 2003; 89(7): 1221–1227. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCurtis RE, Travis LB, Rowlings PA, et al.: Risk of lymphoproliferative disorders after bone marrow transplantation: a multi-institutional study. Blood. 1999; 94(7): 2208–2216. PubMed Abstract\n\nFeldman M, Friedman LS, Brandt LJ: Sleisenger and Fordtran's Gastrointestinal and Liver Disease: Pathophysiology / Diagnosis / Management. Volume 2. 9th edition, Canada: Saunders Elsevier; 2010. Reference Source\n\nJamali FR, Otrock ZK, Soweid AM, et al.: An overview of the pathogenesis and natural history of post-transplant T-cell lymphoma (corrected and republished article originally printed in Leukemia & Lymphoma, June 2007; 48(6): 1237–1241). Leuk Lymphoma. 2007; 48(9): 1780–1784. PubMed Abstract | Publisher Full Text\n\nAllen U, Hébert D, Moore D, et al.: Epstein-Barr virus-related post-transplant lymphoproliferative disease in solid organ transplant recipients, 1988–97: a Canadian multi-centre experience. Pediatr Transplant. 2001; 5(3): 198–203. PubMed Abstract | Publisher Full Text\n\nWalker RC, Marshall WF, Strickler JG, et al.: Pretransplantation assessment of the risk of lymphoproliferative disorder. Clin Infect Dis. 1995; 20(5): 1346–1353. PubMed Abstract | Publisher Full Text\n\nOpelz G, Henderson R: Incidence of non-Hodgkin lymphoma in kidney and heart transplant recipients. Lancet. 1993; 342(8886–8887): 1514–1516. PubMed Abstract | Publisher Full Text\n\nNalesnik MA, Jaffe R, Starzl TE, et al.: The pathology of posttransplant lymphoproliferative disorders occurring in the setting of cyclosporine A-prednisone immunosuppression. Am J Pathol. 1988; 133(1): 173–192. PubMed Abstract | Free Full Text\n\nLai YC, Ni YH, Jou ST, et al.: Post-transplantation lymphoproliferative disorders localizing to the gastrointestinal tract after liver transplantation: report of five pediatric cases. Pediatr Transplant. 2006; 10(3): 390–394. PubMed Abstract | Publisher Full Text\n\nHeo JS, Park JW, Lee KW, et al.: Posttransplantation lymphoproliferative disorder in pediatric liver transplantation. Transplant Proc. 2004; 36(8): 2307–2308. PubMed Abstract | Publisher Full Text\n\nDonnelly LF, Frush DP, Marshall KW, et al.: Lymphoproliferative disorders: CT findings in immunocompromised children. AJR Am J Roentgenol. 1998; 171(3): 725–731. PubMed Abstract | Publisher Full Text\n\nLim GY, Newman B, Kurland G, et al.: Posttransplantation lymphoproliferative disorder: manifestations in pediatric thoracic organ recipients. Radiology. 2002; 222(3): 699–708. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "4391",
"date": "07 May 2014",
"name": "Patrizia Comoli",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting case that highlights the importance of considering PTLD in the differential diagnosis of gastrointestinal bleeding occurring after transplantation. The case report is well written, but requires a few minor revisions.Article contentA few clinical details, important for the diagnosis of PTLD, such as EBV status at transplantation, EBV DNA at diagnosis and at treatment re-evaluation, and LDH at diagnosis, should be inserted. Was a PET scan performed to complete PTLD assessment? In order to increase the clinical value of this report, I believe it is important to add data on patient imaging assessment at diagnosis (small bowel MRI and, if performed, total body PET scan, as figure 2 a and b). If PET scan or CT scan were not performed, add a comment on the reasons for not performing imaging assessment.ReferencesThe reference for 2008 World Health Organization (WHO) classification (cited in the legend to Figure 1) ought to be reported. Reference 3 relates to stem cell rather than solid organ transplantation. The authors need to replace this reference with a more appropriate one, possibly published within the last 10 years. Reference 8 is very outdated. The same author has published a review on the same topic in 2003 (Opelz & Dohler, 2003), thus the authors should replace the 1993 article with the 2003 review.",
"responses": []
},
{
"id": "4390",
"date": "04 Aug 2014",
"name": "David Teachey",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting case report that emphasizes the need to consider the diagnosis of PTLD in post-transplant recipients who present with GI bleeding. It is a well-written report but could be improved by additional details. Readers could benefit from a focused discussion on the treatment of PTLD in the post-transplant setting, including reduction in immune suppression, modulation of immune suppression (e.g. transition from a calcineurin inhibitor to sirolimus), single agent therapy with rituximab, or multi-agent therapy with prednisone, cyclophosphamide, and rituximab. In this case based on the pathology, multi-agent chemotherapy was the appropriate treatment, but in other instances less aggressive measures are sometimes used. Additional details including imaging of his extra-abdominal compartment by CT or PET-CT, EBV viral load, and LDH would be helpful. Also, more details on the length and response to treatment would be of interest (was he reimaged or re-scoped?).",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-88
|
https://f1000research.com/articles/2-272/v1
|
13 Dec 13
|
{
"type": "Method Article",
"title": "The beauty of being (label)-free: sample preparation methods for SWATH-MS and next-generation targeted proteomics",
"authors": [
"Jakob Vowinckel",
"Floriana Capuano",
"Kate Campbell",
"Michael J. Deery",
"Kathryn S. Lilley",
"Markus Ralser",
"Jakob Vowinckel",
"Floriana Capuano",
"Kate Campbell",
"Michael J. Deery",
"Kathryn S. Lilley"
],
"abstract": "The combination of qualitative analysis with label-free quantification has greatly facilitated the throughput and flexibility of novel proteomic techniques. However, such methods rely heavily on robust and reproducible sample preparation procedures. Here, we benchmark a selection of in gel, on filter, and in solution digestion workflows for their application in label-free proteomics. Each procedure was associated with differing advantages and disadvantages. The in gel methods interrogated were cost effective, but were limited in throughput and digest efficiency. Filter-aided sample preparations facilitated reasonable processing times and yielded a balanced representation of membrane proteins, but led to a high signal variation in quantification experiments. Two in solution digest protocols, however, gave optimal performance for label-free proteomics. A protocol based on the detergent RapiGest led to the highest number of detected proteins at second-best signal stability, while a protocol based on acetonitrile-digestion, RapidACN, scored best in throughput and signal stability but came second in protein identification. In addition, we compared label-free data dependent (DDA) and data independent (SWATH) acquisition. While largely similar in protein detection, SWATH outperformed DDA in quantification, reducing signal variation and markedly increasing the number of precisely quantified peptides.",
"keywords": [
"label-free quantitative proteomics",
"in gel digest",
"filter-aided sample preparation",
"RapiGest",
"acetonitrile-based protein digestion",
"SWATH",
"data dependent acquisition"
],
"content": "Introduction\n\nMass spectrometry (MS)-based proteomics facilitates the identification of a large number of proteins in a single experiment1–3. As a result this technique has been established as a powerful complement to the classic tools of protein chemistry, such as western-blotting or enzyme-linked immunosorbent (ELISA) assays, which are of considerably lower throughput and specificity. Where initial proteomic workflows mainly aimed to identify proteins, quantification has become a major focus of much of technology development in recent years4,5. On a quantitative liquid chromatography/mass spectrometry (LC-MS) platform the amount of analyte and the corresponding chromatographic peak area are in linear correlation, hence concentration values are obtained through comparison with reference standards6. A technically powerful approach for protein quantification involves the use of isotope-labelled standards that show a similar structure and chromatographic behaviour to the target molecule, but are distinguished from the target by mass7. When added at an early stage of the quantification workflow, they allow for correction of analyte loss during sample preparation and analysis, hence rendering the quantification experiment robust. However, the requirement for isotope-labelled standards makes proteomics workflows expensive and reduces flexibility, as their production is laborious and applicable only to samples for which these standards can be obtained or generated (please see the Discussion). Moreover, as both the analyte and standard need to be measured, they double the analyte load for the mass spectrometer. Consequently, recent developments that have enabled label-free peptide and protein quantification have attracted much attention8–12. In a label-free experiment, quantification is achieved through comparison of peak areas obtained for an analyte under two or more biological conditions; for instance to compare a wild-type versus a mutant, a compound-exposed versus a control condition, or a biological time series13–16. Upon normalisation, ideally to one or more unaffected internal standards, this approach yields a relative expression value for the target protein. This measure is then used to evaluate whether the expression of the target is altered between the conditions tested. In the case of high sequence coverage, absolute quantities may also be estimated, as peak intensities obtained for the best ionizing peptides correlate in approximation with their absolute concentration10,12.\n\nThe absence of an internal standard spiked early in sample preparation protocols means that label-free methods are sensitive to technical variance, and consequently, label-free proteomics requires high instrument performance and standardization of sample preparation methods. In terms of instrumentation, limitations arise from the linear range of the mass spectrometer and the sample capacity of the liquid chromatography. Moreover, in untargeted proteomics, the stochastic nature of data-dependent acquisition methods, where ions are selected for analysis based on their intensity, reduces the number of quantifiable peptides to only those fragmented in all samples17,18. This problem is a consequence of the high numbers of co-eluting peptides that may considerably exceed the mass spectrometer’s sampling speed when analysing full proteomes, a situation that is amplified by the high number of replicates used in a label-free study. By facilitating data-independent acquisition, where all ions are fragmented irrespective of their intensity, recent studies have demonstrated the possibility of circumventing the need of isolating individual peptides11,17. One such method, pioneered by the Waters Corporation, is termed MSE,11. In this approach fragment ions are assumed to have the same elution profiles as their precursors; this similarity is then used to pair fragments and precursors when a number of parent ions are co-fragmented. Fragment pairs and their corresponding precursor ions are typically retrospectively paired prior to database searching11. More recently, in a workflow termed SWATH, a mass range relevant for peptide-based proteomics (400–1200 m/z) is scanned in 25 m/z windows, in which all ions that fall into that window are simultaneously fragmented (MS/MSall). Quantification is then conducted based on the peak areas of extracted ion chromatograms (XIC), which are computationally reconstituted from the merged spectra on the basis of both experimental and in silico generated spectral information17.\n\nSample preparation techniques are equally important for the performance of a label-free experiment, and easier to optimize on a daily basis than the mass spectrometer’s properties. The main objective for a label-free sample preparation method is to obtain stable peak intensities between replicate sample preparations. Consequently, the ideal workflow avoids processing steps that are prone to stochastic analyte losses, and the LC-MS set up is operated in a way that ensures the dynamic range of the instrument is not exhausted. These objectives may differ to classic shotgun proteomics, where the number of identifiable peptides and proteins is the most important value, and a higher variation in signal intensities is acceptable. Hence, a sample preparation method and LC-MS/MS configuration, which is ideal for identifying a maximum of proteins, may be sub-optimal for label-free quantification, and vice versa. For instance, pre-fractionation of the sample prior to the LC-MS/MS analysis, a popular strategy to improve peptide identification, adds another level of complexity to the sample preparation increasing the signal variability and thus, is avoided wherever possible.\n\nThe main objective of the study presented here is to benchmark proteomic sample preparation methods for their suitability in label-free proteomic studies. We compare popular sample protocols that are based on in gel19, filter-aided20,21 and in solution9,22 digestion procedures. Processing identical proteome samples obtained from budding yeast, and acquiring proteomic data without further prefractionation on two LC-MS/MS platforms, these methods are compared by their performance in sample preparation, their precision in label-free quantification experiments and their effectiveness in terms of time and reagents. Through the analysis of these samples on a 5600 QqTOF23 instrument operating in either a data-dependent mode or SWATH24 mode, this study concludes with an evaluation of data-dependent and data independent acquisition, and suggestions about the optimal protocol selection.\n\n\nExperimental section\n\nFor sample preparation the following reagents were used: Water ULC-MS grade (Greyhound Cat. No. 23214125), formic acid 99% ULC-MS (Greyhound Cat. No. BIO-06914131) and acetonitrile ULC-MS grade (Greyhound Cat. No. Bio-012041-2.5L). Chemicals were obtained from Sigma, with the exception of RapiGest (Waters, Cat. No. 186001861), trypsin (Promega Cat. No. V5111), LysC (Promega, Cat. No. No. V1071), complete EDTA-free protease inhibitor cocktail tablets (used in the eFASP protocol) (Roche Cat. No. 11873580001), dithiothreitol (Melford Cat No. MB1015), ammonium bicarbonate (Fluka Cat. No. 40867-50G-F), sodium dodecyl sulfate (SDS, Melford Cat. No. S1030), 30% acrylamide/0.8% bis-acrylamide (Protogel, Geneflow Limited Cat. No. EC-890), tri-n-butylphosphate (Fluka Cat. No. 90820-100ML) and BCA Protein assay kit (Pierce Cat. No. 23225).\n\nAll experiments were conducted using a single culture derived from a single colony of the yeast strain BY474125. The strain was transferred to yeast peptone dextrose (YPD) media prepared as described in26 and incubated at 30°C at 200 rpm overnight (ON). Subsequently the ON culture was diluted to an optical density (OD600) of 0.2 as measured on an Ultropsec 2000 (Amersham) spectrophotometer, and incubated at 30°C until reaching OD600 = 2. The culture was split into aliquots corresponding to 10 OD600 units, and stored at -80°C until processing.\n\nA detailed protocol for each of the six procedures is available in the Supplementary Materials (found at the end of the document in the offline version) (see Supplementary protocol 1–Supplementary protocol 6). In brief, protein samples were prepared from 30 mg (wet weight) of yeast pellet. For the in gel digest protocols, protein extraction was performed either in 200 µl SDT buffer (4% SDS, 100 mM Tris/HCl pH 7.6, 0.1 M dithiothreitol) or 0.05 M ammonium bicarbonate using a Fast-Prep 24 instrument (MP Biomedicals). Fifty µg of protein was applied onto a denaturing polyacrylamide gel and subjected to electrophoresis (for details please see Supplementary protocol 1 and Supplementary protocol 2). The sample was excised as single band, cut in pieces, and subjected to tryptic digestion27. For the filter-aided protocols (FASP, Supplementary protocol 3 and Supplementary protocol 4) protein extraction was performed either in 200 µl SDT buffer (4% SDS, 100 mM Tris/HCl pH 7.6, 0.1 M dithiothreitol) (FASP, Supplementary protocol 3) or lysis buffer (1% SDS, 10 mM Tris/HCl pH 7.4, 0.15 M NaCl, 1 mM EDTA in PBS) (eFASP, Supplementary protocol 4). For both protocols the digestion was performed directly on filters (Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-3 membrane, Millipore). The FASP procedure (Supplementary protocol 3) involved a treatment with endoproteinase Lys-C (Promega) prior to digestion with trypsin20, while the eFASP Supplementary protocol 4) required protein precipitation using tri-n-butylphosphate/acetone/methanol mix (1:12:1) for lipid removal before digestion21. For in solution digest protocols Supplementary protocol 5 and Supplementary protocol 6) protein extraction was performed either in 200 µl lysis buffer (0.1 M NaOH, 0.05 M EDTA, 2% SDS, 2% β-mercaptoethanol) (RapiGest) or 0.05 M ammonium bicarbonate (RapidACN)20 or using glass-bead lyses using the Fast-Prep 24 instrument (MP Biomedicals), respectively. The in solution digest protocol based on the detergent RapiGest method included a step of protein precipitation for lipid removal through centrifugation prior to trypsin treatment. For the in solution acetonitrile-based digestion protocol, a clean-up step using 3 kDa molecular cut off filters (Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-3 membrane, Millipore) was performed immediately after trypsin digestion9. In order to maximize the proteome depth for the generation of a SWATH ion library, tryptic digests prepared with the RapidACN protocol were separated by high pH reverse phase chromatography before LC-MS/MS analysis. A reverse phase column (Waters, BEH C18, 2.1 × 150 mm, 1.7 µm) was utilised in combination with a 20 mM ammonium formate to 20 mM ammonium formate/80% ACN gradient. Twenty fractions were collected and supplemented with HRM standard peptide kit (Biognosys) prior to analysis.\n\nLC-MS/MS analysis of digested S. cerevisiae lysates was performed on a Tandem Quadrupole Time-of-Flight mass spectrometer (AB/Sciex TripleTOF5600) coupled to a Nanospray III Ion Source (AB/Sciex) and nano-HPLC (Eksigent Ultra 2D) (referred as TripleTOF platform), or hybrid quadrupole orbitrap mass spectrometer (QExactive, Thermo Scientific) coupled to a Dionex Ultimate 3000 and an Easy-spray nanospray ion source (referred as QExactive platform).\n\nOn the TripleTOF platform, peptide separation was carried out by first removing impurities on a pre-column (C18 PepMap100 column NAN75-15-03-C18-PM, Thermo Fisher Scientific Cat. No. 160321) running isocratically at 100% solvent A at a flow rate of 5 μL min-1 for 6 min. Peptides were then eluted onto the analytical column (Zorbax 300SB-C18 column, 75 µm id × 15 cm 3.5 µm, Agilent Technologies Cat. No. 5065-9911), and separated on a linear gradient of 5–35% solvent B for 155 min at a flow rate of 300 nL min-1. Peptides were injected into the mass spectrometer using 10 µm SilicaTip electrospray emitters (New Objective Cat. No. FS360-20-10-N-20-C12), and operating the ion source with the following parameters: ISVF = 2500; GS1 = 12; CUR = 25. The data acquisition mode in the DDA experiments was set to obtain a high resolution TOF-MS scan over a mass range 400–1250 m/z, followed by MS/MS scans of 20 ion candidates per cycle with dynamic background subtraction, operating the instrument in high sensitivity mode. The selection criteria for the parent ions included the intensity, where ions had to be greater than 150 cps, with a charge state between 2 and 4. The dynamic exclusion duration was set for 15 s. Collision-induced dissociation was triggered by rolling collision energy (Supplementary Table 1). The ion accumulation time was set to 250 ms (MS) and to 100 ms (MS/MS). For SWATH MS-based experiments the instrument was tuned to specifically allow a quadrupole resolution of 25 Da/mass selection. An isolation width of 25 Da was set in a looped mode over the full mass range (400–1250 m/z) scan and 32 overlapping windows were constructed28. An accumulation time of 100 ms was set for each fragment ion resulting in a total ion cycle of 3.3 s.\n\nFor LC-MS/MS analysis using the QExactive platform, separation of peptides was performed at a flow rate of 300 nL min-1 using a reverse-phase nano column (Easy-spray, Thermo Scientific PepMap C18, 2 µm particle size, 100 Å pore size, 75 µm i.d. × 50 cm length). Peptides were loaded onto a pre-column (Thermo Scientific PepMap 100 C18, 5 µm particle size, 100 Å pore size, 300 µm i.d. × 5 mm length) from the Ultimate 3000 autosampler (Dionex) with 0.1% formic acid for 3 minutes at a flow rate of 10 µL min-1. Polar impurities were removed by running the system isocratically at 100% Å at a flow rate of 5 μl min-1 for 6 min. Finally, tryptic peptides were loaded onto the analytical column and separated using a linear acetonitrile gradient of 5–35% B for 155 min at a flow rate of 300 nL min-1. The LC eluant was injected into the mass spectrometer by means of an Easy-spray source (Thermo Fisher Scientific). All m/z values of eluting ions were measured in an Orbitrap mass analyzer, set at a resolution of 70000. Data dependent scans were employed to automatically isolate the 20 most abundant ions and generate fragment ions by higher energy collisional dissociation (HCD) in the quadrupole mass analyser. Only peptide ions with charge states of 2+ and above were selected for fragmentation. Finally, the measurement of the resulting fragment ions was performed in the Orbitrap analyser, set at a resolution of 17500.\n\nData acquired in DDA mode was analysed by means of either the Paragon29 (ProteinPilot software, AB/Sciex, v. 4.5.0.0, 1654) or the Mascot search algorithm (Matrix Science, version 2.3.02) using the S. cerevisiae S288C translated ORF database (based on SGD genome version R64-1-130). 156 common contaminant ions (AB/Sciex) were excluded from subsequent analysis. For Paragon searches, we used the following settings: Sample type: Identification; Cys Alkylation: Iodoacetamide; Digestion: Trypsin; Instrument: TripleTOF5600; Special Factors: none; Species: S. cerevisiae; Search effort: Thorough ID; Results Quality: 0.05. Only peptides with a confidence score of > 0.05 were considered for further analysis. For Mascot searches, the data was pre-processed using PeakView (AB/Sciex, Triple TOF v. 1.2.0.3) or Protein Discoverer (Thermo Scientific, v. 1.3) setting carbamidomethylation of cystein (C) as a fixed modification, oxidation of methionine (M) as variable modification and allowing a maximum of 2 missed cleavages. Fragment mass tolerance was set to 0.8 Da, and Instrument type was ESI-TRAP. Peptides under a significance threshold of 0.05 were regarded as acceptable.\n\nFor the extraction of data acquired in SWATH mode, an ion library for yeast was generated from data acquired in data dependent mode. Briefly, a whole proteome yeast digest obtained with the RapidACN procedure was subjected to high pH reverse phase chromatography (Waters, BEH C18, 2.1 × 150 mm, 1.7 mm) and 20 fractions were collected across a linear gradient of 0–56% ACN in 20 mM ammonium formate for 74 min. Solvents were removed by vacuum centrifugation and peptides were resuspended in 5% ACN/0.1% formic acid. Yeast tryptic peptides were then supplemented with 1× iRT standard peptides (Biognosys) and analysed on the TripleTOF platform. Spectral data were acquired in DDA mode and analysed using the Paragon search strategy as described above. Detected peptides were then corrected for retention time shifts, and the corresponding spectra were combined leading to a library containing 2800 unique yeast proteins. For extraction of SWATH data and peptide quantification Spectronaut 3 (Biognosys) and Skyline31 was used. In parallel, Skyline was also used for quantification of peptides from data dependent acquisition experiments. Subsequent data analysis was performed with R, ggplot2 package and custom-built scripts. GO analysis was based on the SGD Gene Ontology Slim Mapper.\n\n\nResults\n\nFor this comparative study we selected an in gel digest method adapted from19, conducted in combination with an SDS-based and native protein extraction, two filter-aided (FASP (Filter Aided Sample Preparation) adapted from32 and a recent enhancement termed eFASP, (adapted from33), and two in solution procedures (RapiGest, adapted from22, and RapidACN adapted from16). There characteristics, as detailed below, are summarized in Figure 1. All procedures are given in lab-protocol format as Supplementary protocol 1 to Supplementary protocol 6.\n\nLeft panel: Schematic overview of the different steps in an LC-Ms/MS sample preparation method Right panel: Main characteristics of the protocols compared in this study. Detailled protocols are given in the Supplementary material. Supplementary protocol 1: In gel/SDS; 2: In gel/ABC; 3: FASP; 4: eFASP; 5: RapiGest; 6: RapidACN.\n\nIn gel digestions are popular sample preparation methods as they are convenient, and offer a simple way of protein pre-fractionation through gel slicing and removal of small or high-molecular contaminants that could interfere with trypsin digestion. These approaches are compatible with multiple sample extraction buffers, can easily be combined with gel staining that does not interfere with protein digestion34–36, and thus provide a visual quality control over the protein sample. However, casting and running the gels render these protocols time consuming; hence the protocols are of relatively low throughput. In this study, we benchmarked in gel digestion in combination with both SDS-containing (Supplementary protocol 1) and SDS-free protein extraction Supplementary protocol 2) (In gel/SDS and In gel/ABC, respectively19, Table 1). SDS-PAGE was however not used as a tool for pre-fractionation. In order to compare in gel methods with filter-aided and in solution digestion, the full mass range was processed and measured at once.\n\nThe second set of assessed protocols involves digestion on filter units. These protocols are popular due to their flexibility and due to the fact that they facilitate a simple handling and require only a modest hands-on time (~3 hrs). The first protocol tested, FASP32 involves a dual protease digest (LysC and trypsin), while the second filter-aided procedure (here called eFASP) is a stepwise-optimized version of FASP by Shevchenko and colleagues21 that involves protein precipitation.\n\nThe final two protocols tested in this study perform protein digestion in solution. The first protocol is based on the proprietary, acid degradable detergent RapiGest (Waters37), included in a protocol derived from Von der Haar et al.22. This protocol involves protein precipitation, which renders the RapiGest procedure more laborious as compared to the second in solution protocol, termed RapidACN. This rather simple method is based upon a tryptic digest in acetonitrile that is combined with a filter-based sample cleanup9. The RapidACN method requires the least number of handling steps and lowest hands-on time (~2 hrs per sample), overall facilitating the highest throughput among the tested procedures.\n\nThe six protocols, provided as detailed protocols in the Supplementary materials, were used to process an identical, full proteome sample of Saccharomyces cerevisiae. This single cellular eukaryote possesses a proteome of medium complexity (6,000–7,000 protein coding genes38) and has served as a reference organism in many landmark proteome studies29,39–41. Here, the use of yeast facilitated sampling from a single culture, bypassing the possibility of biological variability occurring between the samples analyzed. However, once proteins are extracted, these protocols are fully applicable to process samples obtained from other species as well. To process the yeast pellets, the protocols were executed as closely as possible to their original recipes (with unavoidable minor deviations highlighted in the Protocol section), both in full triplicates (= protocol triplicates), and in injection replicates for comparing the acquisition methods (= injection triplicates). Samples were analysed on a hybrid quadrupole time of flight (TripleTOF5600, AB/Sciex) mass spectrometer for DDA and SWATH acquisition, or on a hybrid quadrupole orbitrap mass spectrometer (QExactive, Thermo Scientific) for DDA acquisition. DDA database searches were conducted using Mascot (for TripleTOF5600 and QExactive, Matrix Science,42) or ProteinPilot43 (for TripleTOF5600, AB/Sciex), whilst SWATH data was processed with Skyline31 and Spectronaut44 (Biognosys) software.\n\nIt is noteworthy that in this study the analytical setup was adapted for quantification and not to maximize the number of protein identifications. This involved the injection of low amounts of sample (equalling 1 µg digest per protocol) to prevent column overload and largely overrunning of the dynamic range. Moreover, to allow a direct comparison of the protocols, data was recorded in single injections and samples were not pre-fractionated. This strategy yielded highly reproducible quantification results, achieving up to < 5% coefficient of variance (CV) values in label-free replicate injections for some protocols, as shown below.\n\nAs an indicator of the quality of tryptic digests, we first assessed the relative occurrence of partially cleaved peptides in data obtained from triplicate injections on the TripleTOF platform. All filter-aided and in solution protocols yielded reasonable digestion efficiencies as revealed by an analysis with both Paragon (AB/Sciex, Figure 2a) and Mascot (Matrixscience, data not shown) search engines. Both in solution and the eFASP procedure yielded arginine- and lysine cleavages in a similar ratio as found in the yeast proteome, with the lowest number of spectra assignable to missed cleavage tryptic sites found in the RapiGest dataset (Figure 2a, and Figure 2b). In the fourth protocol, FASP, however, we found lysine cleavages overrepresented compared to arginine cleavages (Figure 2b). This indicates that the presence of LysC in this protocol increased the overall digestion efficiency of lysine residues; however this may introduce a bias in (absolute) quantification experiments, overrating lysine over arginine peptides in quantification. With the employed in gel protocols we obtained a significant higher number of spectra that corresponded to uncleaved peptides (Figure 2a). Incomplete cleavage of peptides can render a sample preparation unsuitable for absolute, but also for relative quantification, as the rate of cleavage may not be reproducible between replicates9. For this reason, we consider the in gel protocol as employed (without prefractionation on the whole-proteome sample) to be potentially erroneous in protein quantification and identification, and excluded the data from the assessment of protein quantification quality. This result however does not exclude the possibility that on other samples, or with modified in gel protocols, acceptable cleavage efficiencies are achieved, and thus, this result should not be interpreted as a critique of in gel methods in general.\n\n(a) Proteolytic digestion efficiencies. Trypsin or LysC/trypsin (FASP) digestion efficiencies expressed as relative occurrence of spectra that could be assigned miscleaved peptides (n = 3). (b) Amino acid specificity of proteolytic digestion. Relative occurrence of identified peptides with C-terminal lysine or arginine, compared to the average frequency of these amino acids across all individual proteins identified (n = 3, Error bars = +/- S.D.) (c) Identified peptides differ per protocol, and correlate with the total peak area as recorded in a DDA experiment. 18 samples derived from the same yeast culture were processed with six protocols in triplicates, and analyzed on a TripleTOF5600 instrument. The number of identified peptides correlates with the total peak area recorded, and indicates the highest identification rate in solution digests, followed by filter-aided, and in gel procedures. (d) Detection of proteins by DDA or SWATH in a label-free experiment. Samples were analyzed in triplicates both for DDA and SWATH acquisition on a TripleTOF5600 instrument, data was searched using paragon (DDA), and Spectronaut (SWATH). SWATH increased the number of detectable proteins in combination with the in solution protocols. In solution protocols RapidACN and RapiGest led to the detection of up to 1000 proteins in single injections, followed by FASP and eFASP, which gave rise to between 250 and 750 proteins, and in gel injections that yielded 300 proteins IDs. Inset: A comparison of protein IDs for the TripleTOF and QExactive platform shows a linear correlation for the protocols investigated. Data was searched using Mascot (n = 3, Error bars = +/- S.D.)).\n\nThe number of detected peptides correlated with the sum of recorded total peak area, confirming that the instrument was operating within its dynamic range (Figure 2c). The yield of detected peptides (Figure 2c) and proteins (Figure 2d) revealed different performance of the tested protocols. For both data dependent (DDA) and SWATH acquisition, the two in solution protocols (RapiGest and RapidACN) gave the highest number of detectable peptides and proteins. Filter-based FASP and eFASP protocols ranked in the middle range, whilst a significantly lower number of proteins were detected from the in gel digests. Of note, SDS-based compared to native protein extraction increased the number of membrane protein detections in the in gel procedure, but in total a higher number of peptides were obtained in the natively extracted samples. To exclude that these results were platform specific, we injected the same samples on a QExactive mass spectrometer, operating with a different HPLC system and column (Dionex Ultimate 3000; 2 µm particle size C18, 75 µm i.d. × 50 cm column, see methods section). However, the number of protein IDs obtained with the two platforms correlated linearly, indicating that the ID performance of the tested protocols is platform independent (Figure 2d, Inset). Additionally, we tested to what extent injecting higher amounts of sample or pre-fractionation would increase the number of identifiable proteins. Single injection of 10 times the RapidACN sample increased the number of identifiable proteins by 34% to 1550 (QExactive), while high-pH RP HPLC pre-fractionation of a RapidACN digest led to the identification of 2800 proteins (TripleTOF). Similar tendencies were observed with the other protocols as well, indicating that when combined with sample pre-fractionation, all protocols and both platforms are suitable ID-optimized experiments, as addressed in other studies.\n\nTo be able to compare data dependent (DDA) and data independent (DIA) acquisition in terms of protein detection, we then analysed the samples using SWATH mode. Overall, when setting the highest quality threshold on SWATH-detected peptides (Spectronaut Q value < 0.01), SWATH and DDA detected a comparable number of proteins for the in gel and FASP procedures. However, SWATH outperformed DDA in the samples with high peptide content, RapiGest and RapidACN, leading to a modest but consistent increase in protein detection numbers (Figure 2d).\n\nNext we used the TripleTOF/DDA data to assess whether the protocols covered a similar set of proteins. A subset of 368 proteins overplayed between protocols 3, 4, 5 & 6 (all filter-aided and in solution protocols), while the filter-aided protocols (Supplementary protocol 3 and Supplementary protocol 4) overlapped for 479 proteins, and the in solution protocols (Supplementary protocol 5 and Supplementary protocol 6) for 915 proteins (Figure 3a). Due to high occurrence of uncleaved peptides, which may affect protein identification, the in gel methods are omitted from this illustration. However, the proteins identified in the in gel samples were to > 95% covered by in gel and in solution methods as well (data not shown.) All other protocols however also covered specific sets of proteins. RapiGest yielded the highest absolute number of unique IDs, while eFASP provided the highest percentage. Hence, in targeted proteome studies, sample preparation with different protocols might be considered in order to increase the probability of quantifying the desired target.\n\n(a) RapiGest and eFASP cover a unique space in the proteome. Identified proteins were visualised in a Venn diagram, excluding the in gel protocols. The RapiGest procedure yielded most unique IDs, followed by eFASP and RapidACN (n = 3). (b) SDS-containing protocols are best suited for the extraction of membrane proteins. For the analysis of annotated functions in each protocol, selected GO terms were expressed as percentages of identified proteins. While cytosolic proteins were not enriched in any protocol, membrane proteins were preferentially detected in the SDS-containing protocols. (n = 3, Error bars = +/- S.D.) (c) Filter-aided sample preparations yield a balanced representation of the proteome. The identified proteins were plotted against the percentage of proteins annotated by the GO term cytosol, in order to illustrate the similarity of extraction properties. The protocol properties required for efficient extraction of membrane and nuclear proteins is inversely correlated with the extraction efficiency for cytosolic proteins, while there is a positive correlation with ribosomal proteins.\n\nWe next assessed whether these differences correlated to the coverage of cellular localisations. The tested protocols gave high coverage of the GO term cytosol, and performed equally on the mitochondrial proteome (Figure 3b, see Supplementary Figure S2 for a complete overview of GO terms). However, different results were obtained for membrane proteins. The lowest relative content of membrane proteins was obtained for those protocols that extract proteins under non-denaturing conditions, namely RapidACN and in gel/ABC. Conversely, most membrane proteins were detected in the detergent-rich protocols, eFASP and RapiGest. Overall, FASP and eFASP yielded the most balanced representation of both the membrane and cytosolic fraction, while RapidACN data exhibited the strongest bias towards cytosolic and against membrane proteins (Figure 2c).\n\nFinally, we tested whether the protocols covered the proteomic mass range and charge state equally. The proteomic mass range was similarly represented by all protocols with a slight positive bias towards large proteins in all protocols (Supplementary Figure 1a). The procedures, however, differed in the representation of proteins with a certain isoelectric point (pI). The best representation of the proteome pI distribution was obtained with RapiGest (deviation coefficient (d) = 2.4), followed by FASP (d = 2.8) and RapidACN (d = 2.9) (Supplementary Figure 1b). In gel procedures scored least as they were negatively biased towards neutral proteins, and achieved a lower d value of 5.3 or 5.9 for in gel/ABC or in gel/SDS, respectively.\n\nNext, we compared the protocols for their consistency in label-free quantification. As illustrated in Figure 2c, the number of identified peptides correlated with the sum of total peak area recorded, hence all procedures in principle lead to quantitative results. To be able to compare the protocols, we expressed the variation of signal intensities obtained from replicate sample preparations as coefficient of variation (CVs), and we plotted the frequency of CVs in two-dimensional distribution histograms (‘violin plots’, Figure 4a). DDA acquisition resulted in a CV maximal likelihood of 20% for eFASP, FASP and RapiGest. Although most peptides showed a variation of this magnitude, it is worth noting that there was a considerable spread of CVs in all three protocols, with some peptides showing as much as 140% variation. By far the highest signal reproducibility with a CV maximal likelihood of 7% was obtained with the RapidACN protocol (Figure 4a), indicating best suitability of this protocol in label-free quantification.\n\n(a) The distribution maximum of coefficients of variation (CV) of the selected protocols varies between 0.075 and 0.2. CV values obtained for protocol triplicates are shown as two-dimensional distribution histograms (‘violin plots’). Quantification in DDA experiments were consistent over the dynamic range, as CV values only marginally changed when filtering by peptides according to their abundance (80%, 60%, 40% or 20%). CV likelihood maxima of all protocols were below 20%, while RapidACN lead to the most reproducible results (CV = 7%) (n = 3). (b) Stability in a quantification experiment is improved by data-independent acquisition. CV values for the same set of peptides measured with SWATH and DDA using the RapiGest protocol, as shown in a two-dimensional distribution histogram. Whereas there was a high signal variation in DDA acquisition, the variation could be largely reduced in SWATH acquisition. (c) In solution protocols yield the highest number of peptides suitable for label-free quantification. The number of peptides with a CV < 0.15 as determined in DDA and SWATH acquisitions. The number of highly reproducible peptides was lower for FASP and eFASP, and SWATH acquisition did not improve the performance in combination with these methods. In solution protocols on the other hand did yield a maximum of about 2500 high-quality peptides using SWATH.\n\nSummary of the main characteristics of the sample preparation methods investigated.\n\nNext, we counted the number of precisely quantified peptides, defined as peptides with a CV < 15%. Also in this measure, the RapidACN procedure outperformed the other methods, while RapiGest, and eFASP performed second and third best, respectively (Figure 4c). Not covered in this benchmark is the performance of the individual protocols in repeated sample preparation over longer periods, i.e. weeks to months. This might be required for particular sets of samples that can not be stored without a protease digest, yet require sampling on different days to address a specific biological question.\n\nFinally, we tested whether SWATH analysis improved label-free quantification. Comparing the CV distribution of peptides detected both in DDA and SWATH data using the RapiGest protocol (Figure 4b), we discovered a much more focussed CV distribution around a maximal likelihood of 5% in SWATH, compared to a maximal likelihood of 20% in DDA mode. When counting the number of precisely quantified peptides (CV < 0.15), SWATH led to an increase of up to a factor of two and five for RapidACN and RapiGest, respectively (Figure 4c). Hence, SWATH acquisition greatly improved the CV stability with label-free acquisition, the result of which is that a substantial number of peptides were precisely quantified.\n\n\n\n\nDiscussion\n\nStable isotope labelling is a popular and reliable strategy in quantitative proteomics, yet it has limitations that arise from an increased analyte load in the precursor ion (MS1) space, and the way standards are produced or incorporated: For instance, targeted protein quantification using AQUA peptides45, achieves absolute quantification though comparison between the peak areas of light and chemically synthesized heavy-isotope labelled peptides of known concentration. However the costs for such peptides limits the number of proteins quantifiable7,45. An alternative strategy is the non-targeted chemical labelling of proteins and peptides with isobaric tags (i.e. iTRAQ, TMT), facilitating multiplexing of proteome samples and providing relative simultaneous quantification of labelled peptides8,46. However, frequent co-selection of the reporter ions reduces both the accuracy and precision of quantification47,48 Such a problem is circumvented when metabolic incorporation of isotope-labelled amino acid residues (i.e. SILAC49, or recent extensions like instance NeuCODE which is based on different nuclear mass dependent on the isotope combination integrated50), is used to create isotope-labelled standards in vivo. However, this approach is limited to heterotrophic species that consume lysine and arginine from the culture medium, and is in practice limited to tissue culture as the attempt to introduce labelling in animal models becomes extremely expensive51.\n\nLabel-free experiments circumvent the use of isotope labelled standards, thus are not affected by the above-mentioned limitations. As such, they are ideal complements when isotope labelling becomes a limitation. However, they lack possibilities to correct for selective sample loss, and hence are more sensitive to variations in sample preparation and instrument performance. The protocols employed thus require more rigorous validation.\n\nOur comparison starts with a classic in gel digestion method19, which is tested in combination with SDS-containing- and SDS-free protein extractions (Supplementary protocol 1 and Supplementary protocol 2). These popular cost-effective procedures are based on the principle that a protein sample is denatured and separated on an SDS-PAGE gel prior to reduction, alkylation and protease digestion that are conducted within the gel matrix. The gel fulfils the function of sample clean up, as it removes positively charged contaminants as well as large macromolecules (i.e. nucleic acids) and small chemical compounds, and is very robustly applied to a large variety of sample types. Furthermore, the excision of individual bands or mass ranges make in gel digestions attractive wherever a simple sample pre-fractionation is required. Proteome pre-fractionation in gel (geLC-MS) has resulted in a significant proteome depth and dynamic range in studies were > 5000 distinct proteins were confidently identified and quantified52,53. Moreover, in gel digests have proven ideal when gel bands resulting from individual proteins are to be identified (i.e. for studying protein complexes). In the present study however, we did not make use of sample pre-fractionation. In order to achieve comparability with the other protocols, the full mass range was processed for the digest (see Methods section, and Supplementary protocol 1 and Supplementary protocol 2). This treatment led to a full representation of the proteomic mass distribution (Supplementary Figure S1). Under these circumstances however, the classic in gel protocol applied proved the least suitable method for label-free quantification. The protocol was the most time consuming, yet yielded a significant number of miscleaved peptides, and we detected the lowest number of proteins and peptides in total. Differences between SDS-free and SDS-containing sample extraction concerned the relative content of membrane proteins identified, which was higher in the latter, whereas the native extraction resulted in a higher number of proteins identified in total. This result should however not be interpreted as a general critique on in gel methods, as in combination with protein pre-fractionation (gel-slicing), they have proven for well-suitable sample preparation methods in ID experiments52,53.\n\nThe dependence on filter units in the two tested filter-aided sample preparation procedures, FASP32, and one of its recent extensions (here called eFASP21), increases the material costs, but has advantages for sample handling and throughput. Indeed, handling of the first protocol, FASP, was efficient and achieved a reasonable throughput with modest hands-on time (Supplementary protocol 1). In protein identification, FASP achieved the highest relative amount of detected membrane proteins. Hence, this protocol might be an ideal choice when membrane proteins are to be studied.\n\nFASP was the only protocol in this study where digestion was carried out using a combination of proteases, LysC and trypsin. Similar to previous reports54, we observed that the addition of LysC increased the relative digestion efficiency. However, this resulted in an over-representation of lysine over arginine containing peptides, which may lead to bias in cases where this protocol is used in an absolute quantification experiment. In label-free quantification, FASP performance was average both in the number of precisely quantified peptides and in the CV values obtained for replicative sample preparations. It is important to mention in this context that the performance of FASP procedures is dependent on the filter units that are available from different manufacturers, but exactly the same filter unit which was used in the original FASP paper32 is no longer available. In this study we have chosen Amicon Ultra-0.5 3k for both FASP based protocols as used in eFASP by Shevchenko et al.21, as their cut-off rate of 3 kDa is the closest to the addressable mass range of the SWATH acquisition (400–1200 m/z). Further work from Wisniewski et al. demonstrated that also larger cut-off rates up to 50k are suitable in combination with the FASP protocol, and can improve the identification rate of larger proteins and peptides55. Moreover, in difference to the other protocols tested in this study, the tryptic digest in FASP is conducted in a very high concentration of urea. A simple protocol adaptation to influence the tryptic digest could thus be to change the buffer conditions, i.e. to a buffer as used in eFASP21 (Supplementary protocol 4).\n\nThe second filter-aided protocol, eFASP, represents a stepwise optimisation of FASP, and contains several alterations compared to its predecessor21 (Supplementary protocol 4). The protease digest is performed using trypsin only, and the protocol includes a lipid removal step and uses n-octyl-D-glucopyranoside (nOGP) as the detergent in sample preparation. The latter might be regarded as an undesirable addition to the sample, as nOGP can interfere with electrospray ionisation. Indeed, despite all washing steps, we could detect traces of nOGP in the MS/MS spectra, and the collection of MS data was reduced at the time a nOGP sodium adduct eluted (data not shown). Despite this, the modifications made for eFASP clearly improved the performance in protein and peptide identification. However, in our hands, they did not improve the precision in label-free quantification, the performance of FASP and eFASP in this measure was comparable (Figure 4). Hence, the main advantage of eFASP over FASP lies in improvements in protein identification and proteome coverage.\n\nThe first method tested (Supplementary protocol 5) is based upon the commercial reagent RapiGest (3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate37 (Waters)), an anionic detergent which is depleted from the sample through acidic cleavage. The established protocol22 contains a step for lipid removal and a precipitation step that renders this procedure more laborious compared to the FASP and RapidACN protocols. However, as it does not involve any filter unit, it was most economic in terms of material costs per sample if one disregards the in gel protocols. Moreover, it yielded the highest number of protein and peptide IDs, and it detected the highest absolute number of membrane proteins. In label-free quantification, it scored third best in the average CV for DDA, and second best in combination with SWATH acquisition. Expressed in absolute quantities, this method yielded the second-highest numbers of precisely quantified peptides. Thus, the RapiGest protocol is a versatile and economic method that may represent the optimal choice in many applications. The only inexplicable issue with this protocol was related to the inefficiency of RapiGest degradation and precipitation in a small subset of samples. Thus care must be taken to avoid its injection in the LC-MS/MS setup.\n\nThe second in solution protocol (termed RapidACN9, Supplementary protocol 6) is detergent-free and based on acetonitrile in sample processing and proteolytic cleavage followed by clearing samples from high-molecular weight contaminants by a final filtration step. As this protocol is based on a native protein extraction, it identified - in relative terms - the lowest number of membrane proteins. Moreover, as it does not contain an intensive pre-digest sample treatment, functionality of this protocol may omit tissue were such a forefront clean up is mandatory. Despite these limitations, RapidACN performed best in the metric most crucial for robust label-free quantification, a low CV value in replicate sample digests and injections. Moreover, compared to the other tested methods, RapidACN was simplest in handling, required the least processing steps and only minimal hands-on time (~2 hrs), while yielding the second highest number of protein and peptide detection both in DDA and SWATH acquisition methods. Hence, RapidACN might be the most suitable solution for a label-free experiment when the focus is not to quantify membrane proteins, or to analyze tissue that requires extensive clean up.\n\nWe chose to perform major parts of this study on a TripleTOF5600 instrument (AB/Sciex), in order to compare data-dependent acquisition (DDA) with data-independent acquisition (DIA). DIA is believed to be advantageous for label-free quantification, as it is not affected by run to run variation, and as MS2 data is reconstructed in chromatograms that resemble selective reaction monitoring (SRM)17. Therefore, this technique appears a desirable choice for the label-free analysis of biological time series, that require many samples (replicates over many time-points) to be compared15. The design of the TripleTOF5600 quadrupole allows precursor ion selection in a rectangular rather than a Gaussian mass selection window as in other instruments, reducing the co-selection of peptides falling in the adjacent mass windows23. In a workflow termed SWATH, the mass range from 400 to 1200 m/z is scanned in 25 Da windows, and the merged data used to reconstruct spectral (MS2) m/z chromatograms17. Processing SWATH data with Spectronaut (V. 3.0.337, Biognosys), we compared the performance of DDA with SWATH in protein detection and label-free quantification. In samples with low peptide content, the number of detected proteins with DDA and SWATH was comparable. However, in the in solution protocols that led to highest IDs, SWATH acquisition gave a slight but significant advantage in terms of peptides detected. This indicates that this approach is advantageous in protein detection when coupled with complex matrices. Significantly improvement of SWATH versus DDA was however observed in label-free quantification. The strongest effect of SWATH acquisition was observed when it was used in conjunction with the RapiGest protocol (Supplementary protocol 5), where the number of precisely quantified peptides increased by a factor of five, followed by the combination with RapidACN (Supplementary protocol 6), where this measure doubled (Figure 4c). Of note, SWATH employed in combination with the latter, resulted in an average CV below 5%, representing a superior value obtained in a label-free experiment. These improvements mainly resulted from a more reliable quantification of peptides in the mid to high abundance range, whereas there was no increased improvement quantification of low abundant spectra over DDA. We assume that this difference could be further optimized by improving the SWATH peak selection algorithms, as noise in the low abundance window results from occasional misassignment of fragment ions to precursors.\n\n\nConclusions\n\nBy facilitating label-free quantification, second-generation proteomics techniques enable flexible proteomic workflows. As the protocols cover different sets of proteins, the main determinant to select the best suitable method and workflow remains the biological question and the set of proteins to be addressed. Despite this, sample preparation methods differ in precision, sensitivity and throughput. Under the conditions of this benchmark, and under the conditions in our laboratory, a combination of in solution digestion protocols RapiGest or RapidACN with SWATH acquisition yielded optimal results for a label-free proteomics experiment. Achieving reliable quantification at reasonable numbers of detected proteins, label-free quantitative proteomics represents a suitable alternative to isotope labelling in addressing a series of biological problems.",
"appendix": "Author contributions\n\n\n\nJakob Vowinckel, Floriana Capuano and Kate Campbell and Michael J. Deery, designed and conducted experiments, Kathryn S. Lilley and Markus Ralser designed experiments, all authors wrote on the paper.\n\n\nCompeting interests\n\n\n\nThe authors declare no competing interests.\n\n\nGrant information\n\nThis work was funded by the Isaac Newton Trust, the Wellcome Trust (RG 093735/Z/10/Z) to MR, the ERC (Starting grant 260809) to MR and the 7th Framework Programme of the European Union (262067- PRIME-XS) to KSL. M.R. is a Wellcome Trust Research Career Development and Wellcome-Beit prize fellow.\n\n\nAcknowledgements\n\nWe thank our lab members for help in this manuscript, and Pavel Shliaha (University of Cambridge) for help with the RapiGest sample preparation procedures.\n\n\nSupplementary material\n\nRAW data: The .wiff files of the DDA and DIA acquision used in Figure 2–Figure 4 are downloadable as Supplementary Data. Supplementary table ST1 lists the different file names.\n\n(ΙCEΙ=(slope)*(m/z)+intercept\n\n(a) The spectrum of protein sizes is well covered for all protocols examined. The number of identified proteins was plotted against the theoretical molecular weight (MW) for each protocol investigated. Although some protocols yielded higher identifications than others, the MW range was well reproduced for all of them when comparing to the whole yeast proteome. (b) Different representations of protein charges. When comparing to the distribution of theoretical protein pI values of the whole proteome, all investigated protocols showed an under-representation of proteins with a pI of 10. When expressing the total deviation as deviation score d, RapiGest, FASP and RapidACN score best. 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{
"id": "3900",
"date": "27 Feb 2014",
"name": "Xianyin Lai",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn their manuscript, Vowinckel et al. evaluated 6 sample preparation methods for label-free quantification, compared DDA and SWATH approaches of TripleTOF5600, and drew two major conclusions. The organization of the manuscript is clear, it is well-written, and the coverage is effective. The first conclusion is supported by their results. However, the second conclusion should be clarified that SWATH outperformed DDA in quantification when a TripleTOF5600 was applied. Without the clarification, it misleads scientists to believe that SWATH outperforms all DDA approaches carried out using other mass spectrometers. Figure 2C shows that DDA of QExactive detected about 40% more proteins than SWATH of TripleTOF5600. It is necessary to compare DDA of QExactive with SWATH of TripleTOF5600. Minor issues:\"Protein Discoverer\" should be \"Proteome Discoverer\"",
"responses": [
{
"c_id": "757",
"date": "07 Apr 2014",
"name": "Markus Ralser",
"role": "Author Response",
"response": "We thank the reviewer for the suggestion and clarify this now. Indeed, the TripleTOF5600 and Qexactive data can only to be compared indirectly/correlative in this study, but not absolutely, as different chromatography, HPLC, and ionisation settings were used. Therefore, the study can not be used to compare the Qexactive and TripleTOF instruments. However, in relative terms the protocols perform equally on both platforms, see Figure 2d, inset. As suggested, we have improved this part in manuscript and abstract, and included the Reviewer's comments."
}
]
},
{
"id": "3833",
"date": "04 Mar 2014",
"name": "Nick Morrice",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well written manuscript and provides an excellent reference for sample preparation ahead of any label free proteomics experiment. The protocols in the supplementary section are very detailed making them very easy for both novice and expert researchers to follow. The manuscript basically compares three different types of sample preparation and describes the positives and negative for each protocol if applied to label free quantification of proteins. This manuscript will be a very useful reference for anyone who wishes to perform label free quantification of proteins as the authors do highlight both the advantages and disadvantages of each approach.The minor revisions I would like to see are in the description of creating a SWATH ion library using offline high pH RP-HPLC. The authors mention the addition of iRT peptides to each fraction, but do not describe why they are added. These retention time reference peptides are added so that any retention time drift from the ion library to the samples can be accounted for by the software used to analyse and quantify the SWATH data. I feel this should be described, especially for novices to the field. Secondly there is a slight complication in using the term eFASP in this manuscript. A very recent publication by Erde, J. et al. (2014) uses the term eFASP to describe a method that differs significantly from the one described here. I think this paper should be referred to in this manuscript just to highlight that the eFASP method used here was based on the method described by Shevchenko et al. (2012) and not to be confused with this more recent publication. Apart from these minor corrections, I have no reservations in approving this article.",
"responses": [
{
"c_id": "756",
"date": "07 Apr 2014",
"name": "Markus Ralser",
"role": "Author Response",
"response": "We thanks for these suggestions, and for spotting out the nomenclature overlap with Erde et al. (2014). As the Erde et al. article was published ~three months after the first version of this article we decided to keep the nomenclature, but add a caveat for clarification on two instances in the paper. An explanation/rationale about the use of retention time normalisation standards is now included as well."
}
]
}
] | 1
|
https://f1000research.com/articles/2-272
|
https://f1000research.com/articles/3-14/v1
|
16 Jan 14
|
{
"type": "Web Tool",
"title": "mfSBA: Multifractal analysis of spatial patterns in ecological communities",
"authors": [
"Leonardo A. Saravia"
],
"abstract": "Multifractals have been applied to characterize complex communities in a spatial context. They were developed for nonlinear systems and are particularly suited to capture multiplicative processes observed in ecological systems. Multifractals characterize variability in a scale-independent way within an experimental range. I have developed an open-source software package to estimate multifractals using a box-counting algorithm (available from https://github.com/lsaravia/mfsba and permanently available at doi: 10.5281/zenodo.7659). The software is specially designed for two dimensional (2D) images such as the ones obtained from remote sensing, but other 2D data types can also be analyzed. Additionally I developed a new metric to analyze multispecies spatial patterns with multifractals: spatial rank surface, which is included in the software.",
"keywords": [
"Multifractals and fractals are related techniques mainly used in physics to characterize the scaling behavior of a system",
"they differ in that fractals look at the geometry of presence/absence patterns",
"while multifractals look at the arrangement of quantities such as population densities or biomass1. Scaling laws are an emergent general feature of ecological systems",
"and there is no a priori reason that power laws apply to ecological communities. If they do apply",
"they reflect constraints in their organization that can provide clues about the underlying mechanisms2",
"3."
],
"content": "Introduction\n\nMultifractals and fractals are related techniques mainly used in physics to characterize the scaling behavior of a system; they differ in that fractals look at the geometry of presence/absence patterns, while multifractals look at the arrangement of quantities such as population densities or biomass1. Scaling laws are an emergent general feature of ecological systems, and there is no a priori reason that power laws apply to ecological communities. If they do apply, they reflect constraints in their organization that can provide clues about the underlying mechanisms2,3.\n\nMultifractals require that the object under study should be statistically self-similar, which means that a power-law could be fitted to data in a range of scales. But that does not mean that the power-law must be the best possible model. We can analyze the data without claiming that it is an exact multifractal. One of the advantages of multifractals is that they require fewer conditions on data than more classical statistics such as autocorrelation and variograms. These usually require isotropy and stationarity4 but multifractals can be used with anisotropic data5 and are inherently non-stationary6,7. Anisotropy and non-stationarity are often seen in spatial ecological distributions8.\n\nMultifractals are associated with systems governed by random multiplicative processes9. In ecological systems, these processes can be given as the interaction of survival probabilities and compound growth10. Moreover, the presence of multiplicative process is argued to produce the log-normal-like shape of species-abundance distributions11. Also, random processes with spatial correlations can generate multifractals9; these kind of processes are part of neutral community models12,13 and are observed in natural communities14. Thus there are a priori reasons to think that multifractals can be applied to spatial ecological data. Indeed, they have been applied to vegetal communities15, tropical forest16, microphytobentos and periphyton biomass patterns1,17, and to the characterization of species-area relationships18–20.\n\nRank-abundance distributions are a representation of species-abundance distributions (SAD) that are a classical description of communities21. These have been used to compare different communities and to compare models and data, but different mechanisms can produce nearly identical SADs22. SADs are often presented using rank-abundance diagrams (RAD) where the log-abundance is plotted on the y-axis vs. rank on the x-axis21. RADs are equivalent to cumulative distributions23 and thus are a robust way to visualize the SAD without losing information24. If the rank of each species is incorporated in its spatial distribution, it forms a surface: the species-rank surface (SRS). This SRS can be analyzed and compared using multifractals.\n\nBut the application of multifractals is not widespread25; one of the reasons being the difficulty of using the available software for quantitative multifractal analysis (MFA). Here I present an open source software package for the application of multifractals, which can be integrated with R statistical software26.\n\n\nMultifractal analysis\n\nSeveral good introductions to multifractal methods applied to ecology are available15,27; thus I will only give a brief overview. Multifractals analyze the scaling properties of quantities distributed in a space that we assume to be two dimensional (a plane), but MFA can be used with one dimensional (time series) or three dimensional data28. A classical way to characterize multifractals is using the generalized dimensions Dq29, also called Renyi dimensions30. Dq has been used to characterize the probabilistic structure of attractors derived from dynamical systems5.\n\nAnother way to characterize multifractals is using the so called spectrum of singularities. This spectrum describes multifractals as interwoven sets each one with a singularity exponent α and a fractal dimension f (α)31. The two multifractal representations are equivalent, they display the same information in a different format. But with the spectrum of singularities, two quantities are estimated (α & f (α)) from data and are obtained with error. Instead, with generalized dimension only one quantity is estimated Dq, thus this method is preferred for statistical comparisons.\n\n\nEstimation\n\nTo estimate multifractal spectra I used the method of moments based on box-counting32. I estimate generalized dimensions and the spectrum of singularities at the same time using the canonical method31. Here I describe only the Dq estimation; the steps for α and f (α) estimation are identical (only the formulae to calculate the quantities are different and can be found in the appendix of Saravia et al. (2012)1).\n\nThe spatial distribution that we are analyzing is covered with a grid, which is divided into N (∈) squares of side ∈. The contents of each square is called μi(∈). Then the so called partition function is computed as:\n\n\n\nWhere q is called moment order. The operation is performed for different values of ∈ and q, within a predetermined range. The generalized dimension is calculated as:\n\n\n\nWhen q = 1, the denominator of the first term in Dq is undefined, so it must be replaced by the following expression:\n\n\n\nIn practical cases, as the limit can not be assessed, the dimensions are estimated as the slope of log(Zq) versus log(∈) in equation (1) (Figure 1). This is done for different q, provided that it is a real number which yields a graph of Dq in terms of q, called the spectrum of generalized dimensions (Figure 2).\n\nIf each species is assigned a number at random (unordered) the Dq is almost flat corresponding to a uniform plus random noise distribution. But when the species rank surface (SRS) is used the Dq spectrum have a wide range of values. The error bars are the standard deviation obtained from the linear regressions used to estimate Dq.\n\nTo be an approximate multifractal, the relationship log(Zq) versus log(∈) should be well described by a linear relationship, although a linear relationship with superimposed oscillations is also acceptable27. A range of q and ∈ is fixed and then Dq is estimated using linear regressions. The coefficient of determination (R2) can be used as a descriptive measure of goodness of fit18.\n\n\nUse of mfSBA software\n\nThe software was built and tested under Ubuntu 12.04 LTS Linux environment, using the GNU C++ compiler (v4.6.3). It requires the libtiff library for reading tiff images. It can be compiled under Windows environments using the GNU compiler and utilities for that operative system, but it was not tested.\n\nYou can download or clone mfSBA from https://github.com/lsaravia/mfsba (using git clone) and build it using the make utility\n\nmake -f mfSBA.mak\n\nYou can run it from the command line using the following command structure:\n\nmfSBA inputFile qFile minBox maxBox numBoxSizes option\n\nthe parameters are:\n\ninputFile: this file can have only two formats: 1) one-layer tiff 2) “sed” file format. Sed is an ascii format I invented to use with my own stochastic cellular automata models to represent a square grid of values. It has a header of two lines: the first line describes the two dimensions X Y of the data, and the second line describes the type of data. For this program, the type must be BI, which means that the values stored in the grid are real numbers with double precision. See the example file with a “.sed” extension.\n\nqFile: this is a sed file with a vector of values representing the q’s used to calculate the multifractal spectrum.\n\nminBox,maxBox,numBoxSizes: Minimum box size, maximum box size and maximum number of box sizes. The program uses box sizes in powers of two: if maxBox is greater than half of the image size, it is set to that value. If the number of boxes between minBox and maxBox is greater than numBoxSizes, the latter number of boxes will be used, discarding the biggest ones.\n\noption: is an upper case character with four possibilities: N,S,D,A.\n\n– N: analyze the input file as is.\n\n– S: normalize the input file then analyze it. Normalization is done summing all the pixels values and dividing each pixel by that total. After that the sum of all values is one.\n\n– D: add 1 to all the pixels then normalize as in S.\n\n– A: normalize as in S and save the normalized image as a sed file.\n\nExamples of input files are included with the source code, thus after compiling you could run the following command assuming a linux system:\n\n./mfSBA b4-991008bio.sed q21.sed 2 256 20 S\n\n\nOutput\n\nThe program generates four output files, attaching a prefix to the original input file name:\n\nt.inputFile: this file has a header line with field names and q + 2 columns. The first two columns are the box sizes and log box sizes used in the estimation. After that, each column corresponds to log (Zq (∈)) of equation (2) with the q’s specified in the qFile. This file could be used to visually check the linearity assumptions to calculate Dq as in Figure 1.\n\na.inputFile & f.inputFile: are similar to the previous file, but used to calculate α and f (α). The formulae are described in the appendix of Saravia et al. (2012)1.\n\ns.inputFile: this file has a header line with field names and 10 columns. The first column is q. The second column is called Tau and is the result of the regression to calculate the limit in equation 2. Thus to obtain Dq we have to divide it by q − 1, except in the case of q = 1 that we take the value of the next column to get Dq. The third column is the value of α and the forth column f (α). After that, columns are the corresponding coefficients of determination R2 and standard deviations.\n\n\nSpecies rank surface\n\nI propose to extend the analysis of SAD attaching the rank of each species to its spatial distribution. In this way, the multivariate spatial distribution of all species can be summarized into a univariate distribution. I called this spatial distribution the species-rank surface (SRS), and it can be analyzed and compared using MFA. To construct the SRS, I first calculate the rank-ordering of the species by their abundance from biggest to smallest, starting from one. Then the rank is assigned to the spatial position of the individuals of each species, forming a surface. This landscape has valleys formed by the most abundant species and peaks determined by the rarest species, and the standard MFA can be applied. The program used to calculate this is called multiSpeciesSBA, and is included with the mfSBA source code. You can compile it using the following command:\n\nmake -f multiSpeciesSBA.mak\n\nThen all the input files and parameters are identical to mfSBA except that the program expects an inputFile containing a multispecies distribution. So the inputFile should be composed of integer numbers each one representing one species. An example of a sed file with a multispecies spatial distribution is given in t64-0100.sed, this file was obtained using a spatially explicit neutral model with 64 species (available at https://github.com/lsaravia/neutral). You can use the following command to perform the MFA:\n\n./multiSpeciesSBA t64-0100.sed q21.sed 2 128 20 N\n\n\nR integration\n\nIncluded with the source is a set of functions as an example to integrate the mfSBA software with the R language. You can load the functions inside R with:\n\nsource('Fun_MFA.r')\n\nand then run the same given examples:\n\ndq1<− calcDq_mfSBA(\"b4-991008bio.sed\",\"q21.sed 2 256 20 S\")\n\nAn interesting example is to compare the Dq from the example multispecies spatial distribution untransformed\n\ndq1<− calcDq_mfSBA(\"t64-0100.sed\",\"q21.sed 2 512 20 S\",T)\n\ndq1$Site <− \"Untransformed\"\n\nwith the Dq from SRS\n\ndq<− calcDq_multiSBA(\"t64-0100.sed\",\"q21.sed 2 512 20 S\",T)\n\ndq$Site <− \"Species Rank Surface\"\n\ndq <− rbind(dq,dq1)\n\nand plot Dq with\n\nplot_DqCI(dq)\n\nIn this plot (Figure 2), we can see that the rank ordering of the species in SRS is crucial to obtain a meaningful result. The Dq calculated from the unordered distribution is nearly flat, this corresponds to an almost constant spatial distribution with uncorrelated random noise.\n\nThe plot of the t.inputFile (Figure 1) gives a visual check of the regressions to obtain Dq:\n\nplotDqFit(\"t.t64-0100.sed\",\"q21.sed\")\n\nadditionally the R2 values could be easily checked:\n\nhist(dq1$R.Dq)\n\nAll the examples and more graphics are included in the file testMFA.r.\n\n\nConclusion\n\nThe multifractal spectrum can be used to describe spatial patterns of biomass, density, height, or any continuous variable. The mfSBA software is especially useful for remote sensing data because it can be used with tiff images. Multifractal patterns could be produced by the existence of multiplicative interaction between species and by spatially correlated random processes such as dispersal and growth9. Plant and animal species are generally aggregated in space thus is very likely that multifractal analysis can be used in a wide range of cases.\n\nThe analysis of SRS using Dq adds a new dimension to the comparison of species spatial distributions, because it can be used to compare spatial distributions of all species at the same time and also the abundances are accounted. An exploration of the results of different spatial patterns should be needed as a continuation of the present work.\n\nThe software presented here is oriented to obtain multifractal spectra for comparisons, rather than to obtain the true value. While the estimation methods used in mfSBA could be improved27,33, it has been used without trouble with the kind of data obtained in ecological studies1,34.\n\nMultifractals can be successfully used to analyze several aspects of community spatial structure. With the advent of the big data era in ecology35 and the use of new technology to acquire spatial data36, new methods to analyze complex data sets are needed and multifractals could be an interesting addition to the ecologist’s toolbox.\n\n\nSoftware availability\n\nZenodo: Multifractal estimation using a standard box-counting algorithm, doi: 10.5281/zenodo.765937\n\nGitHub: Multifractal estimation using a standard box counting algorithm, https://github.com/lsaravia/mfsba",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nI am grateful to Fernando R. Momo for our great conversations about ecological theory and David J. Harris for his language revision.\n\n\nReferences\n\nSaravia LA, Giorgi A, Momo F: Multifractal growth in periphyton communities. Oikos. 2012; 121(11): 1810–1820. Publisher Full Text\n\nBrown JH, Gupta VK, Li BL, et al.: The fractal nature of nature: power laws, ecological complexity and biodiversity. Philos Trans R Soc Lond B Biol Sci. 2002; B 357(1421): 619–626. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSolé RV, Bascompte J: Self-organization in complex ecosystems. Princeton University Press. 2006. Reference Source\n\nFortin MJ, Dale MRT, ver Hoef J: Spatial Analysis in Ecology. Encyclopedia of Environmetrics. John Wiley & Sons, Ltd. 2006. Publisher Full Text\n\nHarte D: Multifractals: Theory and Applications. Chapman & Hall/CRC. 2001; 264. Reference Source\n\nLaurie H, Perrier E: A multifractal model for spatial variation in species richness. Ecological Complexity. 2010; 7(1): 32–35. Publisher Full Text\n\nBez N, Bertrand S: The duality of fractals: roughness and self-similarity. Theoretical Ecology. 2011; 4(3): 371–383. Publisher Full Text\n\nPlotkin JB, Chave J, Ashton PS: Cluster analysis of spatial patterns in Malaysian tree species. Am Nat. 2002; 160(5): 629–644. PubMed Abstract | Publisher Full Text\n\nStanley HE, Meakin P: Multifractal phenomena in physics and chemistry. Nature. 1988; 335: 405–409. Publisher Full Text\n\nMcGill BJ: Strong and weak tests of macroecological theory. Oikos. 2003; 102(3): 679–685. Publisher Full Text\n\nMay RM, Crawley MJ, Sugihara G: Communities: patterns. In: May RM McLean AR editors. Theoretical ecology: principles and applications. New York: Oxford University Press. 2007; 111–131. Reference Source\n\nHouchmandzadeh B, Vallade M: Clustering in neutral ecology. Phys Rev E Stat Nonlin Soft Matter Phys. 2003; 68(6 pt 1): 1–7. PubMed Abstract | Publisher Full Text\n\nYakimov BN, Iudin DI, Solntsev LA, et al.: Multifractal analysis of neutral community spatial structure. J Theor Biol. 2014; 343: 44–53. Publisher Full Text\n\nCondit R, Ashton PS, Baker P, et al.: Spatial patterns in the distribution of tropical tree species. Science. 2000; 288(5470): 1414–1418. PubMed Abstract | Publisher Full Text\n\nScheuring I, Riedi RH: Application of multifractals to the analysis of vegetation pattern. Journal of Vegetation Science. 1994; 5(4): 489–469. Publisher Full Text\n\nManrubia SC, Solé RV: Self-organized criticality in rainforest dynamics. Chaos, Solitons & Fractals. 1996; 7(4): 523–541. Publisher Full Text\n\nSeuront L, Spilmont N: Self-organized criticality in intertidal microphytobenthos patch patterns. Physica A. 2002; 313(3–4): 513–539. Publisher Full Text\n\nBorda-de-Água L, Hubbell SP, McAllister M: Species-area curves, diversity indices, and species abundance distributions: a multifractal analysis. Am Nat. 2002; 159(2): 138–155. PubMed Abstract | Publisher Full Text\n\nYakimov BN, Bossuyt B, Iudin DI, et al.: Multifractal diversity-area relationship at small scales in dune slack plant communities. Oikos. 2008; 117(1): 33–39. Publisher Full Text\n\nLaurie H, Perrier E: Beyond species area curves: application of a scale-free measure for spatial variability of species richness. Oikos. 2011; 120(7): 966–978. Publisher Full Text\n\nMcGill BJ, Etienne RS, Gray JS, et al.: Species abundance distributions: moving beyond single prediction theories to integration within an ecological framework. Ecol Lett. 2007; 10(10): 995–1015. Publisher Full Text\n\nChave J, Muller-Landau HC, Levin SA: Comparing classical community models: theoretical consequences for patterns of diversity. Am Nat. 2002; 159(1): 1–23. PubMed Abstract | Publisher Full Text\n\nNewman MEJ: Power laws, Pareto distributions and Zipf’s law. Contemporary Physics. 2005; 46(5): 323–351. Publisher Full Text\n\nLi L, Alderson D, Doyle JC, et al.: Towards a Theory of Scale-Free Graphs: Definition, Properties, and Implications. Internet Math. 2005; 2(4): 431–523. Publisher Full Text\n\nSeuront L: Fractals and Multifractals in Ecology and Aquatic Science. Taylor & Francis. 2009. Reference Source\n\nR Core Team: R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing. 2013. Reference Source\n\nBorda-de-Água L, Hubbell SP, He F: Scaling biodiversity under neutrality. Scaling biodiversity. 2007; 347–375. Reference Source\n\nLopes R, Betrouni N: Fractal and multifractal analysis: a review. Med Image Anal. 2009; 13(4): 634–649. PubMed Abstract | Publisher Full Text\n\nGrassberger P: Generalized dimensions of strange attractors. Phys Lett A. 1983; 97(6): 227–230. Publisher Full Text\n\nRenyi A: Probability Theory. Amsterdam: North-Holland. 1970. Reference Source\n\nChhabra AB, Jensen RV: Direct determination of the f(alfa) singularity espectrum. Phys Rev Lett. 1989; 62(9): 1327–1330.\n\nHalsey TC, Jensen MH, Kadanoff LP, et al.: Fractal measures and their. singularities: The characterization of strange sets. Phys Rev A. 1986; 33(2): 1141–1151. PubMed Abstract | Publisher Full Text\n\nDe Bartolo SG, Primavera L, Gaudio R, et al.: Fixed-mass multifractal analysis of river networks and braided channels. Phys Rev E Stat Nonlin Soft Matter Phys. 2006; 74(2 pt 2): 26101. PubMed Abstract | Publisher Full Text\n\nSaravia LA, Giorgi A, Momo FR: Multifractal Spatial Patterns and Diversity in an Ecological Succession. PLoS One. 2012; 7: e34096. Publisher Full Text\n\nHampton SE, Strasser CA, Tewksbury JJ, et al.: Big data and the future of ecology. Front Ecol Environ. 2013; 11: 156–162. Publisher Full Text\n\nMichener WK, Jones MB: Ecoinformatics: supporting ecology as a data-intensive science. Trends Ecol Evol. 2012; 27(2): 85–93. PubMed Abstract | Publisher Full Text\n\nSaravia LA: Multifractal estimation using a standard box-counting algorithm. ZENODO. 2014. Data Source"
}
|
[
{
"id": "3170",
"date": "24 Jan 2014",
"name": "Yuxin Zhang",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a useful Web Tool for the researcher, particular the ecologist in the community ecology, who wants to use the multifractal method describing the SAD and point pattern of species distribution. The paper is well written. However, the script (followed with the instruction in the “R integration” section) don't work well in Windows with R 3.0.2 (64bit). The author needs to revise the R scripts or provide detailed instruction on it, before the work is acceptable. Reports on the performance of the software in GNU for the Windows environment are also needed. There are two minor comments:The q is usually an integer in community ecology, because this will directly correspond to the Hill numbers. The range of q in this paper is from -5 to 5. Can the users of this tool set the range of q for their own preference?",
"responses": [
{
"c_id": "704",
"date": "15 Feb 2014",
"name": "Leonardo Saravia",
"role": "Reader Comment",
"response": "I appreciate Dr. Zhang's comments on the manuscript. As the software was developed and tested under Linux it should not be expected to work flawlessly under windows. I agree that it would be a great benefit to release at least a windows working version and I will try to update the software as time and resources are available. But I think that the software should be evaluated as is, and not judged because it's available or not under different operative systems.It will be useful to open issues on github with the required enhancements to facilitate the development of the software: https://github.com/lsaravia/mfsba/issues.The q's can be real numbers or integers in any range and are specified in the qFile as explained on the main text."
}
]
},
{
"id": "3330",
"date": "14 Feb 2014",
"name": "Edmund Hart",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI think this software package will make a needed contribution to ecology. It meets are criterion for technical merit. However I believe the paper can still be substantially improved. There are two aspects which will benefit from revision.Main textIntroduction:I think the introduction would benefit greatly from clear examples of the benefits and uses of mfSBA. It seems that the author should address both the benefits of multifractal analysis as well as where the SRS improves upon comparisons made by SAD's. It seems that the author has already made some publications that could address this, and it would be useful to have a brief explanation. In general I found the Introduction lacking specificity. For instance the author states in the first paragraph: \"If they do apply, they reflect constraints in their organization that can provide clues about the underlying mechanism\" What constraints? What underlying mechanism is the author referring to? How does it provide clues about this these mechanisms?I finished reading the introduction and was unclear both about what multifractal were, and why I would want to use them.Methods and conclusionI think the most important part here is a clear explanation of the quantities of interest that I can get from mfSBA, and how to interpret them. While parameters like slope and intercept seem obvious to most people now, I had no idea how to interpret q or D_q, or how I would use them. Furthermore the author focuses on the ability of this new method to make comparisons, stating: \"The analysis of SRS using Dq adds a new dimension to the comparison of species spatial distributions, because it can be used to compare spatial distributions of all species at the same time and also the abundances are accounted.\"However, the comparison the author makes is very unclear. It would be nice to see a comparison where simulation conditions were clearly made, and perhaps even performed in R so users could tweak them. Figure 2 provides output for a comparison, but it's unclear how to interpret that comparison. In summary the main text would benefit from a clearer presentation of use cases, what parameters are being estimated, and how to understand those parameters in different scenarios, both simulated and real data.Software packaging.I think the average ecologist will find this package still rather intimidating. It requires the installation of secondary libraries as well as a C compiler. I wouldn't even know how to begin to install this on a windows machine. I would suggest the author create an R package with precompiled binary libraries for windows and mac. This combined with an improved manuscript will provide ecologists with a powerful tool.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-14
|
https://f1000research.com/articles/3-87/v1
|
04 Apr 14
|
{
"type": "Opinion Article",
"title": "The putative mechanisms underlying testosterone and cardiovascular risk",
"authors": [
"Avinash Maganty",
"Jason R. Kovac",
"Ranjith Ramasamy",
"Avinash Maganty",
"Jason R. Kovac"
],
"abstract": "The use of testosterone supplementation therapy (TST) is increasing primarily in men with symptomatic hypogonadism. While TST has been shown to have numerous benefits, as its use increases, the role on cardiovascular health must be explored. Previous evidence showed no adverse cardiovascular risks associated with TST use; however, more recent studies suggest that there may be an associated risk. The exact mechanism by which TST may contribute to cardiovascular risk has not been elucidated. Numerous mechanisms have been proposed which include testosterone’s effect on thromboxane A2 receptors, vascular adhesion molecule 1 receptors, erythropoiesis, and obstructive sleep apnea, all of which can ultimately lead to atherogenesis and increased cardiovascular risk.",
"keywords": [
"Testosterone supplementation therapy (TST) is used to treat patients who suffer from symptomatic hypogonadism. The benefits of TST are numerous and include improved sexual function",
"bone mineral density",
"muscle mass and strength1. Currently",
"there is no direct evidence linking TST with cardiovascular risk factors",
"however",
"several small studies have theorized potential mechanisms (Figure 1) by which TST contributes to cardiovascular risk and these are summarized in this article."
],
"content": "\n\nTestosterone supplementation therapy (TST) is used to treat patients who suffer from symptomatic hypogonadism. The benefits of TST are numerous and include improved sexual function, bone mineral density, muscle mass and strength1. Currently, there is no direct evidence linking TST with cardiovascular risk factors; however, several small studies have theorized potential mechanisms (Figure 1) by which TST contributes to cardiovascular risk and these are summarized in this article.\n\nTST increases TXA2 receptor expression, VCAM1 expression, erythropoiesis, and worsens sleep disorder breathing, all of which ultimately contribute to atherogenesis and worsening of cardiovascular health. VCAM1 - vascular cell adhesion molecule 1.\n\nOne proposed mechanism by which cardiovascular risk could be affected is in the regulation of platelet thromboxane A2 (TXA2) receptor expression by testosterone2. TXA2, by acting on membrane receptors, helps in platelet aggregation and vascular smooth muscle contraction3. TXA2 synthesis is increased in numerous thrombotic cardiovascular events4. Ajayi et al.2 examined the relationship between testosterone and TXA2 by measuring platelet TXA2 receptor density in response to TST in healthy men2. Men were given testosterone cypionate 200 mg IM or placebo at day 1 and day 14 of the 10-week study period. Platelet TXA2 receptor density and dissociation constant then were measured. Testosterone supplementation was associated with increased TXA2 receptor density compared with placebo, suggesting that testosterone regulates the expression of platelet TXA2 receptors. The mechanism by which this occurs is not completely understood. However, it has been shown that inhibition of both transcription and translation of TXA2 attenuates testosterone’s effects on TXA2 receptor density in multiple cell lines5. This finding suggests that testosterone may increase TXA2 receptor density by acting at the genomic level to stimulate its synthesis.\n\nTestosterone has also been shown to contribute to atherosclerotic lesions by promoting monocyte adhesion6. Atherosclerotic lesions are due to low density lipoprotein (LDL) infiltration into the arterial intima layer that subsequently results in endothelial cell activation and monocyte recruitment7. The endothelial cells express vascular cell adhesion molecule 1 (VCAM1) which allows for monocyte adhesion before transmigration through endothelial junctions7. These monocytes then release local inflammatory cytokines and metalloproteinases. The inflammatory cytokines promote smooth muscle proliferation that contributes to local plaque formation. Several studies have explored the role of dihydrotestosterone (DHT), a potent testosterone metabolite, in the various aspects of atherosclerotic plaque development8. McCrohon et al. studied the effect of DHT in male umbilical vein endothelial cells9. They demonstrated increased VCAM1 expression with increased monocyte adhesion. Similar findings have been observed using arterial endothelial cells9.\n\nTestosterone also plays a role in increasing hematocrit levels. Several studies have reported increased hematocrit levels following TST administration10,11. This correlation is likely to be secondary to the stimulation of erythropoiesis by testosterone. Increased hematocrit levels, or polycythemia, can itself contribute to adverse cardiovascular events. For example, an increased number of red blood cells can result in increased blood viscosity and predispose to thrombosis. A recent study by Marchioloi et al. analyzed the cardiovascular risks in patients with polycythemia vera12. Patients, whose mean age was 64 (62% males), were divided into either a less intensive treatment group in which the target hematocrit was 45–50% or an intensive treatment group in which the target hematocrit was 45–50%. The choice of therapeutic approach was left to the investigator. The primary endpoint studied was the time until death from a cardiovascular cause or a major thrombotic event. The authors found that those whose hematocrit was maintained below 45% had significantly lower rate of cardiovascular death and major thrombotic events compared to those who had a hematocrit between 45–50%12. Additionally, Kunnas et al. evaluated the association between hematocrit and coronary heart disease (CHD) in men over 5513. This study’s conclusions were similar to that previously discussed, in that men with a hematocrit greater than 50% were 1.8 times (1.1–2.7) more likely to die from CHD compared with men with a hematocrit of less than 50% after adjusting for coronary risk factors13.\n\nTestosterone may also contribute to cardiovascular disease by worsening pre-existing obstructive sleep apnea (OSA). Androgen deficiency is often observed in men who are obese or who have OSA14–16. Obese men have lower serum testosterone compared to age-matched non-obese men17. This may be secondary to hypothalamic dysfunction or increased metabolic clearance as a result of central obesity17. Similarly, previous studies have shown that men with OSA have low systemic testosterone levels, independent of increasing age or obesity, that correlate with the severity of hypoxia during sleeping hours16,18,19. Additionally, the low systemic testosterone levels in these men have been shown to be reversible with nasal continuous positive airways pressure therapy (CPAP)16,19. The mechanism of reduced testosterone in men with OSA is likely to occur through OSA mediated dysfunction of the pituitary-gonadal axis17,20. Although controversial, TST has been used in men with severe OSA, given that men may remain androgen deficient if they are unable to comply with continuous positive airway pressure (CPAP) or lose weight21. Hoyos et al. studied the effects of TST by conducting a randomized, double-blind, placebo controlled trial in 67 men with severe OSA. Testosterone treatment worsened the oxygen desaturation index and nocturnal hypoxemia compared to placebo, suggesting worsening of OSA14. OSA itself is an independent risk factor for cardiovascular disease, and is associated with myocardial infarction and stroke22. OSA likely contributes to disease by worsening atherosclerosis by affecting multiple atherogenic pathways such as hypertension, insulin resistance, dyslipidemia, endothelial dysfunction, and oxidative stress22.\n\nWhile testosterone has not been definitively linked to cardiovascular risk, these proposed mechanisms provide some insights into TST’s physiological effect on the cardiovascular system. Despite recent studies demonstrating an increased cardiovascular risk associated with TST23, there is a large body of literature demonstrating the benefits of testosterone therapy. In fact testosterone replacement has demonstrated to decrease mortality24,25. Until the results of randomized trials are available, appropriate patient counseling and an emphasis on the importance of compliance with follow-up are essential prior to initiating testosterone replacement.",
"appendix": "Author contributions\n\n\n\nAll the authors contributed equally to the writing of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nVigen R, O’Donnell CI, Barón AE, et al.: Association of testosterone therapy with mortality, myocardial infarction, and stroke in men with low testosterone levels. JAMA. 2013; 310(17): 1829–36. PubMed Abstract | Publisher Full Text\n\nAjayi AA, Mathur R, Halushka PV: Testosterone increases human platelet thromboxane A2 receptor density and aggregation responses. Circulation. 1995; 91(11): 2742–7. PubMed Abstract | Publisher Full Text\n\nHalushka PV, Mais DE, Mayeux PR, et al.: Thromboxane, prostaglandin and leukotriene receptors. Annu Rev Pharmacol Toxicol. 1989; 29: 213–39. PubMed Abstract | Publisher Full Text\n\nFitzgerald DJ, Roy L, Catella F, et al.: Platelet activation in unstable coronary disease. N Engl J Med. 1986; 315(16): 983–9. PubMed Abstract | Publisher Full Text\n\nMatsuda K, Mathur RS, Duzic E, et al.: Androgen regulation of thromboxane A2/prostaglandin H2 receptor expression in human erythroleukemia cells. Am J Physiol. 1993; 265(6 Pt 1): E928–E34. PubMed Abstract\n\nMcCrohon JA, Jessup W, Handelsman DJ, et al.: Androgen exposure increases human monocyte adhesion to vascular endothelium and endothelial cell expression of vascular cell adhesion molecule-1. Circulation. 1999; 99(17): 2317–22. PubMed Abstract | Publisher Full Text\n\nHansson GK: Inflammation, atherosclerosis, and coronary atery disease. N Engl J Med. 2005; 352(16): 1685–95. PubMed Abstract | Publisher Full Text\n\nDeath AK, McGrath KC, Sader MA, et al.: Dihydrotestosterone promotes vascular cell adhesion molecule-1 expression in male human endothelial cells via a nuclear factor-kappaB-dependent pathway. Endocrinology. 2004; 145(4): 1889–97. PubMed Abstract | Publisher Full Text\n\nMcCrohon JA, Jessup W, Handelsman DJ, et al.: Androgen exposure increases human monocyte adhesion to vascular endothelium and endothelial cell expression of vascular cell adhesion molecule-1. Circulation. 1999; 99(17): 2317–22. PubMed Abstract | Publisher Full Text\n\nHaddad RM, Kennedy CC, Caples SM, et al.: Testosterone and cardiovascular risk in: a systematic review and meta-analysis of randomized placebo-controlled trials. Mayo Clin Proc. 2007; 82(1): 29–39. PubMed Abstract | Publisher Full Text\n\nBasaria S, Coviello AD, Travison TG, et al.: Adverse events associated with testosterone administration. N Engl J Med. 2010; 363(2): 109–22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarchioli R, Finazzi G, Specchia G, et al.: Cardiovascular events and intensity of treatment in polycythemia vera. N Engl J Med. 2013; 368(1): 22–33. PubMed Abstract | Publisher Full Text\n\nKunnas T, Solakivi T, Huuskonen K, et al.: Hematocrit and the risk of coronary heart disease mortality in the TAMRISK study, a 28-year follow-up. Prev Med. 2009; 49(1): 45–7. PubMed Abstract | Publisher Full Text\n\nHoyos CM, Killick R, Yee BJ, et al.: Effects of testosterone therapy on sleep and breathing in obese men with severe obstructive sleep apnea: a randomized placebo-controlled trial. Clin Endocrinol (Oxf). 2012; 77(4): 599–607. PubMed Abstract | Publisher Full Text\n\nWu FCW, Tajar A, Beynon JM, et al.: Identification of late-onset hypogonadism in middle-aged and elderly men. N Engl J Med. 2010; 363(2): 123–35. PubMed Abstract | Publisher Full Text\n\nGrunstein RR, Handelsman DJ, Lawrence SJ, et al.: Neuroendocrine dysfunction in sleep apnea: reversal by continuous positive airways pressure therapy. J Clin Endocrinol Metab. 1989; 68(2): 352–8. PubMed Abstract | Publisher Full Text\n\nLiu PY, Caterson ID, Grunstein RR, et al.: Androgens, obesity, and sleep-disordered breathing in men. Endocrinol Metab Clin North Am. 2007; 36(2): 349–63. PubMed Abstract | Publisher Full Text\n\nGambineri A, Pelusi C, Pasquali R: Testosterone levels in obese male patients with obstructive sleep apnea syndrome: relation to oxygen desaturation, body weight, fat distribution, and metabolic parameters. J Endocrinol Invest. 2003; 26(6): 493–8. PubMed Abstract\n\nMeston N, Davies RJ, Mullins R, et al.: Endocrine effects of nasal continuous positive airway pressure in male patients with obstructive sleep apnoea. J Intern Med. 2003; 254(5): 447–54. PubMed Abstract | Publisher Full Text\n\nLuboshitzky R, Aviv A, Hefetz A, et al.: Decreased pituitary-gonadal secretion in men with obstructive sleep apnea. J Clin Endocrinol Metab. 2002; 87(7): 3394–8. PubMed Abstract | Publisher Full Text\n\nBhasin S, Cunningham GR, Hayes FJ, et al.: Testosterone therapy in men with androgen deficiency syndromes: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2010; 95(6): 2536–59. PubMed Abstract | Publisher Full Text\n\nDrager LF, Polotsky VY, Lorenzi-Filho G: Obstructive sleep apnea: An emerging risk factor for atherosclerosis. Chest. 2011; 140(2): 534–42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFinkle WD, Greenland S, Ridgeway GK, et al.: Increased risk of non-fatal myocardial infarction following testosterone therapy prescription in men. PLoS One. 2014;9(1): e85805. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMuraleedharan V, Marsh H, Kapoor D, et al.: Testosterone deficiency is associated with increased risk of mortality and testosterone replacement improves survival in men with type 2 diabetes. Eur J Endocrinol. 2013; 169(6): 725–33. PubMed Abstract | Publisher Full Text\n\nShores MM, Smith NL, Forsberg CW, et al.: Testosterone treatment and mortality in men with low testosterone levels. J Clin Endocrinol Metab. 2012; 97(6): 2050–8. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "4383",
"date": "15 Apr 2014",
"name": "M. Emre Bakırcıoğlu",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript - 'The putative mechanisms underlying testosterone and cardiovascular risk' - gives valuable information about the mechanisms of cardiovascular effects of testosterone supplementation therapy in hypogonadal men. This opinion article is well written and deserving of indexation.",
"responses": []
},
{
"id": "4378",
"date": "15 Apr 2014",
"name": "Matthew S. Wosnitzer",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a comprehensive summary of the potential mechanisms (i.e. platelet thromboxane A2 receptor, monocyte adhesion via endothelial cell vascular cell adhesion molecule 1, increased hematocrit level) underlying the possible association of testosterone supplementation therapy (TST) with cardiovascular outcomes. The discussion of possible mechanisms is important in light of recent questions about the cardiovascular side effects of TST, with studies showing both decreased and increased cardiovascular and mortality risk. An ideal study would be prospective and include additional measurements of hematocrit (or TXA2, VCAM1 for example) which could be related to cardiovascular outcomes and help to better select which men may derive the most benefit and least risk from TST. Additionally, a recent by Yassin et al.1 adds more data for the safety of TST (intramuscular testosterone undecanoate) in older men with late-onset hypogonadism and erectile dysfunction (mean age 59 years with mean treatment duration of over 4 years). No cardiovascular side effects were noted during the study and there was improvement of metabolic syndrome-related characteristics including obesity, blood glucose, cholesterol, and BP. Subjects were mostly were overweight or obese, and a minority had baseline cardiovascular comorbidities (12%), DM2 (31%) or HTN (45%) issues (which are similar to - or increased compared with - the baseline characteristics of the Finkle et al. study2 – reference 23 in the article). In the future, more studies are needed to confirm these findings, especially investigating the mechanisms by which TST may affect cardiovascular outcomes.",
"responses": []
},
{
"id": "4380",
"date": "22 Apr 2014",
"name": "Boback Berookhim",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMaganty and colleagues provide a timely and interesting report on a rather controversial topic which has been the subject of a number of recent, large, population-based, retrospective studies that have received media attention. The role of testosterone replacement therapy in worsening cardiovascular disease remains unanswered, and will likely require extensive, well-matched, randomized, placebo-controlled trials to prove its effect - whether protective, or aggravating - in cardiovascular disease. The authors, however, provide a well researched review of the putative mechanisms underlying this possible, though unproven, relationship. While the thromboxane A2, monocyte activation, polycythemia vera and obstructive sleep apnea pathways proposed are plausible, there is - to date - no clear evidence that any of these contribute to increased cardiovascular risk in men. Platelet activation and increasing atherosclerotic intimal plaque would be interesting topics for further research in testosterone replacement therapy, and will help to better elucidate this issue.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-87
|
https://f1000research.com/articles/3-86/v1
|
04 Apr 14
|
{
"type": "Research Article",
"title": "Chewing through the Miocene: an examination of the feeding musculature in the ground sloth Hapalops from South America (Mammalia: Pilosa)",
"authors": [
"Virginia L. Naples",
"Robert K. McAfee",
"Virginia L. Naples"
],
"abstract": "Hapalops, a smaller-sized and early sloth of the Megatheroidea, appeared in the middle Miocene Santa Cruz formation of Argentina. This genus is part of the group from which later, larger megatheroids arose, i.e., Nothrotheriops and Megatherium. Many cranial characters support this idea; however Hapalops is not merely a smaller antecedent of the later forms. Specifically, Hapalops retains short anterior caniniform teeth, and a temporomandibular joint elevated above the cheek tooth row; a combination distinct among sloths. An elevated temporomandibular joint occurs in Bradypus, a tree sloth with anterior chisel-shaped teeth instead of caniniforms, and the tree sloth Choloepus, which is aligned with the megalonychids, has anterior caniniforms. Hapalops has an elongated zygomatic ascending process that is reminiscent of that in Bradypus; however, the Bradypus skull is extremely foreshortened while that of Hapalops is elongated, as in nothrotheres, but not deepened as in megatheres. Previous work identified many sloth cranial character complexes, and functional limitations on skull feature combinations. The unique Hapalops character patterns indicate a selective feeder with a mediolaterally oriented grinding stroke during mastication.",
"keywords": [
"Hapalops",
"Megatheroidea",
"Bradypus",
"Choloepus",
"feeding strategy",
"mastication",
"muscle reconstruction",
"Pilosa"
],
"content": "Introduction\n\nThe Xenarthra has two divisions, the Cingulata and the Pilosa. The cingulates have carapaces, i.e., the extinct glyptodonts and giant armadillos, and the living armadillos. Pilosans instead are covered with hair, although some retain bony dermal ossicles. This group contains sloths and anteaters with strikingly different body forms and habits. Four sloth families have extinct representatives, the Mylodontidae, the Megalonychidae, the Megatheriidae and the Nothrotheriidae, and one has only extant members, the Bradypodidae. The anteaters are united in a single family, the Myrmecophagidae (Scott, 1937; Simpson, 1945; McKenna & Bell, 1997).\n\nMany early sloths were small and appeared in the Oligocene already as obligate herbivores, albeit retaining morphologic influences from insectivorous or myrmecophagous antecedents. Myrmecophagy specializations in sloth progenitors have been suggested as restricting later group members from developing a feeding apparatus typical of other herbivores (Winge, 1941; Hirschfeld & Webb, 1968; Patterson & Pascual, 1972; Hirschfeld, 1976; Webb, 1985). Some more unusual herbivore characters inherited by sloths are: (1) narrow, greatly elongated skulls, (2) zygomatic arches missing the anteroposterior jugal-squamosal temporal process connection, (3) decreased numbers and types of teeth, (4) simplified shapes for retained teeth, (5) enamel loss, (6) ever-growing teeth, (7) fewer anterior teeth, and (8) a small buccal opening. All of these characters, advantageous to myrmecophages, are disadvantageous to the typical herbivore condition. Another important character inferred in extinct sloths because of their large hyoid apparatuses (Naples, 1987; Naples, 1989), and by comparison with Recent tree sloths (Naples, 1986) and xenarthran anteaters, are large, long and highly mobile tongues which are also characteristic of insectivorous forms (Owen, 1862; MacAlister, 1875; Reeve, 1940; Montgomery, 1985; Naples, 1999).\n\nXenarthrans also vary widely in cranial specializations, perhaps reflecting bulk versus selective feeding strategies. Characters typifying bulk feeding, as in Lestodon and Glossotherium, include a wide a muzzle and predental spout. Sloths considered selective feeders have narrow muzzles and predental spouts longer than wide, such as the scelidotheres (Bargo et al., 2006a; Bargo et al., 2006b). Further specialization for aquatic feeding is noted for Thalassocnus (Muizon et al., 2004).\n\nIn spite of an odd herbivore morphology, the combined structural and functional features among sloth lineages succeeded sufficiently to permit small sloths to evolve into moderate and large forms. Megatheroids display this tendency most strikingly, with genera ranging from small (sheep-sized) early genera such as Hapalops, to moderate (bear-sized) forms such as Nothrotheriops, and finally, to the giant (elephant-sized) megatheres such as Megatherium and Eremotherium. Recent tree sloths are obligate suspensory arboreal folivores, whereas extinct sloths are considered “ground” sloths. The unique inverted tree sloth locomotion pattern has been cited as evidence of a close phylogenetic relationship between these genera (Webb, 1985). However, uniting the tree sloths in Bradypodidae is now viewed as incorrect, with each genus taxonomically distinct at the family level (Patterson & Pascual, 1972; Webb, 1985; Gaudin, 2004). As such, Bradypus is retained within Bradypodidae while Choleopus is in Megalonychidae. Extinct sloths have been divided into four families, Megalonychidae, Megatheriidae, Nothrotheriidae and Mylodontidae, although these associations partially assume that all are terrestrial (Gaudin & McDonald, 2008). However, some of the small sized extinct species including Hapalops, may have been at least partially arboreal (White, 1993; Pujos et al., 2007). Larger sloths, such as Nothrotheriops, might have been semiarboreal, or as capable of climbing as are bears. Perhaps the young of even the largest species could climb. The living South American Andean spectacled bear, Tremarctos ornatus, may weigh up to 80 kilograms. It is the most arboreal ursid, living mostly in trees, including building nests and sleeping arboreally (Nowak, 1999). Other authors also suggest that some extinct sloths might have been arboreal (Anthony, 1916; Matthew & Paula Couto, 1959; Romer, 1966; Hirschfeld & Webb, 1968; Paula Couto, 1971; Mayo, 1980; Scillato-Yane, 1980; Coombs, 1983; Sherman, 1984; Hirschfeld, 1985; Webb, 1985; White, 1993; Pujos et al., 2007).\n\nMany mammalian insectivorous or omnivorous basal stocks evolved specialized lineages; therefore, it is unsurprising that at least one xenarthran group gave rise to herbivorous forms (Winge, 1941; Janis et al., 1998). However, fossil record gaps prevent direct observation of such changes, and the four fossil sloth families are distinct from their earliest appearance. Lacking direct evidence, reconstruction of morphologies that allowed sloth divergence may be modeled by study of evolutionary trends in known xenarthran lineages. Goals of this study include identification of related structural characters, their association into functional complexes, and evaluation of the potential feeding habits of Hapalops.\n\n\nMaterials and methods\n\nStudies of Hapalops are often hampered by taxonomic issues as over twenty species were historically assigned to the genus (De Iuliis and Pujos, 2006). Many such species were created with little diagnostic description or character explanation (Ameghino 1889; Scott, 1903–1904; Hirschfeld, 1985). Therefore, a contemporary taxonomic revision will likely result in a reduced number of Hapalops species. As review and revision of the systematics of Hapalops and closely related taxa is beyond the scope of this paper, only currently identified generic characters are recognized here. Typical Hapalops species features were grouped to establish an overall generic level character suite. Anatomical reconstructions were also created as a means of studying other extinct sloths (Naples, 1987; Naples, 1989; Naples, 1990; Muizon et al., 2004; Bargo et al., 2006b), especially when complete specimens were unavailable. Additionally, the variability among Hapalops specimens studied here is less than what is considered intraspecific variation among anatomical characters in a single living species.\n\nSpecimens examined were skulls and jaws of Hapalops from the Field Museum of Natural History, Department of Geology, the Museum of Natural History at the University of Kansas, and the American Museum of Natural History. All specimens (Table 1) were considered “adult”, based on partial sutural fusion, cortical bone density, and being in the largest size range (Naples, 1982; Anderson & Handley, 2001). Cranial scars indicating origin and insertion of facial, masticatory, lingual, pharyngeal, cervical, intrinsic and extrinsic back muscles were examined.\n\nThe names of museums are represented by the following acronyms: American Museum of Natural History (AMNH), Field Museum of Natural History (FMNH) and University of Kansas Natural History Museum (KU).\n\nStudy of soft tissues of living species allows modeling of patterns in related extinct species, permitting a hypothetical soft tissue reconstruction, and therefore, an estimate of the appearance and function of tissues of fossils. Tree sloth muscles, which show convergent patterns, were identified by tracing innervations during dissection of preserved specimens of Bradypus and Choloepus (Naples, 1985a; Naples, 1985b). As the closest living relatives to extinct sloths, we use them to infer similar muscle arrangement patterns despite their disparate phylogenetic associations (Gaudin, 2004). Terminology agrees with prior studies of tree sloths and fossil sloths (Naples, 1982; Naples, 1986; Naples, 1987; Naples, 1989), as well as the Nomina Anatomica Veterinaria (2005). Muscle and muscle segment names have been homologized with those in the older literature, and references to muscle heads or slips indicate origins and insertions, respectively, for muscles composed of more than one part or belly.\n\nAdditionally, dental occlusion, wear and mandibular movement patterns were determined and compared to those of Bradypus, Choloepus, Eucholaeops, Nothrotheriops and Thalassocnus (Naples, 1982; Naples, 1985a; Naples, 1987; Naples, 1995; Muizon et al., 2004; Bargo et al., 2009). No Hapalops crania or mandibles were sufficiently complete or uncrushed to allow articulation, and few cranial and mandibular specimens were unequivocally associated. Therefore, the illustrations have been drawn as composites, approximating cranial feature relationships as they would have been in the intact sloth. All illustrations were drawn by one of the authors (VLN).\n\n\nResults\n\nSloth crania differ from those of other xenarthrans in having: (1) reduced edentulous premaxillae that may retain unfused sutures even in aged adults, (2) elongated maxillae, (3) zygomatic arches with elaborated ascending and descending jugal processes, (4) pterygoid bones elongated into flanges or inflated sinuses, (5) fused mandibular symphyses lacking sutural traces, and (6) an elongate predental spout. These characters show different forms, and not all occur in all sloths. Nevertheless, previous sloth cranial studies (Naples, 1982; Naples, 1987; Naples, 1990) showed characters associated into complexes or suites. Although Hapalops shows many such sloth synapomorphies, this genus also displays unique character complexes indicative of the transitions necessary to revert to an herbivorous lifestyle.\n\nPremaxilla—Sloth skulls show great shape diversity, but in the earliest genera, as well as some later forms, they tend to be elongate, as is the case for Hapalops (Figure 1, Figure 2). Premaxillae are rarely preserved and, even if present, generally not in situ, although one Hapalops reutmeyeri specimen (KU 145120) shows intact premaxillae loosely articulated to the maxillae. Dorsally, these bones are narrow, elongate, pointed anteriorly, and perforated by a premaxillary foramen. They widen posteriorly to equal the nasals at the dorsal premaxillary-nasal suture, characterized by a simple, straight abutting joint. Sloths have sometimes been restored with a bony extension forming a nasal ring from the premaxillae dorsally to the nasals (Wible et al., 1993; Bargo et al., 2006b); confirmed in Mylodon [=Grypotherium] darwini, a sloth with very large nasal openings (Reinhardt, 1879). No specimen we examined has this area preserved. In Hapalops the anterior ends of the ventral, horizontal portion of the premaxillae are shallow, and curve upward (Figure 1B). They are slightly rugose, indicating the presence of an anterovertical nasal extension and large nasal openings, typical of all sloths and anteaters (Naples, 1982; Naples, 1985a; Naples, 1999). The interpremaxillary sutures remain unfused, even in adults where most other sutures are fused, and in some cases, at least partially obliterated. Extreme premaxillar slenderness contributed to distortion of available specimens; therefore, these bones have been idealized in illustrations of the reconstructed skull (Figure 1, Figure 2).\n\nThe skull of Hapalops, dorsal view (A) and lateral view (B), with skeletal and dental features discussed in the text labeled. The scars of muscle origin are shown by shaded areas. The anterior portion of the zygomatic arch has been removed from B to show the rugosity of the articular surface, and to reveal structures on the side of the skull that would otherwise be hidden.\n\nThe premaxilla is attached only to the palatal and slightly to the lateral portions of the maxilla, although the missing vertical portion, sometimes also termed a septomaxilla, is represented by a shaded area in Figure 1B (Wible et al., 1993). The skull slopes gently posterodorsally from the nasals, unaltered by the bulges for the anterior zygomatic arch root and the postorbital process. A slight dorsal parietal swelling in some specimens allowed for a larger braincase. The occiput is gently rounded, the occipital condyle profile round, and the condyles almost entirely ventral to the skull.\n\nNasal—Dorsally (Figure 1A), the nasal is an elongate rectangle, flared slightly posteriorly with the widening of the anterior zygomatic arch root and slightly pointed anteriorly. The anterior edges are roughened, as for attachment of a bony premaxilla and/or septomaxilla. Posteriorly, the nasofrontal suture is curved, simple and convex, and lack the interdigitating complexity that increases surface area in most sutures to allow stronger bone-bone junctions (Herring, 1972; Herring & Teng, 1995). Anteriorly each shows a convex mediolateral curve and slight depression of the internasal suture.\n\nFrontal—This bone approximates a rectangle dorsally (Figure 1A), and the simple, straight interfrontal suture remains unfused and visible in specimens of all ages. Anteriorly, the frontals flare broadly to form the anterior origin for the zygomatic arch. Further posteriorly, a second frontal bulge forms the short, rounded overhanging postorbital process bearing an anteroposteriorly elongated depression housing the large and obvious cranial foramina, including the foramen rotundum, sphenoidal fissure and the optic canal (Figure 1B). In some specimens, a prominent ridge posterior to the postorbital process makes a broadly curved “V” with the apex pointing posteriorly. This shape forms the anterior margin of the extensively variable M. temporalis origin (Figure 1). In some specimens, the “V” points posteriorly along the parietals, producing an elevated sagittal table. In other specimens, a narrow sagittal crest occurs even though the “V”-shaped ridge does not. Alternately, there is no sagittal crest. These differences reflect the relative degree of development of the anterior limit of the temporal fossa, and therefore, the anterior extent of the M. temporalis. Such differences may result from age or sex differences, or possibly species level differences.\n\nParietal—This bone curves in a gentle convexity for the laterally bulging braincase, the widest skull region in dorsal view (Figure 1A). The parietal-temporal suture shows a raised ridge, and the typical convoluted mammalian pattern (Figure 1B). Laterally, the bone bulges posterodorsally. Most of the temporal fossa, i.e. the M. temporalis origin, is from the outer temporal surface, marking the dorsal boundary of muscle’s origin. Animals with a farther lateral superior temporal line are probably younger, even though they may be adult in size and overall skull shape (Naples, 1982; Anderson & Handley, 2001).\n\nOccipital—This bone is visible dorsally, and the parieto-occipital suture is complex and “C”-shaped, with a posterior facing open end. Dorsally, the occipital condyles are invisible (Figure 1A). This bone bears markings for muscles originating on the prominent nuchal crest, i.e. those that control the position of the head on the neck.\n\nMaxilla—The bone forms much of the skull anterolaterally, and bears the upper dentition (Figure 1, Figure 2). It has a rounded, rugose lateral projection with many pits and projections that attach to the jugal bone. The maxillary-frontal suture is complex.\n\nLacrimal—This bone is a prominent and rounded, rectangular shape on the lateral skull surface, ventral to the nasal and frontal (Figure 1). It has a prominent lateral-facing lacrimal foramen, a feature shared by sloths and anteaters, but not obvious in other xenathrans or other mammalian orders.\n\nJugal (= Malar, Zygomatic)—This bone forms the anterior part of the zygomatic arch (Figure 1, Figure 3). Many specimens examined here, as is common in fossil sloths, lack the anterior zygomatic arch root. This large and slender projection is vulnerable to breakage during fossilization, and the maxillolacrimal-jugal suture never fuses, even in the oldest and largest adults. Because this connection is weak, the entire jugal is typically lost. In contrast, in most mammals, the maxillolacrimal-jugal suture fuses, and therefore a portion of the anterior root of the zygomatic arch usually remains attached to the skull, even if the middle region is broken, crushed or missing.\n\nThe cranium of Hapalops in lateral view showing the restored cranial ligaments (A). Enlargements of the lateral (B) and medial (C) surfaces of both the anterior and posterior portions of the zygomatic arch in Hapalops are also shown, along with the zygomatic arch ligament, and the locations of the scars of muscle and muscle head origins that are indicated by differentially shaded areas.\n\nThe zygomatic arch lacks an anteroposterior bony connection in Hapalops, as in the living tree sloths Bradypus and Choloepus, and some of the smaller extinct genera [(Matthew & Paula Couto, 1959; Paula Couto, 1971; Naples, 1982) Figures 1A; 3]. In living forms, the anterior portion is bound to the posterior (i.e., the anteriorly projecting process of the temporal bone) by a ligamentous sheet; therefore a similar connecting ligament has been restored in Hapalops. The jugal in this sloth shows both dorsal and ventral processes. The dorsal process is elongate and posterodorsally oriented, as in Bradypus. The ventral process has lateral rugosities and depressions for origin of the M. masseter superficialis heads (Figure 3B). The medial zygomatic surface is rugose, with several depressions, reflecting the origin of the M. masseter profunda and the M. zygomaticomandibularis (Figure 3C). The latter likely also arose, in part, from the anteroposterior connecting ligament, as well as from an anteromedial depression of the anteriorly projecting squamosal process of the temporal. This arrangement allows the zygomatic arch to act as a single functional unit, as in other mammals.\n\nSphenoid—This bone is often considered to consist of several elements, including orbitosphenoid, alisphenoid, presphenoid and basisphenoid (Wible & Gaudin, 2004). However, in Hapalops, all of these fuse, or are indistinguishable in available specimens. In juvenile tree sloths these bones are distinct, with the orbito- and alisphenoid visible laterally, and the pre- and basisphenoid visible ventrally. The composite bone occupies the ventral portion of the lateral skull wall, including the orbit posteriorly and the anterior and ventral portion of the temporal fossa. It meets its opposite at the ventral midline posterior to the choanae. The four major foramina of the orbital region pass through this bone (Figure 1B).\n\nPalatine—This bone forms the anterior portion of a thin flange extending ventrally, which is completed posteriorly by the pterygoid and dorsally by the orbito- and alisphenoid components of the composite sphenoid (Figure 1B, Figure 2). In previously published literature, these projections have been designated as pterygoid flanges, because those bones constitute the greater portion of this feature (Parker, 1885; Naples, 1982; Gaudin, 2004).\n\nPterygoid—This bone extends ventrally as a thin flange posteriorly from the anterior palatine and alisphenoid portions of the sphenoid (Figure 1B, Figure 2). It serves as the origin for the M. pterygoideus medialis and the M. pterygoideus lateralis, upper and lower slips. The flanges are elongate, but not inflated in Hapalops, as in the tree sloth, Bradypus (Naples, 1982).\n\nTemporal—This element has small squamous and large zygomatic portions, with an anterior process forming the posterior zygomatic arch root. The anterior tip, medial and lateral margins of the process are sharp. A lateral triangular concavity renders the process wedge-shaped (Figure 1B). A dorsal triangular concavity also serves as the origin of the most posteroventral fibers of the M. temporalis (Figure 1A). It flares to the widest point at the widest part of the braincase. Ventral to the posterior zygomatic root merging with the braincase is the large, external auditory meatus, which has a large, rounded, laterally facing opening ventral to the posterior zygomatic root origin. The mastoid process is a broad descending projection with a pointed tip, near the posterior aspect of the skull.\n\nMost mammalian sutures are complex; however, in Hapalops, the interpremaxillary, premaxilla-nasal, and the nasal-frontal sutures are simple, abutting approximately linearly. Retaining these open sutures, even in aged adults, appears to be common in sloths. However, not all of the sutures in Hapalops are simple in structure. The most anterior suture showing the typical complexity is the frontoparietal. In one specimen (FMNH P13141), bilateral sutural bones occur between the frontals and parietals, further increasing sutural complexity in this area [(Herring, 1972; Herring, 1974; Jaslow, 1989; Jaslow, 1990; Herring & Mucci, 1991; Herring & Teng, 1995; Herring, 1997) Figure 1]. Other, especially more posterior sutures, become increasingly complex.\n\nPalate—The anterior premaxillary portion is pointed and narrow, widening greatly at the maxillary suture anterior to the caniniform tooth alveoli (Figure 2, Figure 4A). Posterior to the caniniforms the palate constricts, widening again anterior to the molariform toothrow. The hard palate ends at the posterior margin of the last upper molariform. It is rugose, with many pits and nutrient foramina in a double row parallel to the midpalatal suture. In some specimens the medial suture is partially fused, and when so, it is complex. The palate narrows posteriorly, ending in a “C” shape with the open ends posterior at the choanae (Figure 2). The origin scar of M. uvulae is a pair of elongate ridges anterior to the posterior margin of the bones that mark the anterior soft palate edge, which continues farther posteriorly. The pterygoid hamulus is a small tubercle for the anterior origin of M. constrictor superioris, which may continue posteriorly along the ventral margin of the pterygoid flanges, although the continuation of this attachment is indistinct. Space between the palatine and pterygoid flanges is wide for the length of the region. The maxillary-palatine suture is only sometimes discernible. Anterior to the ridges and medially, two elongate depresssions serve as the anterior origins of M. tensor veli palatini. In specimens with well-preserved interpterygoid regions, a pair of large depressions with anteroposteriorly oriented long axes occurs medial to the palatine-pterygoid suture. Posterior to these depressions is a second shallower pair. Farther posteriorly is a pair of raised rounded ridges that are the M. tensor veli palatini origins. A second pair of rugose oval depressions with anteroposterior long axes occurs posterior to the tensor veli palatini ridges where the M. levator veli palatini inserts.\n\nThe maxillary (A) and mandibular (B) dentitions of Hapalops are shown. The shaded and unshaded surfaces of maxillary teeth occlude with corresponding surfaces of mandibular teeth. Arrows indicate the orientation of the masticatory power stroke which emphasizes the mediolateral component of mandibular movement.\n\nTemporal—The glenoid fossae are smooth, ovate depressions with mediolateral longer axes, and without prominent pre- or postglenoid processes. Anterior to the external auditory meatus is the elongate insertion scar for the posterior slip of M. levator veli palatine (Figure 2).\n\nBasioccipital—On either side of the midline is the roughened raised basilar tubercle that continues posteriorly as a ridge ending immediately anterior to the raised lip of the anterior edge of foramen magnum (Figure 2). This feature is the posterior origin of M. constrictor superioris. Posterolateral to the tubercles and anterior to the occipital condyles is an ovate depression for the M. longis capitis insertion. The foramen magnum is a large oval with a horizontal longer axis. It opens posteroventrally, and is most visible ventrally. There are no tympanic bullae. There is a prominent stylohyal pit, which serves as the stylohyoid bone articulation. The stylohyal pit is far posterior on the skull, anterior to the lateral aspects of the occipital condyles. The prominent mastoid processes are lateral to this feature.\n\nThe nuchal crest is prominent with deep, triangular scars for insertion of the M. trapezius; a broader, shallower depression is the insertion of the M. splenius capitis, and posteroventrally a sharp-edged process is the M. sternocleidomastoideus origin (Figure 5). Ventral to the nuchal crest are roughened scars for insertion of the M. semispinalis capitis, M. rectus dorsalis posterior major et minor, and the M. obliquus capitis cranialis. Scars for the M. rectus capitis lateralis insertion occur lateral to the M. rectus dorsalis posterior major et minor insertions. The posteromedial aspect of the descending ridge, the M. sternocleidomastoideus origin, shows a small oval depression bounded by a roughened ridge; this is the origin of the M. digastricus posterior belly. The occipital condyles are large for the skull size, as is common in long headed sloths. They are widely spaced mediolaterally, with the rounded articular surfaces extending dorsally beyond the vertical, covering about 200 degrees. Condylar convexity in the horizontal plane allows about 150 degrees of mediolateral head movement.\n\nScars of origin of the suboccipital muscles are indicted by shading.\n\nSloths have ever-growing teeth. Hapalops has a dental formula reduced compared to other mammals, but with the maximum number of tooth types for sloths; anterior upper and lower caniniform and four upper and three lower molariform teeth (Figure 4). The upper caniniforms are rounded, recurved triangles with a single flat wear facet facing posteriorly (Figure 1B). Sloth teeth lack enamel, but have a harder outer and a softer inner dentine layer, with central tooth basins formed from wear (Naples, 1987). The caniniforms occur at the widest part of the palate, with roots large enough to cause labial bulges in the maxilla. A diastema separates these teeth from the more posterior molariforms. The first molariform is a rounded rectangle, wider labiolingually. The harder outer dentine “shell” is thinner than that of the caniniform, and the central basin is approximately transverse; slightly more anterior lingually (Naples, 1982; Naples, 1987; Naples, 1990). In our specimens, the harder outer dentine was worn through into the softer inner layer, as is true in all but the youngest sloths (Naples, 1982; Naples, 1987). Flat wear surfaces angled anterodorsally and posterodorsally occur on both anterior and posterior faces of the first three molariforms. The second molariform is also a rounded rectangle, separated from the first by approximately 25% of the anteroposterior length of the first tooth, a spacing also shown between the more posterior molariforms. The second molariform is the largest, approximately rectangular, but with more rounded anterior and labial faces. The third molariform is a more rounded trapezoid than the first two, with the widest face anterior. In several specimens, the central basin is directly transverse rather than slightly anterior labially. The fourth molariform occlusal surface is half the anteroposterior diameter of the third, and about 80% as wide, with a single large anterodorsal wear facet and a smaller posterodorsal one. Even though the molariforms are different in size and labiolingual width, the interdental palatal width is equal anteroposteriorly. Occlusion in Hapalops (Figure 4) resembles that in Choloepus (Naples, 1982).\n\nDorsal view characters—The predental spout dorsal edge is sharp, and the outer surface rugose (Figure 4B) with a smooth lingual “U-shaped” depression. The symphysis is broadly fused and anteroposteriorly deep, with no traces of the intermandibular suture. There is a shallow depression anterior to the lower caniniform, to accommodate the tip of the upper caniniform at full dental occlusion. It is a small, triangular-shaped area, with the apex directed anteriorly. There is a flattened rugose area between the caniniform tooth and the first molariform, i.e., the diastema. The dorsal surface of the bone surrounding the molariforms is slightly roughened and more porous than that on the labial or lingual aspects of the mandible. A depression dorsal to the M. buccinator attachment marks the gingival attachment, and parallels the toothrow lateral to the molariform alveoli (Figure 6A). The alveoli are bounded labiolingually by sharp ridges that converge and continue posteriorly as a single, sharp-edged ridge, lingual to the coronoid process. The coronoid process is thicker anteriorly, and thins posteriorly. The mandibular condyles are ovoid, with a greater lingual flare and a gentler lingual slope.\n\nThe mandible of Hapalops is shown in lateral view (A) and lingual view (B). The scars of muscle origin and insertion are labeled and shaded.\n\nLateral view characters—In profile, the dorsal surface of the predental spout varies from rounded to a sharply pointed anterior tip (Figure 6), and is elevated at an anteriorly directed angle from the dentition. Especially marked in specimens with rounded, blunt anterior predental spout profiles (AMNH 9222 and KU 145120), a shallow “V-shaped” notch occurs on the anterodorsal tip when the mandible is viewed anteriorly. Regardless of shape, a thickened bony border reinforces the dorsal rim (Figure 4B). Ventral to the rim, the spout shows a slight concavity (Figure 4B, Figure 6A), as seen in other sloths with caniniforms laterally displaced from the main axis of the tooth-row (McAfee, 2007). The shallow depression for the upper caniniform is also visible labially. About halfway between the tip of the spout and the caniniform is a large mental foramen; a smaller, additional one sometimes occurs near the tip (Figure 6A). These openings allow passage of one or more large mental nerve branches that innervate a large, flexible lower lip. The ridged, thick dorsal boundary of the spout continues posteriorly the length of the toothrow. Ventral to it is a deep depression between the caniniform and first molariform that ends in a ridge formed by the root of the posterior portion of the first molariform (Figure 4B). This tooth is tilted at 30 degrees anterior to vertical. The posterior ridge border is bounded by a second, smaller depression. A second similarly inclined ridge occurs labial to the bulge of the second molariform, followed by a shallower depression anterior to the third tooth. The ridge labial to the third molariform is less pronounced, and more vertical than previous ones. Grooved ventral borders of the depressions mark the labial attachment of the oral gingiva, underlain by the M. buccinatorius (Figure 6A). Posterior to the posterior end of this scar, at the base of the coronoid process, and posterior to the third tooth, is a large elongate and oval condylar foramen with a labially directed opening (Figure 6A). This character is an autapomorphy for sloths, and among other mammals has only been observed in the platypus, Ornithorynchus aculeatus (personal observation). The coronoid process is large and broad anteroposteriorly, with a convex anterior edge and a rounded apex. The posterior face descends to a broad, shallow condylar notch. A ridge that continues dorsally along the labial surface begins dorsal to the condylar foramen, parallels, and then moves onto the leading edge. A second ridge runs dorsally, parallel to the more anterior one. A third ridge marks the posteroventral boundary of the M. temporalis insertion, from opposite the toothrow level to slightly dorsal to the coronoid notch (Figure 6A).\n\nThe mandibular condyle is gently convex, with some articular surface visible labially (Figure 6A). A labial ridge from the posterior edge buttresses the condyle, extending anteroventrally, fading labially. Depressions and rugosities, separated by ridges, mark labial masticatory muscle insertions. The angular process is large, convex ventrally, and projects posteriorly farther than the condyle. The dorsal aspect is ovate, with the long axis anterolateral to posteromedial. The articular surface with the glenoid fossa is convex, and “C-shaped” with the open arms facing anteriorly.\n\nLingual view characters—As in other sloths, the mental symphysis in Hapalops is fused (Figure 4B). Nevertheless, fossil sloth mandibles are often represented by a single ramus, with the break at or near the symphysis, perhaps because despite its robustness, it is weaker than the mandibular body housing the large ever-growing molariforms. The symphysis is thick in cross-section, particularly ventrally, thinning dorsally, toward the anterior tip of the spout. The mandibular lingual surface is smooth, although marked by the mylohyoid line, the M. mylohyoideus origin from the posterior symphysis, level with the mental spine. It trends posterodorsally, ending posteriorly ventral to the tubercle where the anterior M. constrictor superioris originates. Posterior to the last molariform, this tubercle may continue anteroposteriorly as a ridge, projecting lingually at the base of the anterior part of the coronoid process (Figure 6B). A third ridge marks the posteroventral boundary of the M. temporalis insertion from opposite the toothrow to slightly dorsal to the coronoid notch. The lingual coronoid surface bears anterior and posterior insertion scars for M. temporalis slips (Figure 6B). The M. pterygoideus medius insertion is large, and occupies most of the angular process. Unusual in comparison to other sloths, in Hapalops this region is subdivided by two posteroventrally curving parallel ridges.\n\nPosterior to the coronoid notch, the mandibular condyle is convex. The ventral articular surface, visible lingually, is sculpted by rugose, dorsal and ventral ovoid insertion scars with longer anteroposterior axes; the lower more elongate, for the M. pterygoideus lateralis. In some cases, the lower scar is oriented toward the groove that leads anteriorly to the mandibular foramen. This unusually large foramen is a sloth autapomorphy, and in Hapalops a broad, deeply incised groove leads to it, proceeding anteroventrally from the condylar notch dorsally, and the ventral border of the condylar process ventrally.\n\nVentral view characters—The predental spoutis elongate, slender and pointed, with a sharp midventral ridge (Figure 7A, 7B). The ventral mandibular surface is rugose with oval scars for the M. genioglossus origin on each side of mental spines at the midline. Posterior to this feature is a larger, oval, ridge-bounded rugosity that is the M. geniohyoideus origin. Posteriorly a much larger ridge-bounded roughened rectangle with rounded corners extends distally as far as the bulge housing the molariform roots in the mandibular body. This scar is for the M. digastricus, anterior belly insertion. It is prominent in Hapalops, but smaller than in the tree sloths Bradypus and Choloepus (Naples, 1985a).\n\nVentral views of the mandible of Hapalops showing suprahyoid muscle scars (A) and with areas of muscle origins shaded (B); 2 cm scale. These muscles are reconstructed in ventral view (C) and lateral view (D, E). In D and E, the tongue position has been estimated to show it relative to that of the oral musculature; 1 cm scale.\n\nMandibular dentition—In adult sloths tooth root diameters equal the erupted portions, and extend almost to the ventral aspect of the mandibular body (Naples, 1982; Naples, 1990). They are surrounded by thick alveolar walls, and together cause labially and lingually rounded bulges in the mandibular body, similar to maxillary bulges for the upper caniniforms (Figure 4B). The first lower molariform grows vertically, but the second and third are increasingly oblique, with the occlusal surfaces more lingual. The increasingly oblique orientations toward the posterior aspect of the toothrow, as well as their spacing form the wear-induced dental occlusal surface shapes [(Naples, 1985a) Figure 4]. Oblique tooth orientation allows the toothrow width to remain equal anteroposteriorly, while allowing increased space posteroventrally for housing the tongue base and other soft oropharyngeal tissues.\n\nEach ramus has four teeth, an anterior caniniform and three posterior molariforms (Figure 4B). The caniniforms are rounded triangles, with a single articular facet slanted anteroventrally, separated by a diastema and slightly laterally displaced from the longitudinal toothrow axis. The first and second molariforms are rectangular, with an anterior anterodorsally facing facet and a middle basin, bounded posteriorly by a posterodorsally facing facet. All molariforms are separated by large interdental spaces, an unusual feature in herbivores but common in sloths. The second molariform occlusal surface is slightly larger than the first. The third molariform surface is generally smaller, but more variable, ranging from diamond shaped, with the long axis oblique, labial side more anterior, to rectangular. In a few animals it is larger than the more anterior molariforms.\n\nTooth occlusion—Occlusion in Hapalops is similar to the pattern in other sloths studied [(Naples, 1982; Naples, 1985; Naples, 1987; Naples, 1989) Figure 4]. Caniniforms occlude with the maxillary tooth anterior to the mandibular, each with a single facet. This is opposite to the canine occlusion pattern in other mammals; hence these teeth are unlikely canine homologues. Molariform tooth homologies are also unknown, as no sloth has a deciduous dentition to allow study of tooth eruption sequences. As with the caniniforms, the upper molariforms occlude half a tooth length anterior to the mandibular molariforms with the anterior faces of the maxillary tooth central basins occluding with the anterior faces of similarly numbered mandibular molariform teeth. The posterior sides of the central basins of the upper molariforms occlude with the anterior faces of the central basins of the corresponding lower molariform. The single anteriorly facing facet of the upper fourth molariform occludes against the posterior face of the lower third molariform. The tooth positions suggest that when the caniniforms are in occlusion, the molariforms are not, as is the case in tree sloths [(Naples, 1982: Figures 4C, 4D) Figures 4; 8].\n\nOther than a few mammals with complete bony postorbital bars, such as primates and a few felids (Romer, 1966; Naples, 1982; Naples & Martin, 2000a; Naples & Martin, 2000b; Kardong, 2005), or a postorbital wall, as in humans and derived barbourofelins (Naples & Martin, 2000b; Standring et al., 2005), the usual condition is a bony postorbital process partially separating the orbit from the temporal fossa. In sloths, as most other mammals, a ligament connects the posteroventrally projecting bony supraorbital process to a corresponding posterodorsally projecting edge or process of the jugal. In sloths a ligament between the anterior and posterior portions completes the zygomatic arch and serves in lieu of the complete bony arch (Kardong, 2005). There is also a second ligament, the postorbital, as in most mammals, which connects the postorbital process to the jugal bone. The third set of cranial ligaments surrounds the craniomandibular joint, constraining the direction and degrees of freedom of movement of the mandibular condyle in the temporal glenoid fossa.\n\nZygomatic arch ligament—An unfused zygomatic arch is a derived character that distinguishes pilosans from most other mammals (Naples, 1999), but is plesiomorphic within the group. The form varies among the living and extinct sloths; however, it is partly affected by overall cranial size and proportion. Observations from tree sloth dissections and reconstructions of medium-sized fossil sloths illustrate how skull proportions and zygomatic morphologies differ from Hapalops (Naples, 1982; Naples, 1985a; Naples, 1987; Naples, 1989). Specifically, the ascending jugal process is more vertically oriented than that of all other sloths studied herein, except for Bradypus [(Naples, 1982) Figures 1B; 3A; 8B)]. However, this feature differs from Bradypus because the cranium in Hapalops is elongate, while tree sloth’s is the most anteroposteriorly shortened among known sloth lineages. Therefore, both the postorbital ligament and the zygomatic arch ligament in Hapalops are shallower dorsoventrally than in Bradypus; these positions allow a shorter vertical but anteroposteriorly broader area for attachment of the portions of the masseter and temporalis muscles arising from them. These differences in the origins and insertions of these muscle segments do not resemble those of Bradypus, despite similarities of the shape and orientation of the zygomatic arch. These dissimilarities determine some of the biomechanical differences between Hapalops and all other sloths for which these characters have been elucidated (Naples, 1982; Naples, 1987; Naples, 1989).\n\nPostorbital ligament—In Hapalops, the postorbital process connects to the anterodorsal edge of the ascending process of the jugal by a ligament. This ligament forms the posterior limit of the orbit and overlies the anteriormost fibers of M. temporalis (Figure 9). Ligament calcification occurs progressively as sloths age, causing the postorbital process of the frontal to lengthen and increase in robustness at the base (Naples, 1982; Naples, 1985a; Naples, 1985b; McAfee, 2007).\n\nCraniomandibular joint ligaments—These ligaments in Hapalops can be inferred from rugosities surrounding the glenoid fossa and condylar process neck, and by comparing these features to dissections of Bradypus and Choloepus. In both tree sloths, the joint is surrounded by a continuous sheet of fibrous connective tissue, interrupted only at the anteromedial “corner” by the tendons of origin of the two portions of the lateral pterygoid muscle.\n\nPrevious studies (Naples, 1985a) discussed tree sloth cranial musculature, and the details of their anatomy can be used to infer the arrangement of the same muscle groups in Hapalops. Muscle-bone attachments leave characteristic marks, such as rugose surfaces bounded by raised ridges. Divisions of individual muscles are also marked by elevated ridges, allowing estimation of the relative surface area of attachment of different muscle segments, as well as of entire muscles. These features are more prominent as animals increase in size, and Bradypus and Choloepus show the typical masticatory muscle pattern, including the same innervation (Trigeminal nerve, V3) as in other mammals (Naples, 1985b). The musculature of the tongue, throat and hyoid regions also conforms to the typical mammalian pattern of divisions and innervations, such as discussed in: Sisson & Grossman, 1953; Crouch, 1969; Evans, 1993; Williams et al., 1989; Anderson & Anderson, 1994; De Iuliis & Pulera, 2007; Schuenke et al., 2007. Nerve branches in Hapalops could not be traced; however, the positions of cranial foramina are similar to those of the tree sloths.\n\nThe masseter musculature—The masseter muscle in living sloths is complex and uniquely subdivided into five superficial portions, or parts, and a single deep portion, separated by tendons and fascial sheets (Naples, 1985a). Each of these divisions is easily discerned because it arises from a clearly marked region of the ventral process and posterior base of the dorsal process of the jugal and the ligament that connects the jugal and squamosal. A similar arrangement occurred in Hapalops (Figure 3, Figure 8, Figure 9), although the relative positions and orientations of muscle parts in this sloth differ from those in the tree sloths.\n\nThe cranium of Hapalops is shown in lateral view (B), illustrating the differential height of the craniomandibular joint for the skull and mandible. In A, the lines of action of the five individual parts, and a composite line of action of the M. masseter superficialis, and the anterior and posterior as well as a composite line of action for the M. temporalis are illustrated.\n\nThe five distinct parts of the superficial masseter muscle of Hapalops are shown in lateral view (A–E), with a composite muscle restored in F. The muscle has been reconstructed based on the scars of origin and insertion, and in relation to those dissected in the tree sloths, Bradypus and Choloepus.\n\nM. masseter superficialis, part 1—This muscle arose from a rectangular depression with rounded edges, oriented approximately 45 degrees posterior to vertical. The fibers trend posteroventrally to insert into a crescent-shaped, lateral depression of the mandibular coronoid process. This superficial masseter muscle segment insertion is the most posterodorsal.\n\nM. masseter superficialis, part 2—This segment originates from a “U-shaped” depression that covers most of the lateral root surface of the ascending jugal process. The muscle inserts into a triangular depression anterior and slightly ventral to that of part 1, and has a similar orientation.\n\nM. masseter superficialis, part 3—This segment’s origin encompasses the largest elongated oval depression on the lateral surface of the descending jugal process, from an elongate depression ventral to the horizontal ridge thickening the transverse portion of the jugal passing ventral to the orbit. This muscle’s insertion is from the posterior mandibular body edge to the angular process tip, immediately ventral to the part 2insertion, and shows a line of action similar to parts 1 and 2.\n\nM. masseter superficialis, part 4—This portion arises anterior to part 3, continuing distally along the descending jugal process to the tip, which it encompasses. The insertion occupies a crescent-shaped depression immediately ventral to the ridge of attachment for the most ventral tendon bounding part 3, continuing along the ventral mandibular margin. This segment’s line of action is more horizontal than the muscle average.\n\nM. masseter superficialis, part 5—The dorsal part of the origin is anterior to parts 3 and 4, continuing distally from an elongate, narrow ovate depression paralleling the anterior descending jugal process edge. The insertion is into a crescentic depression that echoes the concavity between the mandibular body and the angular process, wrapping around the ventral edge. The relative size, orientation and position of these attachments determine the degree to which the mandible can open because this segment is stretched to the greatest extent upon mandibular depression. The line of action of this segment is the most horizontal. Together, the five M. masseter superficialis segments show a wide range of individual lines of action, therefore functioning at different parts of the total range; their average also shows the overall muscle orientation (Figure 10B), which is more horizontal in Hapalops than Bradypus.\n\nReconstructions of the M. temporalis in dorsal view (A) and lateral view (B), the M. masseter profunda (C), M. zygomaticomandibularis (D), M. pterygoideus medius (E) and M. pterygoideus lateralis (F). In B the mandibular coronoid process is represented as if the muscle inserting on it was transparent. In C and D, the zygomatic arch and cranial ligaments are shown as if transparent. In E, both the anterior and posterior portions of the zygomatic arch are cut (indicated by shading), as well as the ascending portion of the coronoid process. The mandible is depicted as transparent to show the insertion of the muscle on the medial side. In F the portions of the zygomatic arch are cut as in E, and the shape of the pterygoids flanges are shaded grey. The mandible is also depicted as transparent, with the insertion of the M. pterygoideus lateralis shows attaching to the medial side of the condylar process. This muscle has been restored with two heads as is the case in tree sloths.\n\nM. masseter profundus—In Hapalops, the origin is from an elongate oval depression on the medial aspect of the lower two-thirds of the ascending portion of the jugal and the proximal half of the descending process. It inserts into an oval depression with a longer anteroposterior axis on the mandibular body dorsal to M. masseter superficialis, part 5 (Figure 3B, Figure 10C).\n\nIn Hapalops the muscle arises from an anteroposteriorly elongated oval fossa on the lateral braincase surface (squamosal and parietal) and the posterior dorsolateral walls of the frontal. The muscle does not meet at the dorsal midline, as is true of other sloths that have been studied; the superior temporal line is clearly marked on the lateral sides of the braincase (Figure 1). It is not possible to determine whether the origin was divided into superficial and deep portions; however, because of the shape of the temporal fossa it is a relatively large muscle. The insertion covers muscle scars on the anterior, lateral and medial portions of the large, prominent coronoid process (Figure 6, Figure 9, Figure 10B). Although the average muscle orientation trends anteroventrally, the great anteroposterior temporal fossa length indicates that there is a range of lines of action, from nearly vertical anteriorly to almost horizontal posteriorly (Figure 9A).\n\nM. zygomaticomandibularis (= M. temporalis, deep portion)—This muscle arises on the deep zygomatic arch face and the ligament uniting it, from an ovate scar which is broader dorsally than ventrally (Figure 3B, Figure 6A, Figure 10D).\n\nThese muscles are large relative to the pterygoid mass in other mammals, although they show the same pattern of division (Toldt, 1905; Toldt, 1907; Toldt, 1908; Edgeworth, 1935; Turnbull, 1970; Naples, 1985a).\n\nM. pterygoideus medius—This muscle is relatively large and arises from an oval depression on the ventrolateral pterygoid flange (Figure 1B, Figure 6B, Figure 10E). The muscle inserts on the rugose medial surface of the mandibular ramus and the large, posteriorly extended angular process.\n\nM. pterygoideus lateralis—This musclehas dorsal and ventral heads, arising from ovate depressions anterolateral on the pterygoid flange. The upper head originates anterodorsal to the lower. These muscles insert separately onto the medial mandibular surfaces, anteroventral to the mandibular condyle (Naples, 1982). The interruption of the joint capsule, the site of the insertions of the two tendons in Hapalops, is marked by a pair of adjacent depressions (Figure 6B). In the tree sloth Choloepus, orientation of the M. pterygoideus lateralis heads is posteroventral, as in other mammals, but in Bradypus the heads are posterodorsally oriented (Naples, 1985a). This arrangement is similar in Hapalops (Figure 1B, Figure 6B, Figure 10F).\n\nM. digastricus—In most mammals, this muscle has two bellies; the anterior has a small, ventral, fleshy origin from the mandible, and the posterior arises from the medial aspect of the mastoid process. The two bellies are united by a thick, short tendon. The bellies have different innervations (Mandibular Nerve V3 for the anterior and Facial Nerve VII for the posterior), confirming that they develop from different branchial arches. The anterior belly is often more robust, and in tree sloths it expands greatly, with a less robust posterior belly (Naples, 1986). In Hapalops, the origin scar of the anterior belly is larger and more robust than in most other mammals, occupying a rectangular area on the ventral mandible. The posterior belly origin is from a small tubercle medially on the mastoid process (Figure 5, Figure 7C, 7D), and from a thick tendon.\n\nM. stylohyoideus—This muscle is absent in the tree sloths (Naples, 1986), as is likewise for Hapalops. In the sloths studied, a stylohyal pit indicates that the stylohyal bone articulated directly into this basicranial depression on the anteromedial opening for the external auditory meatus (Naples, 1982).\n\nM. mylohyoideus—In most mammals, this muscle forms the oral floor, meeting at a ventral midline raphe. In Hapalops this muscle originated from a prominent lingual ridge of the mandibular ramus. The mandible is deep at the mental symphysis, and the anterior scar limit is near the ventral symphyseal surface. The mylohyoid ridge angles posterodorsally along the lingual mandibular body, continuing anteroventrally to the bony prominence of the posterior terminus of the labial ridge where it is joined by the ascending medial ridge on the condylar process (Figure 6B, Figure 7B, 7C, 7E). No Hapalops hyoid elements were available for study; therefore the extent of the attachment of this muscle to the basihyal was not possible to determine, and it is assumed that the muscle was united by a midline raphe as in other mammals (Naples, 1986).\n\nM. geniohyoideus—In most mammals, this muscle arises from the mandibular midline dorsal to the mylohyoideus and inserts on the basihyal. It protracts the hyoid with a fixed mandibular symphysis, or assists with mandibular opening if the hyoid is fixed. In Hapalops, it originates from a robust, oval scar with a wider mesiodistal axis, lateral to the ventral aspect of the mental symphysis and posterior to the M. genioglossus scar (Figure 6B, Figure 7B, 7E).\n\nM. buccinatorius—This muscle shows many patterns among mammals; complex, multilayered or simple. In tree sloths, it is simple, single-layered, with dorsoventral fibers from the maxilla labially to the mandible (Naples, 1986). In Hapalops, a depression marks the labial maxillary surface anterior to the caniniform, continuing dorsally and then ventrally to pass posteriorly between the ventral aspect of the molariform roots. The muscle scar continues posterior to M4, then trends ventrally toward the maxillary-palatal surface (Figure 1B, Figure 6A). The anterior mandibular attachment begins anterior to the lower caniniform and continues posteriorly to a short distance posterior to m3. The anteroposterior extent of origin and insertion scars suggest Hapalops had a long oral cavity, as is corroborated by other features (Figure 2B, Figure 6A).\n\nIn most mammals this muscle group consists of four muscles that primarily extend the head at the atlanto-occipital joint (M. rectus capitis dorsalis major et minor), and rotate the head at the atlanto-axial joint (M. obliquus capitis cranialis and caudalis). These muscle scars in Hapalops seem relatively larger than in other mammals, and occupy most of the rugose surface of the posterior occiput (Figure 5).\n\nMuscles of this group originate outside the tongue and insert within it. They control tongue position with respect to the mandible and hyoid apparatus. Of the two muscles of this group, M. styloglossus was unable to be reconstructed.\n\nM. genioglossus—This muscle arises from an oval scar with the main axis mesiodistal, anterior to the larger M. geniohyoideus origin scar (Figure 6B, Figure 7B, 7E).\n\nThis muscle group supports the hyoid bones and larynx, and forms the pharyngeal tube and esophagus. Muscle scars for M. stylopharyngeus were not discernable for reconstruction.\n\nM. constrictor superioris—In most mammals, this muscle is quadrilateral, originating from the pterygoid hamulus and plate, the pterygomandibular raphe, the posterior end of the mylohyoid line and by an aponeurosis to the pharyngeal basioccipital tubercle. The muscle is a sphincter and provides peristaltic contractions for swallowing. In Hapalops a prominent tubercle for the anterior origin occurs at the posterior end of the mylohyoid line (Figure 6B). A pterygoid flange attachment is also possible, but damage to the bone surface in this area in all available specimens precluded observation. Nevertheless, the pterygoid flange shape eliminates the space for either a pterygoid hamulus or pterygomandibular raphe. The posterior origin for this muscle can be restored in Hapalops, however, from paired, rounded basioccipital tubercles (Figure 2).\n\nThis muscle group controls the soft palate position and facilitates. Only muscles for which origin or insertion scars were discernable are discussed. The unrestorable muscles include: M. palatoglossus, M. palatopharyngeus, and M. salpingopharyngeus.\n\nM. tensor veli palatine—In most mammals, this muscle is a sheet originating from the sphenoid bone horizontal plate, posterior to the curved palatine crest and on the anterior aspect of the lateral pterygoid plate (Figure 2). The insertion is into the uvula base, and acts to move the uvula laterally to the same side, or to tense the soft palate when both sides contract simultaneously. In Hapalops, a depression marks the anterior palatal attachment posterior to a distinct palatine crest; anterior to the crescent-shaped depression that is the M. uvulae origin. The posterior origin is from the lateral surface of the anterior portion of the pterygoid flanges posterodorsal to the M. pterygoideus medius origin. As in other mammals, this muscle was a thin sheet trending anteroventrally from the posterior origin, joining with fibers from the palatal origin, to insert into the soft palate and the uvula laterally. The scars marking the origins of the muscle in Hapalops are large, suggesting that the soft palate was elongate anteroposteriorly.\n\nM. levator veli palatine—In most mammals, this muscle origin surrounds the carotid canal on the medial, anterior, and lateral surfaces as a crescentric temporal bone scar. It inserts into the soft palate laterally on each side, elevating this structure. In Hapalops, the carotid canal is large, round, and has a large triangular, anteromedial scar (Figure 2). This suggests the muscle originating from it was also large, and projected anteroventrally to merge with the soft palate.\n\nM. uvulae—In most mammals, this muscle forms the substance of the uvula, the soft tissue projection arising from the posterior midline of the hard palate and traversing the soft palate posteriorly as a plug to complete the oral seal prior to swallowing. The attachment in Hapalops is typical of other mammals, although particularly large (Figure 2).\n\n\nDiscussion\n\nThe characters reconstructed suggest that Hapalops was a selective feeding herbivore. The initial movement of the masticatory cycle would be determined by combined masseter and temporalis muscle actions in elevating the mandible until dental occlusion occurred. Once the dentition engaged, the mandible would have completed the stroke mediolaterally. This masticatory cycle is supported by the mediolaterally elongated glenoid fossa and the wear facets of the teeth. It would have been achieved by alternating contractions between the right and left components of the medial and lateral pterygoid muscles. Thus, while the initial movement of the masticatory cycle could produce a degree of crushing force, the greater amount of food processing could be achieved by the secondary half of the cycle provided by the grinding forces. A preliminary and unpublished assessment of bite force ratios for a few small-bodied sloths by one of the authors (RKM) showed the moment arm for M. masseter to have the greatest effect upon mandibular elevation. The greatest ratios for relative bite force were for the posterior dentition, which is common for many herbivores, and indicates a large input of initial force that could be then applied to the mediolaterally oriented grinding stroke.\n\nDivergence of pilosans into anteater and sloth lineages likely occurred in the early Eocene of South America (Delsuc et al., 2004). Eocene habitat and geography suggest that pilosan ancestors were small, arboreal insectivores living in forest canopies (Pascual & Ortiz Jaureguizar, 1990; Marshall & Sempere, 1993). Sloths originated from these ancestors by shifting their ecological niche toward herbivory in conjuction with climatic changes that created opportunities for arboreal and terrestrial browsers. The fossil record, in contrast to the molecular data, that links sloths to anteaters is poor, although sloths show many structural influences retained from myrmecophagous antecedents, such as elongate, tubular heads, small oral openings, long tongues, an anteroposteriorly lengthened oral cavity, unfused zygomatic arch, and large occipital condyles extending posterior to the skull that allow great cranial freedom of movement in all directions.\n\nA challenge facing early megatheroiids would have been food processing, as adaptations for insectivory select for reduced masticatory musculature and dentition, exemplified in early anteaters. An adaptive shift toward herbivory for such animals would be complicated by reduced muscle origins and insertions from the incomplete zygomatic arch. This condition occurs in only a few other terrestrial mammal orders, such as pholidotes and insectivores (Mivart, 1871; Parker, 1885; Nowak, 1999), and the extinct multituberculates (Carroll, 1988); none of which are herbivorous. The remaining dentition is reduced relative to that of buccinator-pilosan ancestors in the simple, enameless dentine structure that is plesiomorphic for xenarthrans. The transitional protosloth ancestor is currently unknown, but the adaptive changes necessary to fill an herbivore niche had been achieved by the time Hapalops and all other sloth families first appeared in the Miocene fossil record of South America. While many conditions in Hapalops are derived beyond those in a hypothetical protosloth, changes relative to increasing masticatory efficiency that differ from anteater lineages can be examined.\n\nSloth features allowing herbivory include elaboration and elongation of the jugal processes and bridging of the incomplete zygomatic arch. The increased length, dorsoventral and anteroposterior orientations of these flanges initiate masticatory efficiency improvement by increasing surface area for support of a five-part superficial masseter, relatively larger and reoriented deep masseter and zygomaticomandibularis muscles. Correlated changes include ventral pterygoid flange extension for pterygoideus muscle insertion and increased temporal fossa length and depth for temporalis origin. In the mandible, mental symphysis fusion and anterior elongation to form a predental spout as a channel for protrusion of the long, slender tongue was retained from earlier forms. A deep mandibular body provided additional space for ever-growing tooth roots and modification of mandibular condylar elevation relative to the mandibular toothrow affected gape and extent of the oral opening.\n\nIn addition to taxonomic relationships, characters in Hapalops provide specific insights concerning potential functional behaviors. Cranial anatomy can be used to infer locomotor capabilities and feeding strategies, which supports the phylogenetic characters associated with the transition toward becoming a unique herbivore, as well as those developed by later megatheroiids. Hapalops has been proposed as able to engage in arboreal or semiarboreal locomotor postures (Scott, 1903; White, 1993), both of which require different structural and functional capabilities than terrestrial postures. Although it should be possible to identify structures correlated with habitat usage and locomotor patterns, the most important and probably earliest adaptations that would allow a change of feeding ecology to herbivory, would occur in the cranial and cervical regions. These changes would be reflected in the position and shapes of cranial bones and soft tissues of the laryngeal and pharyngeal regions; including structures involved in feeding, deglutition and respiration (Reidenberg & Laitman, 1991). Change in feeding habits in Hapalops from ancestral forms likely resulted from evolution of new or different feeding strategies. These correlate with head and neck movement capabilities, making the anatomy of these regions appropriate indices for denoting change in habitus. Arboreal or semiarboreal animals must orient the vertebral column vertically or nearly so to climb trees, requiring a head and neck that can be held at up to ninety degrees to the body.\n\nOsteological adaptations and correlations—In Hapalops, cranial changes for masticatory muscles in jugal proceses, pterygoid flanges and the temporal fossa produced lines of action directing the masticatory stoke labiolingually. Masticatory muscle origins and insertions differed in later sloths, but the labiolingual masticatory stroke orientation was retained. Further changes among sloth lineages in masticatory muscle orientation are variations on the theme of increasing the efficiency of this novel approach to herbivory. The typical mammalian herbivore has a complete zygomatic arch-musculoskeletal complex, whereas sloths adapted an incomplete zygomatic arch into a unique functional complex by regaining the ligament between anterior and posterior portions (Figure 3A). This ligament also serves as the origin, permitting expansion of deep masseter and zygomaticomandibularis muscles in Hapalops (Figure 3). In later, larger sloths a bony connection forms, but remains unfused in all but the largest megatheriids, because the trend toward connection is related to allometric pressures for increased body size (Naples, 1985a; Naples, 1987; Naples, 1989). Smaller, later ground sloths and tree sloths retain separate arch portions.\n\nThe anterior sutures between the premaxillae, nasals, frontals, maxillae, and zygomatic bones in Hapalops are simple, and the anterior root of the zygomatic arch never fuses, even in the largest, most rugose and robust specimens. Because sutures between the more posterior skull bones show the typical complex interlocking mammalian pattern, simple anterior sutures indicate greater flexibility in this region, possibly an example of cranial kinesis; a rare phenomenon in mammals. Bones that abut along a straight line allow more “give” than do entirely immobile interdigitating sutures (Badoux, 1966; Buckland-Wright, 1972; Buckland-Wright, 1978; Herring, 1972; Herring, 1974; Herring, 1997; Herring & Mucci, 1991; Herring & Teng, 1995). The presence of simple sutures may be important in distributing twisting forces generated on the elongate anterior face by facial muscles, and contraction of the masseter musculature pulling on the anterior portion of the zygomatic arch (Thomason, 1991).\n\nMandibular structure in Hapalops and other megatheroids is derived when compared to the anatomy of other sloth families and a hypothesized protosloth. These differences include a mandibular condyle elevated above the occlusal plane and a deep ventral bowing of the mandibular body. In contrast, the mandibular condyle level is more even with the mandibular toothrow in mylodontids, and slightly to greatly elevated in megalonychids. Ventral bowing of the mandibular body also occurs in non-megatheroid sloths, but to a lesser degree. These differences, along with the abundant variation in predental spout shape and length, suggest that the mandible was less prone to phylogenetic influences from myrmecophagous ancestors.\n\nIn spite of their obligate herbivory, the dentition in all sloths is reduced in tooth number and type, as is true in Hapalops. The anterior caniniform teeth are small and project no farther from the occlusal plane than the molariforms. As with other sloths, the relative size, shape, orientation and spacing of the cheek teeth in conjunction with the anteromedially directed masticatory stroke determine the shape of the occlusal wear surfaces.\n\nIn many mammals, such as Choleopus and carnviorans, elongate teeth serve as occlusal guides because once the tips engage, mandibular movement is restricted to the direction allowed by dental overlap. Additionally, elongate canine teeth that require precise piercing point alignment are constrained to a path of motion that emphasizes movement in the vertical plane. Such an occlusal path requires the craniomandibular joint level to equal that of the maxillary-mandibular plane (Greaves, 1973; Greaves, 1974; Greaves, 1980; Naples, 1982; Naples, 1985a), but does not allow an increased mechanical advantage for the superficial masseter, the main masticatory muscle of herbivores (Turnbull, 1970; Greaves, 1980). However, the caniniforms in Hapalops are short and have minimal interlock. As such, they are occlusal guides for only a small percentage of the masticatory stroke, allowing greater flexibility in masticatory muscle arrangement, and are unencumbered by the elevated mandibular condyle.\n\nThe hard palate in Hapalops is elongated and of constant anteroposterior width, beginning at the pointed anterior premaxillary apex and continuing posteriorly with the maxilla and palatines. The marginal ridge on the posterior edge of the palate and reduced nasal spine are likely origins for M. uvulae and M. tensor veli uccinat. Together, these muscles elevate the uvula and tense the soft palate, sealing the oral cavity during food intake and mastication, and assist in pharyngeal opening during initiation of swallowing. The large origins of these muscles suggest they were of greater importance in Hapalops because of the great anteroposterior length of the oral cavity and posterior displacement of the pharyngeal entrance. Further evidence of an elongated oral cavity in Hapalops is demonstrated by the lengthy region between the pterygoid flanges and the paired ovate depressions marking the posterior origins of the superior pharyngeal constrictor muscles. The posterior origin is from the basilar tubercles anterior to foramen magnum; these tubercles in Hapalops and other sloths are apomorphies as this feature is usually single in mammals. Together, these attachments anchor the pharyngeal tube musculature, demonstrating it to be anteroposteriorly lengthened. This morphology indicates there was sufficient space for a long tongue with a great ability to protrude and retract. Elongation of this part of the muscular tube leading to the esophageal and glottal openings also suggests that Hapalops had great freedom of movement to elevate and depress the head; movements helpful for feeding and alteration of body orientation (Reeve, 1940; Naples, 1999).\n\nThe scar for gingival attachment in Hapalops is more ventral on the labial side of the mandible than in many other animals. This indicates presence of a large buccal pocket labial to the molariforms, allowing the sloth large, flexible cheeks. This permits a large buccal cavity for manipulation of vegetation, as well as sufficient space to house a large, elongate tongue.\n\nThe narrow palate and pointed predental spout suggest Hapalops was a selective feeder, as opposed to a bulk or grazing feeder as are herbivores with wide muzzles. The anteriorly pointed premaxillae further suggest a nipping/cropping action as the most likely method for food acquisition. The delicate nature of the premaxillae reduces the likelihood of Hapalops having strong, prehensile lips, but the rugose predental spout could have served as a sort of cutting board for vegetation. Most sloths need a cropping mechanism, as a result of the far posterior placement of the dentition. However, unpublished data determining relative bite force by one of the authors (RKM) indicates that the amount of force generated during the initial masticatory stroke in Hapalops to be much weaker than that of the similarly sized buccinators, Nematherium. With the lack of strong, prehensile lips, this places a greater emphasis on the need for Hapalops to engage in selective feeding, possibly on softer foods. The oblique wear facets between the caniniforms, along with the mandibular depression anterior to the lower caniniforms for the upper caniniforms could indicate an ability to shear more difficult foods if ingested laterally, and could constitute an additional means for cutting food. Shortness of the hard palate posterior to the last upper molariforms also contradicts a bulk feeding strategy. Animals ingesting a larger bolus need an increased palatal region for crushing food before swallowing. Hapalops, with a narrow palate, could only process small food boluses intraorally, although the capacious buccal cavity would have permitted shifting of food transversely during repetitive mediolateral chewing cycles. All of these factors contribute to this animal being a selective browser.\n\nMany mammals have a styloid process, an elongated bony spur, but no direct connection between skull and hyoid bones. Hapalops lacks a stylohyoid process, instead showing a pit for articulation of the stylohyoid bone of the hyoid apparatus. This feature is located far posteriorly on the ventral cranial surface, also maximizing possible anteroposterior lingual excursion.\n\nHapalops possesses relatively large, round occipital condyles positioned farther under the occiput than in many larger sloths. This orientation provides great ability to flex the head on the neck, correlating with upright posture. The articulation is oriented so that the atlas and more posterior cervical vertebrae face posteroventrally, and may indicate that these sloths spent at least some of their time in a semivertical position; a necessity for arboreal or semiarboreal animals. This contrasts with the more dorsoventrally flattened posteriorly projecting occipital condyles in later megatheriids, such as Eremotherium and Megatherium; animals with more horizontal cervical vertebral articulations that were terrestrial. This capability would allow Hapalops to move its head from a position aligned anterior to the neck to nearly ninety degrees. Such head mobility would allow the sloth to feed while walking or standing quadrupedally, as well as while assuming a bipedal stance (Scott, 1937: Figure 408).\n\nMuscular adaptations and correlations—The main sloth masticatory muscles used in cutting vegetation are the superficial masseter and the medial pterygoid, and sloth ancestry explains their unique arrangements, bony attachments and lines of action. Mandibular movements are also effected by deep masseter and temporalis muscles in most herbivores, although generally of lesser importance (Turnbull, 1970). In sloths, the attachments and lines of actions of the latter group have been reoriented to compliment superficial masseter and medial pterygoid movements. Although the superficial masseter is positioned more like that of Bradypus than Choloepus, because the ascending portion of the jugal is more posteriorly oriented, the line of action is more horizontal as is that of Choloepus, and the reconstruction of the same segment in Paramylodon (=Glossotherium harlani in Naples, 1989) (Naples, 1982; Naples, 1985a; Naples, 1987). The line of action of the medial pterygoid in Hapalops is more horizontal than in the tree sloths, Nothrotheriops, and Paramylodon (=Glossotherium) (Naples, 1982; Naples, 1985a; Naples, 1987; Naples, 1989). Specifically, the summed lines of action of superficial masseter and medial pterygoid are approximately 105 degrees to those of deep masseter, temporalis, and zygomaticomandibularis muscles (Figure 8). In contrast, the average line of action in Bradypus is approximately 80 degrees, while that of Choloepus is approximately 100 degrees (Naples, 1982).\n\nMuscle reorientation turns the elongate cranium in Hapalops into an advantage by allowing a larger temporalis muscle with a greater arc of rotation, albeit with a more horizontal line of action. The significance of a more horizontal line of action for the temporalis, deep masseter, and zygomaticomandibularis muscles is an emphasis on the anteroposterior component of motion. The orientation for the line of action of temporalis is similar to that of Choloepus and Paramylodon, but less vertical than in Bradypus (Naples, 1982; Naples, 1985a; Naples, 1989). The deep masseter orientation is approximately 45 degrees anterior to vertical, and reminiscent of the orientation in Bradypus and Paramylodon, but is relatively steeper than the more horizontal orientation in Choloepus and opposite to the posteriorly inclined muscle in Nothrotheriops (Naples, 1982; Naples, 1985a; Naples, 1987; Naples, 1989). The zygomaticomandiularis line of action in Hapalops is more vertical than in any of the other sloths studied, but is not anteriorly oriented as in Bradypus (Naples, 1982; Naples, 1985a; Naples, 1987; Naples, 1989). This arrangement sets these muscles in opposition to the increase of the vertical component of the line of action that would increase the mechanical advantage of the superficial masseter and medial pterygoid muscles. Bradypus has an increased the mechanical advantage for these muscles by elevation of the glenoid fossa and lowering of the insertion from the deepening of the mandibular ramus. Both Bradypus and Hapalops show posterior elongation of the mandibular angular process to a greater extent than in many other sloths. This feature and ventral pterygoid flange expansion allow an increased attachment area for the medial pterygoids, despite simultaneously increasing the anteroposterior component of mandibular motion. Such a mandibular motion in Hapalops would have become even greater if the craniomandibular joint remained at the level of the occlusal plane (approaching 120 degrees), but this angle has been reduced because this sloth has only slight craniomandibular elevation (Figure 8A).\n\nAs active and balancing side masticatory muscles counteract lateral components of motion, superficial masseter and medial pterygoid muscle contraction with temporalis, deep masseter, and zygomaticomandibularis muscles cancel much of the anteroposterior component of motion. This results in mandibular elevation and allows a primarily mediolaterally oriented masticatory stroke. Mediolateral movements maximize the chewing stroke along the long axes of the teeth in Hapalops. Loss of a large anteroposterior component of motion, while decreasing masticatory efficiency, is less critical to Hapalops than other herbivores because of the large masticatory muscle mass and the small degree of gape. In Hapalops these are compensated during food ingestion by inheritance of a large, elongate tongue. Tongue usage was compensated for by the buccinator muscle, by allowing the mandible to be maintained in a slightly open position, such that the tongue could be rapidly and/or forcefully extended and retracted. The mylohyoid line is located in an unusually low position on the medial mandibular surface, confirming that there was a long and deep intraoral space. A long oral cavity is a liability offset by an elongated buccinator muscle. Large buccal cavities increase masticatory efficiency by accommodating and controlling the mediolateral movement of masses of vegetation during each masticatory cycle. Such an accommodation would also benefit Hapalops.\n\nEstimation of gape—The degree to which an animal can gape correlates with the manner of feeding (Herring & Herring, 1974; Herring, 1975). Sloths arose from insectivorous forms, and retain elongate crania and small buccal openings. Other factors that affect the masticatory pattern include the dental occlusal pattern and the shape, position and orientation of the mandibular condyle. Gape in Hapalops was estimated by comparison of the anterior and posterior maxima to which the condyles could rock in the glenoid fossa, and was found to be no more than 45 degrees. A wider opening would cause mandibular dislocation, and the angular processes to bump against neck tissues. Condylar convexity also permits some mediolateral rocking, providing the ability to swing the mandible labiolingually, as would be required to complete the lingual component of the anterolingual masticatory stroke indicated by the wear facet pattern in these sloths (Greaves, 1973; Naples, 1990). The craniomandibular joint in Hapalops is only slightly elevated above the occlusal plane (Figure 10A). This joint arrangement emphasizes movement in the mediolateral plane, as demonstrated by the shape and orientation of the dental occlusal facets (Figure 4).\n\nGiven the cranial musculoskeletal arrangement of Hapalops, the initial masticatory cycle movement would have been mandibular elevation until dental occlusion occurred by combined masseter and temporalis muscle action. Because Hapalops only gapes to a small degree, mandibular elevation with a great force would have been possible. This degree of force is supported by an unpublished assessment of bite force ratios for a number of small-bodied sloths, including Hapalops (by RKM), which shows that the moment arm for M. masseter has the greatest effect upon mandibular elevation, and that the greatest bite forces occurred at the posterior dentition. Once the molariform teeth engaged, the mandible would complete the stroke cycle in a mediolateral direction before briefly depressing the jaw and repeating. The mediolaterally elongated glenoid fossa provided the space to allow the mandibular condyle to pivot, in the mediolateral swing provided by pterygoid muscles. Whereas the initial masticatory cycle movement could produce a suitable degree of crushing force, greater food processing would be achieved by grinding forces produced during the second half of the cycle. Because the greatest ratios for relative bite force were at the posterior dentition, as is common in herbivores, large initial forces would then be applied to the mediolaterally oriented grinding stroke.\n\n\nConclusions\n\nAdaptive shifts in the proto-sloth lineage from insectivory or myrmecophagy toward obligate herbivory resulted in a masticatory musculature rearrangement in Hapalops that reflects the typical herbivore pattern, although achieved by a unique combination of cranial musculoskeletal features. As the masticatory muscles and their cranial attachments were reduced in sloth ancestors that emphasized tongue-based food ingestion, sloths were free to develop these re-emphasized structures unconstrained by patterns inherited by other herbivores. Thus, in spite of the phylogenetic constraints of myrmecophagous ancestors, pilosans became successful, albeit atypical, herbivores. These features in Hapalops also occur in later megatheriids and other large-bodied sloths, and selective pressures resulting in the diversity of form in sloths are variations upon the theme of increased masticatory efficiency for herbivory. As such, the reconstruction of the cranial morphology and functions of Hapalops presented here can be used as a basis for understanding the selective pressures channeling the evolution of later megatheroids, and the relative contribution of phylogenetic history and functional constraints to the trend toward increased size and possible changes in locomotor habits and habitats.\n\nReconstruction of cranial osteology and masticatory musculature demonstrates that the appearance of the head of Hapalops probably differed significantly from that of other small-bodied sloths. The zygomatic arches flare widely, making sloth heads wider at this location as well as longer than usually depicted (Figure 1A, Figure 2, Figure 11). Likewise, the pointed anterior tips of the premaxilla and predental spout suggest an extreme narrowness of the anterior part of the muzzle, which was not appreciated in previous studies.\n\nReconstruction of the appearance of the head of Hapalops in dorsal view (A), showing the narrowness of the muzzle, and the much greater posterior width of the head, and in lateral view (B), showing the greatly elongated anterior portion of the cranium in this animal. This reconstruction results from new anatomical data generated in this study.",
"appendix": "Author contributions\n\n\n\nData collected by Naples; manuscript writing and data analysis shared between both authors; figures initially drawn/created by Naples and further tweaked by McAfee.\n\n\nCompeting interests\n\n\n\nThe authors declare that there are no competing interests for this work.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe authors are grateful to the team members of the American Museum of Natural History, Field Museum of Natural History and University of Kansas for all of their help and assistance with accessing those collections housing the specimens used in this study. We would also like to dedicate this to the memory of Dr. Larry D. Martin. The many years of shared friendship and collaboration are deeply missed, as well as the hospitality showed our visits to study specimens at the University of Kansas and write portions of the manuscript.\n\n\nReferences\n\nAnderson WD, Anderson BG: Atlas of canine anatomy. Lea and Febinger, Philadelphia, Pennsylvania, 1994; pp. 1230. Reference Source\n\nAnderson RP, Handley CO: A new species of three-toed sloth (Mammalia: Xenarthra) from Panama, with a review of the genus Bradypus. Proceedings of the Biological Society of Washington. 2001; 114(1): 1–33. Reference Source\n\nAnthony HE: Preliminary report on fossil mammals from Porto Rico, with descriptions of a new genus of ground sloth and two new genera of hystricomorph rodents. Annals of the New York Academy of Science. 1916; 27(1): 193–203. Publisher Full Text\n\nBadoux DM: Framed structures in the mammalian skull. Acta Morphol Neerl Scand. 1966; 6(3): 239–250. 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Publisher Full Text\n\nNaples VL, Martin L: Restoration of the superficial facial musculature in Nimravids. Zool J Linn Soc. 2000b; 130(1): 55–81. Publisher Full Text\n\nNowak RM: Walker’s Mammals of the World, 6th ed. Johns Hopkins University Press, Baltimore, Maryland, 1999; pp. 1936. Reference Source\n\nOwen R: On the anatomy of the great anteater (Myrmecophaga jubata, Linn.). Transactions of the Zoological Society of London. 1862; 4: 117–140. Reference Source\n\nParker WK: On the structure and development of the skull in the Mammalia. Part II. Edentata. Philosophical Transactions of the Royal Society of London. 1885; 176: 1–119. Publisher Full Text\n\nPascual R, Ortiz Jaureguizar E: Evolving climates and mammal faunas in Cenozoic South America. J Hum Evol. 1990; 19(1–2): 23–60. Publisher Full Text\n\nPatterson B, Pascual R: The fossil mammal fauna of South America. In A. Keast, F. C. Erk and B. Glass (eds.), Evolution, Mammals and Southern Continents. State University of New York Press, Albany, New York, 1972; pp. 246–309.\n\nPaula Couto C: On two small Pleistocene ground sloths. Anais Acad Bras Cienc. 1971; 43: 500–513. Reference Source\n\nPujos F, De Iullis G, Argot C, et al.: A peculiar climbing Megalonychidae from the Pleistocene of Peru and its implication for sloth history. Zool J Linn Soc. 2007; 149(2): 179–235. Publisher Full Text\n\nReeve ECR: Relative growth in the snout of anteaters. A Study in the Application of Quantitative Methods to Systematica. Proceedings of the Zoological Society of London, series A. 1940; 110(1–2): 47–79. Publisher Full Text\n\nReidenberg JS, Laitman JT: Effect of basicranial flexion on larynx and hyoid position in rats: An experimental study of skull and soft tissue interactions. Anat Rec. 1991; 230(4): 557–569. PubMed Abstract | Publisher Full Text\n\nReinhardt JT: Beskrivelse af Hovedskallen af et Kaempedovendyr Grypotherium darwinii. 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Ph.D. dissertation, University of Chicago, Chicago, Illinois, 1984; pp. 478. Reference Source\n\nSimpson GG: The principles of classification and a classification of the mammals. Bulletin of the American Museum of Natural History. 1945; 85: 1–350. Reference Source\n\nSisson S, Grossman JD: The Anatomy of the Domestic Animals, 4th ed. Saunders, Philadelphia, Pennsylvania, 1953; pp. 972. Reference Source\n\nStandring S, Ellis H, Berkovitz BKB: Gray’s anatomy: The anatomical basis of clinical practice, 39th ed. Elsevier Churchill Livingstone, London, United Kingdom, 2005; pp. 1672. Reference Source\n\nThomason JJ: Cranial strength in relation to estimated biting forces in some mammals. Can J Zool. 1991; 69(9): 2326–2333. Publisher Full Text\n\nvon Toldt C: Der Winkleforsatz des Unterkiefers bein Menschen und bei den Saugetieren und die Beziehungen der Kaumuskeln zu demselben. Sitzungsberichte der Akademie der Wissenschaften Wien, Mathematisch-naturwissenschaftliche Klasse, II Teil. 1905; 114: 315–476. Reference Source\n\nvon Toldt C, Der vordere Bauch des M: Digastricus mandibulae und seine variationen bein Menchen. Sitzungsberichte der Akademie der Wissenschaften Wien, Mathematisch-naturwissenschaftliche Klasse, 1907; 116: 1–373.\n\nvon Toldt C, Der vordere Bauch des M: Digastricus mandibulae und seine variationen bein Menchen. II. Sitzungsberichte der Akademie der Wissenschaften Wien, Mathematisch-naturwissenschaftliche Klasse, 1908; 117: 229–231. Reference Source\n\nTurnbull WD: Mammalian masticatory apparatus. Fieldiana: Geology. 1970; 18(2): 147–356. Reference Source\n\nWebb SD: The interrelationships of tree sloths and ground sloths. In G. G. Montgomery (ed.), The Evolution and Ecology of Armadillos, Sloths and Vermilinguas. Smithsonian Institution Press, Washington D.C., 1985; pp. 105–112. 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}
|
[
{
"id": "4369",
"date": "06 May 2014",
"name": "Jennifer White",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall Impression:Hapalops is an important genus in sloth evolution: as a Miocene sloth it is old enough to have given rise to many later specialized genera of sloths, it exhibits cranial characters associated with both insectivory and herbivory, it is well represented in the fossil record, and there is substantial evidence that this genus was arboreal or semi-arboreal. Therefore, a detailed study of cranial morphology and inferred feeding habits is of great interest. This study parallels and expands upon previous studies by the first author on masticatory anatomy and function of other xenarthrans such as living tree sloths, the extinct sloths Nothrotheriops and Glossotherium, and living anteaters. The descriptive morphology is thorough, the illustrations are precise, the interpretations appear to be sound, and the conclusions are justified. Although the paper is well written, I have a number of comments and suggestions for minor revision outlined below.Abstract:The phrasing in this sentence is awkward and should be revised: “An elevated temporomandibular joint occurs in Bradypus, a tree sloth with anterior chisel-shaped teeth instead of caniniforms, and the tree sloth Choloepus, which is aligned with the megalonychids, has anterior caniniforms.\"Introduction:The statement “Perhaps the young of even the largest species could climb” is speculative; without evidence from fossil juvenile material why make a statement about it?White (1997) and White (2012) also presented evidence of arboreality in extinct sloths:[White, J. L. 2012. Phylogenetic and functional diversity of West Indian sloths. In: R. Borroto-Paez, C. A. Woods, and F. E. Sergile (eds.), Terrestrial Mammals of the West Indies; Contributions. Gainesville, FL: Florida Museum of Natural History Press, pp. 317-336.][White, J. L. 1997. Locomotor adaptations in Miocene xenarthrans. In: R. F. Kay, R. H. Madden, R. L. Cifelli, J. J. Flynn (eds.), Vertebrate Paleontology in the Neotropics: The Miocene Fauna of La Venta, Colombia. Washington: Smithsonian Institution Press, pp. 246-264.]Muizon et al. 2004 (cited regarding Thalassocnus) does not appear in the reference list.The authors should reference Bargo et al. (2009), which provides an analysis of cranial morphology and feeding mechanics of Eucholaeops, another Miocene megatherioid sloth.[Bargo et al. 2009. Predominance of orthal masticatory movements in the Early Miocene Eucholaeops (Mammalia, Xenarthra, Tardigrada, Megalonychidae) and other megatherioid sloths. J. Vertebrate Paleontology 29(3): 870-880.]Materials and Methods:Bradypus and Choloepus were used to infer muscle position in Hapalops. These sloths have very different facial morphologies, so how was it determined which to use for comparison?The authors state “No Hapalops crania or mandibles were sufficiently complete or uncrushed to allow articulation”. Table 1 lists four specimens with both skull and mandible; were these in such poor condition that they could not be articulated?An explanation of how the line of action of muscles was determined should be included in Methods.Results:Figure 2: the caption has a spelling error in the word “osteological”.The statement “Animals with a farther lateral superior temporal line are probably younger, …” is again speculative; is there other evidence to support a juvenile status, such as suture morphology?Section on Mandibular Bony FeaturesReferences should be provided for statements about sloth autapomorphies (under lingual and lateral view characters).Reiss (1997) discusses plesiomorphic states in the feeding apparatus for xenarthrans, along with derived conditions in myrmecophagids – perhaps this paper deserves to be cited.[Reiss K.Z. 1997. Myology of the feeding apparatus of Myrmecophagid anteaters (Xenarthra: Myrmecophagidae). J. Mammalian Evolution 4(2): 87-117.]Regarding the section on mandibular lateral view characters: the mental nerve is a branch of the mandibular division of trigeminal (V3) which supplies motor innervation to muscles of mastication and carries sensory innervation from the lips; motor innervation to muscles of the lips (e.g. M. orbicularis oris, M. depressor labii inferioris) is supplied by the facial nerve.Regarding the section on mandibular dentition: a reference is needed for the statement that large interdental spaces are unusual in herbivores, and for the statement that unfused zygomatic arches distinguish pilosans from most other mammals.Reconstruction of Cranial MusculatureFigure 8: How is it determined exactly where lines of action of muscles are? Is this simply based on the apparent mid-point of each muscle scar?In the discussion of M. temporalis, the last sentence of the first paragraph should refer to Figure 10B (not 9A).A reference is needed for the statement about M. buccinatorius having many patterns among mammals.Figure 5: Where is the distinction between M. rectus capitis posterior major et minor? Is this muscle called M. rectus capitis dorsalis in the text, but posterior in the figure? Figure 5: Does M. obliquus superioris (as labeled in the figure) represent M. obliquus capitis cranialis and caudalis (as referenced in text)?The first sentence under Palatine Musculature is incomplete.Discussion:McAfee (2011) should be cited as the paper discusses characters associated with herbivorous feeding in sloths and derives bite force estimates.[McAfee, R.K. 2011. Feeding mechanics and dietary implications in Neocnus (Mammalia) Xenarthra: Megalonychidae from Haiti. J. Morphology 272 (10): 1204-1216.]What is the functional significance of pterygoid flange extension?White (1997) [see citation above] also proposed arboreality in Hapalops.Section on osteological adaptations and correlations:In the phrase “In many mammals, such as Choloepus and carnviorans,…”, the word carnivorans is misspelled.The phrase “…much weaker than that of the similarly sized buccinators, Nematherium” – is unclear. What is meant by “buccinators”?Section on osteological adaptations and correlations:I question how accurate the estimate of 105 degrees (summed lines of action of superficial masseter and medial pterygoid) is when it is unclear how the lines of actions of muscles were derived. Also, what is the functional significance of this value for these muscles in Hapalops in comparison to those of the living tree sloths?It is difficult to evaluate the validity of the bite force estimates in Hapalops that are derived from unpublished analyses by McAfee.",
"responses": []
},
{
"id": "4376",
"date": "11 Jun 2014",
"name": "David Burnham",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a very good paper and the science is sound. We suggest some minor technical writing issues that need to be addressed (see below). We think the figures could be much improved by making them less busy. Some suggestions were handled as comments, but overall many of the figures could be improved by shifting words out of the figure and into the captions or supporting text. The first two figures in particular have many words, and we think some of the labelled anatomical structures are either unnecessary to label or could be abbreviated to make the figure appear less busy. That being said, the figures are very good and help considerably in making the arguments outlined in the paperSpecifics:Page 2, First Paragraph – The second sentence reads “i.e., the extinct glyptodonts and giant armadillos, and the living armadillos”, instead it should read “i.e., the extinct glyptodonts, extinct giant armadillos, and the living armadillos”. Page 2, Fourth Paragraph – The seventh sentence (“Extinct sloths have been divided…”) is redundant with the first paragraph of the introduction and should be rewritten. Page 2, Fourth Paragraph – In the eighth sentence, “small sized” should be “small-sized”. Page 2, Seventh Paragraph – In the first sentence, it is not clear to what institution “Department of Geology” refers. Furthermore, Table 1 contains a reference to YPM that is not explained in this paragraph. Page 2, Seventh Paragraph – In the second sentence, “adult” does not require quotation marks. Page 3, Table 1 – see previous comment about no explanation for the abbreviation YPM. It will also need to be added to the table caption. Page 4, Figure 1 – The label pointing out the zygomatic arch in Fig. 1A may be unnecessary for the intended audience, and removing the label will clean up the diagram. Furthermore, the label “Temporal” in the lower portion of Fig. 1A doesn’t seem to refer to anything since it lacks an arrow and it is not contained inside the line drawing. Page 7. Figure 4 – The sentence in the figure about the shaded surfaces indicating occlusion is also included in the figure caption and can be removed from the figure itself. Page 12, Figure 8 – I would prefer to read the explanations of the arrows in the figure caption rather than overlaying the figure. Page 13, Sixth Paragraph – The third sentence reads “The relative size, orientation and position of these …” should read “The relative size, orientation, and position of these…”. Page 14, Seventh Paragraph – In the first sentence, “musclehas” should be “muscle has”. Page 15, Twelfth Paragraph – The first sentence (“This muscle group controls the soft palate position and facilitates.”) is an incomplete sentence. Page 16, Fifth Paragraph – The fourth sentence “The fossil record…” should be referenced. Page 17, First Paragraph – The first sentence ends with “denoting change in habitus”. Is the word “habitus” really the word that was intended? I think the word habit would be preferable.",
"responses": []
}
] | 1
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https://f1000research.com/articles/3-86
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https://f1000research.com/articles/3-84/v1
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03 Apr 14
|
{
"type": "Research Article",
"title": "Trace amines inhibit insect odorant receptor function through antagonism of the co-receptor subunit",
"authors": [
"Sisi Chen",
"Charles W. Luetje",
"Sisi Chen"
],
"abstract": "Many insect behaviors are driven by olfaction, making insect olfactory receptors (ORs) appealing targets for insect control. Insect ORs are odorant-gated ion channels, with each receptor thought to be composed of a representative from a large, variable family of odorant binding subunits and a highly conserved co-receptor subunit (Orco), assembled in an unknown stoichiometry. Synthetic Orco directed agonists and antagonists have recently been identified. Several Orco antagonists have been shown to act via an allosteric mechanism to inhibit OR activation by odorants. The high degree of conservation of Orco across insect species results in Orco antagonists having broad activity at ORs from a variety of insect species and suggests that the binding site for Orco ligands may serve as a modulatory site for compounds endogenous to insects or may be a target of exogenous compounds, such as those produced by plants. To test this idea, we screened a series of biogenic and trace amines, identifying several as Orco antagonists. Of particular interest were tryptamine, a plant-produced amine, and tyramine, an amine endogenous to the insect nervous system. Tryptamine was found to be a potent antagonist of Orco, able to block Orco activation by an Orco agonist and to allosterically inhibit activation of ORs by odorants. Tyramine had effects similar to those of tryptamine, but was less potent. Importantly, both tryptamine and tyramine displayed broad activity, inhibiting odorant activation of ORs of species from three different insect orders (Diptera, Lepidoptera and Coleoptera), as well as odorant activation of six diverse ORs from a single species (the human malaria vector mosquito, Anopheles gambiae). Our results suggest that endogenous and exogenous natural compounds serve as Orco ligands modulating insect olfaction and that Orco can be an important target for the development of novel insect repellants.",
"keywords": [
"Insects have positive and negative impacts on humans",
"in terms of health",
"economy",
"and food stores. Insects pollinate plants to increase global food production",
"with 35% of global production of crops depending on animal pollinators1",
"2. Insects also cause significant destruction of crops and food stores3–5. Insects can also transmit fatal diseases such as dengue fever6",
"malaria7",
"yellow fever and epidemic typhus8. Insects use olfaction to sense their surroundings and to guide important activities",
"including feeding",
"mating and oviposition. This makes the insect olfactory system receptors an attractive target for the chemical control of deleterious insect species."
],
"content": "Introduction\n\nInsects have positive and negative impacts on humans, in terms of health, economy, and food stores. Insects pollinate plants to increase global food production, with 35% of global production of crops depending on animal pollinators1,2. Insects also cause significant destruction of crops and food stores3–5. Insects can also transmit fatal diseases such as dengue fever6, malaria7, yellow fever and epidemic typhus8. Insects use olfaction to sense their surroundings and to guide important activities, including feeding, mating and oviposition. This makes the insect olfactory system receptors an attractive target for the chemical control of deleterious insect species.\n\nInsects use odorant receptors (ORs) to recognize and distinguish a diverse range of odorants9,10. Each OR is composed of two functionally essential parts: a highly conserved co-receptor subunit (Orco) and one of a large number of variable odorant-binding (or “tuning”) subunits11–17. These subunits associate in an unknown stoichiometry to form an odorant-gated ion channel18,19. ORs have also been proposed to initiate, or be modified by, second messenger cascades13,18. While the odorant-binding subunit is responsible for interacting with odorants9,20,21, both the odorant-binding subunits and Orco are involved in forming the ion channel pore21,22. Insect ORs are not related to the receptors and channels of humans and other tetrapods15, suggesting that control of detrimental insect activity may be possible through the development of insect OR selective compounds. A current approach to developing these compounds is to identify the particular odorant binding subunits that recognize behaviorally important odorants10,23–26 and then conduct large scale ligand screens27,28, but high diversity among the odorant binding subunit repertoires of different species makes this approach exceptionally labor intensive29,30.\n\nThe recent identification of the synthetic compound VUAA1 as a novel OR agonist that acts directly on Orco27, suggests that manipulation of insect behavior might be achieved by targeting Orco. Based on the VUAA1 structure, several additional synthetic Orco agonists and a larger, more diverse series of synthetic Orco antagonists have been identified31–33. Importantly, several of these Orco antagonists were shown to inhibit odorant activation of ORs through a non-competitive mechanism31–33. These findings suggest that Orco antagonists might be useful in altering insect behavior.\n\nOrco subunits are highly conserved across insect species, suggesting that Orco serves an essential function common to all insect ORs15,34. This high conservation underlies observations that Orco subunits from different species are functionally interchangeable; an Orco subunit from one species can form functional ORs with an odorant-binding subunit from a different species21,22. As the “pharmacology” of synthetic Orco agonists and antagonists has expanded, it has also become clear that Orco subunits from disparate insect species have very similar sensitivities to known Orco ligands27,31–33,35. This suggested to us that the binding site for Orco ligands may serve as a modulatory site for compounds endogenous to the insects or may be a target of exogenous compounds, such as those generated by plants. Insects use a variety of amines as neurotransmitters and neuromodulators36–39. Plants also generate a variety of amines that may play a role in resistance to insect herbivores40–42. For these reasons, we screened a panel of biogenic and trace amines for agonist and antagonist activity at insect Orco subunits. We found tryptamine to be a potent Orco antagonist with broad activity at Orco subunits from different species. Tyramine and phenethylamine also function as Orco antagonists, but were substantially less potent than tryptamine. Importantly, we found that tryptamine, acting through Orco, could inhibit odorant activation of a wide range of ORs from a variety of insect species. Our findings suggest a role for Orco as a modulatory site common to all insect ORs and support the development of Orco-directed compounds that can be used to manipulate insect behavior.\n\n\nMethods\n\nXenopus laevis frogs were purchased from Nasco (Fort Atkinson, WI). The care and use of Xenopus laevis frogs in this study were approved by the University of Miami Animal Research Committee (Animal Welfare Assurance #A-3224-01, Protocol #13-056) and meet the guidelines of the US National Institutes of Health. All experimentation was conducted on cultured oocytes after surgical removal from the frogs (see below). The amines screened in this study (Figure 1), odorants (L-fenchone, acetophenone, geranyl acetate, 6-methyl-5-hepten-2-one, 2-nonanone and eugenol), OLC12 and other chemicals were from Sigma-Aldrich. Cqui\\Orco (from Culex quinquefasciatus), Onub\\Or6, Onub\\Orco (from Ostrinia nubilalis), Mcar\\Or5 and Mcar\\Orco (from Megacyllene caryae) were cloned and inserted into the pGEMHE vector43 as previously described23,24,44,45. Dmel\\Or35a and Dmel\\Orco (from Drosophila melanogaster) were generously provided by J. Carlson and L. Vosshall, respectively. Agam\\Or27, Agam\\Or28, Agam\\Or31, Agam\\Or39, Agam\\Or48, Agam\\Or65 and Agam\\Orco (from Anopheles gambiae) were generously provided by L. Zweibel.\n\nMature Xenopus laevis frogs were anesthetized by submersion in 0.1% 3-aminobenzoic acid ethyl ester. Depth of anesthesia was judged by loss of nasal flare and swallow reflexes. Oocytes were surgically removed. The incision was treated with gentamicin sulfate (two subcutaneous injections of 0.1 mL 10 mg/mL gentamycin at the surgical site) and sutured. Immediately following surgery (and before recovery from anesthesia), as an analgesia agent, one subcutaneous injection of Meloxicam solution (0.1 mg/mL) (0.1 mg/kg body weight) was administered to the dorsal lymph sac of the frogs. The frogs were allowed to recover from surgery in a humid chamber before being placed back in the holding tank. Surgeries were performed on individual frogs no more often than once every 3 months. Following the fourth surgery, frogs were anesthetized as described above and then pithed.\n\nFollicle cells were removed by treatment with collagenase B (Boehringer Mannheim) for 2 hours at room temperature. Capped cRNA encoding each OR subunit was generated using mMessage mMachine kits (Ambion). For heteromeric ORs, 25 ng of cRNA encoding each OR subunit was injected into Stage V-VI Xenopus oocytes. For expression of Orco homomers, 50 ng of cRNA was injected. Oocytes were incubated at 18°C in Barth's saline (in mM: 88 NaCl, 1 KCl, 2.4 NaHCO3, 0.3 CaNO3, 0.41 CaCl2, 0.82 MgSO4, 15 HEPES, pH 7.6, and 150µg/ml ceftazidime) for 2–5 days prior to electrophysiological recording.\n\nOdorant and Orco ligand induced currents were recorded under two-electrode voltage clamp, using an automated parallel electrophysiology system (OpusExpress 6000A, Molecular Devices). Oocytes were perfused with ND96 (in mM: 96 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 5 HEPES, pH 7.5). Orco ligands were prepared as 50 or 100 mM stock solutions in DMSO and then diluted into ND96 on the day of the experiment. Odorants were prepared as 100 mM stock solutions in DMSO and then diluted into ND96. Unless otherwise noted, applications were for 60 sec at a flow rate of 1.0 ml/min, with extensive washing in ND96 at 4.6 ml/min between applications. Micropipettes were filled with 3 M KCl and had resistances of 0.2–2.0 MΩ. The holding potential was -70 mV. Current responses, filtered (4-pole, Bessel, low pass) at 20 Hz (-3 db) and sampled at 100 Hz, were captured and stored using OpusXpress 1.1 software (Molecular Devices).\n\nTo screen for agonist activity, oocytes were exposed to 30 sec applications of candidate compounds with 5 min washes between applications (Figure 2A). For the concentration-response protocol (Table 1), applications were for 20 sec at a flow rate of 1.65 ml/min. To measure antagonist activity at Orco (Figure 2B, 2C, Figure 3, Figure 4A and Figure 5A), oocytes were exposed to two 60 sec applications of the synthetic Orco agonist OLC12 (2-((4-Ethyl-5-(4-pyridinyl)-4H-1,2,4-triazol-3-yl)sulfanyl)-N-(4-isopropylphenyl)acetamide) with 4 min washes between applications. Oocytes were then exposed to a 90 sec application of antagonist candidate, immediately followed by a 60 sec co-application of antagonist candidate and OLC12. The current response in the presence of antagonist candidate was compared to the mean of the preceding two responses to OLC12 alone and is presented as a percentage.\n\nConcentration-response data was fit as described in Methods. nH is the apparent Hill coefficient. Values are presented as mean ± SEM (n = 3-14).\n\nA) The tested amines do not display Orco agonist activity. Oocytes expressing Cqui\\Orco were challenged with 30 sec applications of 100µM gramine, tyramine, tryptamine and melatonin (top trace), phenethylamine, serotonin, octopamine and dopamine (middle trace), or histamine, epinephrine and norepinephrine (bottom trace), with 5 min washes between applications. 30µM OLC12 (Orco agonist) was applied at the end of each trace. B) Tryptamine and tyramine are antagonists of Cqui\\Orco. Oocytes expressing Cqui\\Orco were exposed to 60 sec applications of 30µM OLC12 with 4 min washes between applications. 100µM tryptamine (top trace), tyramine (middle trace), or octopamine (bottom trace) were applied and incubated for 90 sec preceding the third application of OLC12 and then co-applied during the OLC12 application. C) Screen of 11 amines for Orco antagonism. Responses of Cqui\\Orco to 30µM OLC12 (~EC5) in the presence of 100µM of each compound are presented as a percentage of the average of two preceding responses to OLC12 alone (mean ± SEM, n = 3-10). Statistical significance was assessed by one-way ANOVA, followed by Dunnett's post-test comparing to sham treated oocytes (*p<0.01; **p<0.001).\n\nA) Concentration-inhibition curves for tryptamine, tyramine and phenethylamine inhibition of Cqui\\Orco activated by 30µM OLC12. B) Altering the concentration of Orco agonist (OLC12) shifts the tryptamine inhibition curve. The IC50 for tryptamine inhibition of Cqui\\Orco activation by 30µM OLC12 (4.7 ± 0.7µM, n = 5) is significantly different (p<0.0001, F-test) from the IC50 for tryptamine inhibition of Cqui\\Orco activation by 100µM OLC12 (143 ± 18µM, n = 6).\n\nA) Oocytes expressing Orco from each of three different species were activated by the indicated concentration of OLC12. For Cqui\\Orco from Cx. quinquefasciatus, 30µM is the ~EC5; for Agam\\Orco from An. gambiae, 30µM is the ~EC3; for Dmel\\Orco from D. melanogaster, 20µM is the ~EC10. Current responses in the presence of 10µM tryptamine were compared to the average of two preceding responses to OLC12 and are presented as mean ± SEM (n = 4-9). B–C) Tryptamine and tyramine inhibit odorant activation of heteromeric ORs from different insect species. Oocytes expressing an OR from An. gambiae (Agam\\Orco+Agam\\Or65) were activated by 100nM eugenol, oocytes expressing an OR from O. nubilalis (Onub\\Orco+Onub\\Or6) were activated by 1µM Z11-14:OAc, oocytes expressing an OR from M. caryae (Mcar\\Orco+Mcar\\Or5) were activated by 150µM 2-phenylethanol. Current responses in the presence of 10µM tryptamine (B) or 100µM tyramine (C) were compared to the preceding response to odorant alone and are presented as mean ± SEM (n = 3).\n\nA) Tryptamine competitively inhibits OLC12 activation of Agam\\Orco+Agam\\Or65. Altering the concentration of Orco agonist (OLC12) shifts the tryptamine inhibition curve. The IC50 for tryptamine inhibition of Agam\\Orco+Agam\\Or65 activation by 20µM OLC12 (2.9 ± 0.5µM, n = 3) is significantly different (p<0.0001, F-test) from the IC50 for tryptamine inhibition of Agam\\Orco+Agam\\Or65 activation by 100µM OLC12 (8.5 ± 1.1µM, n = 3). B) Tryptamine non-competitively inhibits odorant activation of Agam\\Orco+Agam\\Or65. Altering odorant (eugenol) concentration fails to shift the tryptamine inhibition curve. The IC50 values for tryptamine inhibition of responses to 10nM eugenol (3.1 ± 0.4µM, n = 4), and 100nM eugenol (3.2 ± 0.3µM, n = 3) did not differ (p=0.7172, F-test).\n\nTo measure inhibition of odorant activation of heteromeric ORs (Figures 4B, 4C, Figure 5B and Figure 6), oocytes were exposed to a 30 sec application of odorant followed by a 10 min wash. Oocytes were then exposed to a 90 sec application of tryptamine or tyramine, immediately followed by a 30 sec co-application of tryptamine or tyramine and odorant. The current response in the presence of antagonist candidate was compared to the preceding response to odorant alone and expressed as a percentage. In our previous work, we found that repeated odorant applications to some ORs could cause a progressive decrease in response amplitude31,33. For this reason, we then re-normalized antagonism data to the value obtained when the assay was run in the absence of antagonist candidate (sham). In the “sham” assay, oocytes were exposed to a 30 sec application of odorant followed by a 10 min wash and then exposed to a 90 sec application of ND96 (no antagonist candidate), immediately followed by a 30 sec application of odorant. The second odorant response was compared to the first response and expressed as a percentage. In Figure 4B, 4C, Figure 5B and Figure 6, the sham value for 100nM eugenol was 57 ± 3% (mean ± SEM, n = 3). In Figure 5B, the sham value for 10nM eugenol was 93 ± 4% (n = 4). In Figure 4B and C, the sham value for 1µM Z11-14:OAc was 82 ± 6% (n = 6) and the sham value for 150µM 2-phenylethanol was 92 ± 2% (n = 3). In Figure 6, the sham value for 3µM l-fenchone was 83 ± 1% (n = 3), the sham value for 40µM acetophenone was 92 ± 1% (n = 3), the sham value for 70µM geranyl acetate was 97 ± 1% (n = 3), the sham value for 10µM 6-methyl-5-hepten-2-one was 94 ± 2% (n = 3) and the sham value for 3µM 2-nonanone was 81 ± 1% (n = 3).\n\nCurrent responses of oocytes expressing Agam\\Orco+Agam\\Or27 (activated by 3µM L-fenchone), Agam\\Orco+Agam\\Or28 (activated by 40µM acetophenone), Agam\\Orco+Agam\\Or31 (activated by 70µM geranyl acetate), Agam\\Orco+Agam\\Or39 (activated by 10µM 6-methyl-5-hepten-2-one), Agam\\Orco+Agam\\Or48 (activated by 3µM 2-nonanone), or Agam\\Orco+Agam\\Or65 (activated by 100nM eugenol) in the presence of 10µM tryptamine (A) or 100µM tyramine (B) were compared to the preceding response to odorant alone and are presented as mean ± SEM (n = 3). Odorant structures are shown.\n\nInitial analysis of electrophysiological data was done using Clampfit 9.1 software (Molecular Devices). Curve fitting and statistical analyses were done using Prism 5 (Graphpad). Concentration-inhibition data were fit to the equation: I = Imax/(1+ (X/IC50)n) where I represents the current response at a given concentration of inhibitor, X; Imax is the maximal response in the absence of inhibitor; IC50 is the concentration of inhibitor present that still allows a half maximal response from odorant; n is the apparent Hill coefficient. Concentration-response data were fit to the equation: I = Imax/(1+(EC50/X)n) where I represents the current response at a given concentration of odorant, X; Imax is the maximal response; EC50 is the concentration of agonist yielding a half maximal response; n is the apparent Hill coefficient. Statistical significance (p<0.05) was assessed using a two-tailed unpaired t test, an F test, or a one-way analysis of variance followed by the Dunnett's post-test, as appropriate.\n\n\nResults\n\nTo screen a panel of biogenic and trace amines (Figure 1), we expressed Orco from Culex quinquefasciatus (Southern House Mosquito) in Xenopus oocytes and recorded ligand-induced current responses using two-electrode voltage clamp electrophysiology (see Methods). Orco subunits from several species, including Cqui\\Orco, have been shown to form homomeric channels when heterologously expressed in the absence of odorant-binding subunits27,33. This convenient property of Orco allowed us to perform the initial screen without potentially confounding interactions with odorant-binding subunits. Successful functional expression of Cqui\\Orco was confirmed by application of OLC12, a previously identified Orco specific agonist31. While OLC12 elicited robust current responses, none of the amines displayed agonist activity at Cqui\\Orco (Figure 2A). Next we screened the amines for antagonist activity by applying 30µM OLC12 (∼EC5) to activate Cqui\\Orco and co-applying 100µM of each amine (Figure 2B, C). Several amines were able to inhibit OLC12 activation of Cqui\\Orco. Tryptamine was the most effective antagonist, blocking more than 90% of the OLC12 response (92 ± 2% inhibition). Highly significant inhibition (p<0.001) was also observed for phenethylamine (41 ± 1%), tyramine (40 ± 5%), gramine (30 ± 4%) and serotonin 23 ± 3%), but the extent of inhibition was less than 50%, suggesting relatively low affinity interactions. Histamine (16 ± 8%), melatonin (13 ± 1%) and epinephrine (9 ± 3%) also displayed significant (p<0.01), but modest, inhibition of the OLC12 current. Octopamine, dopamine and norepinephrine were inactive in this assay.\n\nIn Figure 3A, we constructed concentration-inhibition curves for block of Cqui\\Orco activity in order to quantitatively evaluate the inhibitory potency of tryptamine, as well as phenethylamine and tyramine, representing the less effective amines. Tryptamine was clearly the most potent of these antagonists, inhibiting Cqui\\Orco with an IC50 of 4.7 ± 0.7µM, a value similar to that of the most potent synthetic Orco antagonists that we identified in our previous work33. Phenethylamine (IC50 = 117 ± 12µM) and tyramine (IC50 = 157 ± 22µM) were substantially less potent than tryptamine (25-fold and 33-fold, respectively). Previously identified Orco antagonists inhibited OLC12 activation of Orco through a competitive mechanism31,33. To determine whether tryptamine was also a competitive antagonist of Orco, we measured blockade of Cqui\\Orco achieved by tryptamine when the OLC12 concentration was increased from 30µM to 100µM (Figure 3B). Tryptamine was significantly less effective at inhibiting responses to 100µM OLC12 (IC50 = 143 ± 18µM, p<0.0001, F-test), indicating that tryptamine is a competitive antagonist of Cqui\\Orco.\n\nWe next asked whether tryptamine could also inhibit Orco from other insect species. In addition to Cqui\\Orco, we tested Agam\\Orco from An. gambiae (human malaria vector mosquito) and Dmel\\Orco from D. melanogaster. Co-application of 10µM tryptamine inhibited OLC12 activation of Orco from each of these three insect species (Figure 4A). We then wondered whether tryptamine could also inhibit odorant activation of heteromeric insect ORs containing both Orco and odorant binding subunits. We chose ORs from three insect orders: Agam\\Orco+Agam\\Or65 from An. gambiae (Order Diptera) that responds to the eugenol25; Onub\\Orco+Onub\\Or6 from O. nubilalis (European Corn Borer, Order Lepidoptera) that responds to the pheromone Z11-14:OAc45; and Mcar\\Orco+Mcar\\Or5 from M. caryae (Long-Horned Beetle, Order Coleoptera) that responds to 2-phenylethanol44. We chose to proceed with an OR from An. gambiae instead of Cx. quinquefasciatus for two reasons. The best characterized of the Cqui\\Or subunits respond to indoles23,24, which are structurally related to tryptamine and might confound our experiments. Also, the Agam\\Or subunit family has been more extensively characterized10,25, offering more options for OR expression (see below). Each odorant was applied at or near the EC50 concentration (Table 1,44,45). Co-application of 10µM tryptamine resulted in substantial inhibition of each receptor (Figure 4B). We also examined tyramine. While tyramine is a low potency Orco antagonist (Figure 3A), it is a major neurotransmitter in insects37. Tyramine was also able to reduce odorant activation of these ORs, but was less effective than tryptamine (Figure 4C). These results suggest that tryptamine and tyramine are broadly active antagonists of insect ORs.\n\nSeveral previously identified Orco antagonists have been shown to inhibit odorant activation of insect ORs through a non-competitive mechanism31–33. To determine whether the tryptamine inhibition of odorant activation that we observed in Figure 4 was also non-competitive, we examined the effect of tryptamine on activation of the heteromeric Agam\\Orco+Agam\\Or65 in more detail (Figure 5). When the concentration of Orco directed agonist (OLC12) was increased, the tryptamine inhibition curve was significantly shifted to the right (Figure 5A). However, when the concentration of odorant agonist (eugenol) was increased, the tryptamine inhibition curve did not shift (Figure 5B). These results indicate that, similar to previously identified synthetic Orco antagonist compounds, tryptamine is a competitive antagonist of direct activation of Orco and a non-competitive antagonist of odorant activation of the OR.\n\nThe ability of tryptamine to interact with Orco and exert a non-competitive inhibitory effect on odorant activation of a heteromeric OR (Figure 5) suggests that tryptamine should be able to inhibit activation of a variety of ORs activated by diverse odorants. To examine this possibility, we tested the ability of tryptamine to inhibit odorant activation of ORs formed by Agam\\Orco and each of six different odorant-binding subunits chosen from across the An. gambiae OR gene family46. We activated each OR with a previously identified cognate odorant25 at a concentration at or near the EC50 (Table 1,25). In addition to Agam\\Orco+Agam\\Or65 (activated by eugenol), we tested Agam\\Orco+Agam\\Or27 (activated by L-fenchone), Agam\\Orco+Agam\\Or28 (activated acetophenone), Agam\\Orco+Agam\\Or31 (activated by geranyl acetate), Agam\\Orco+Agam\\Or39 (activated by 6-methyl-5-hepten-2-one) and Agam\\Orco+Agam\\Or48 (activated by 2-nonanone). With the exception of Agam\\Or39 and Agam\\Or48, which display overlapping odorant specificities at 4 odorants, there is little or no similarity among the odorant specificities of these six odorant-binding subunits25. In each case, 10µM tryptamine was able to inhibit odorant activation of the receptor, despite the disparate odorant-binding subunits and diverse odorant structures (Figure 6). Tyramine was also able to inhibit odorant activation of each of these receptors, but was less effective than tryptamine (note that tyramine is applied at 100µM). We conclude that tryptamine and tyramine are general antagonists of insect ORs.\n\n\nDiscussion\n\nAnimals use a variety of biogenic and trace amines as neurotransmitters and neuromodulators. These include compounds derived from tyrosine (dopamine, norepinephrine, epinephrine, tyramine, octopamine and phenethylamine), tryptophan (serotonin, melatonin and tryptamine) and histidine (histamine)47. Dopamine and serotonin play a variety of roles in the insect nervous system48–50. In addition, insects use octopamine, histamine and tyramine as neurotransmitters48–53. Melatonin also appears to exert neuromodulatory effects in insects54,55. Interestingly, many of these amines modulate the olfactory system56–58.\n\nRecent reports27,32, together with our previous findings31,33, have revealed the existence of a ligand-binding site on the Orco subunit and that inhibition of odorant activation through a non-competitive mechanism may be a general property of Orco-directed antagonists. Our current results suggest that endogenous and exogenous natural compounds serve as Orco ligands and modulate insect olfaction. While tyramine is a major neurotransmitter in insects53, its low potency in our assay (Figure 3) suggests that it might not serve as an endogenous OR modulator. However, the function of an endogenous Orco antagonist is unlikely to be the complete block of OR function. Rather, an endogenous Orco antagonist might be used to diminish olfactory sensitivity by inhibiting a fraction of the available receptors. For tyramine, such inhibition could occur at concentrations ranging from 10µM to 30µM. Alternatively, there may be additional, more potent, but as yet uncharacterized, endogenous Orco antagonists that can decrease olfactory sensitivity at lower concentrations.\n\nIn contrast to the low potency of tyramine, we found tryptamine to be a high potency Orco antagonist. Tryptamine inhibited odorant activation of an OR with an IC50 in the low micromolar range (Figure 5). While it is currently unclear whether tryptamine is endogenous to insects, tryptamine and similar compounds, such as gramine, are produced by a variety of plants and are thought to serve as a defense against insect herbivores42,59. Various tryptamine analogs have been proposed as larvicides60 and when tryptamine is caused to accumulate in poplar and tobacco, through ectopic expression of tryptophan decarboxylase, the feeding behavior of insects that target these plants is altered41. Tryptamine-based structures also act on various receptors and transporters, particularly those involved in serotonergic neurotransmission, exerting psychedelic effects in humans. Indeed, many plant derived and synthetic hallucinogens are based on the tryptamine and phenethylamine scaffolds61–63. Interestingly, the potency that we observed for tryptamine inhibition of odorant activation of an insect OR (Figure 5) is similar to the potency for tryptamine inhibition of the D. melanogaster serotonin transporter64.\n\nMight there also be natural endogenous or exogenous Orco agonists? An endogenous Orco agonist could serve to increase olfactory sensitivity, perhaps in a circadian fashion, to alter behavior during critical foraging or mating periods. An exogenous, plant-derived Orco agonist would, by activating all ORs through Orco, serve as an olfactory “confusant” and might alter the feeding behavior of insect herbivores. The limited screen of 11 compounds that we conducted here did not identify any Orco agonists, but more extensive screening is clearly warranted.\n\nSeveral synthetic Orco antagonists have been shown to inhibit odorant activation of ORs through an allosteric mechanism31–33. The ability of these compounds to inhibit multiple ORs from a variety of species is likely due to the high conservation of Orco across the insects12. Similarly, we found that tryptamine and tyramine, acting as Orco antagonists, could inhibit odorant activation of ORs from insect species chosen from three different orders: Diptera (An. gambiae), Lepidoptera (O. nubilalis) and Coleoptera (M. caryae). Furthermore, when we examined multiple ORs from a single species (An. gambiae), we found that tryptamine and tyramine blocked odorant activation of each receptor. The action of these compounds through Orco allowed blockade to occur despite the highly diverse odorant-binding subunits used to form the receptors and the different odorant structures used to activate the receptors. Interestingly, while all six receptors were inhibited, the extent of inhibition varied depending on the odorant-binding subunit present and the pattern of variation was similar for tryptamine and tyramine. This suggests differences in allosteric coupling between Orco and the various odorant-binding subunits. Also, while we showed that tryptamine is a potent inhibitor of odorant activation of Agam\\Or65+Agam\\Orco, the results we present in Figure 6 suggest that tryptamine is even more potent at other ORs, such as those formed by Agam\\Or27, Agam\\Or31 and Agam\\Or39. Our current results with naturally occurring amines, together with previous reports with synthetic compounds27,31–33,35 strongly suggest that: 1) allosteric antagonism of odorant activation of ORs is a general property of Orco antagonists; 2) Orco antagonists are broadly active at ORs of many insect species; and 3) Orco is an important target for the development of novel insect repellants. The broad activity of Orco directed compounds across many insect species that has been observed to date suggests that these compounds may have limited agricultural utility, since both pests and pollinators could be affected. Determining whether species-specific Orco ligands can be developed will require further effort. What is clear, however, is that the pursuit of new, synthetic Orco directed ligands (both agonists and antagonists) is a promising direction for the development of new, more effective insect repellants that can aid in controlling the spread of insect-borne diseases.\n\n\nData availability\n\nfigshare: Inhibition of odorant and Orco agonist initiated current responses of oocytes expressing insect odorant receptors by various amines, doi: 10.6084/m9.figshare.97779165",
"appendix": "Author contributions\n\n\n\nSC and CWL conceived the study. SC and CWL designed the experiments. SC performed the experiments. SC and CWL analyzed the data. SC and CWL wrote the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by a grant from the National Institutes of Health (RO1 DC011091 to CWL).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe thank B. Sherman and A. Castro for Xenopus care and oocyte preparation.\n\n\nReferences\n\nKlein AM, Vaissière BE, Cane JH, et al.: Importance of pollinators in changing landscapes for world crops. Proc Biol Sci. 2007; 274(1608): 303–313. 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Publisher Full Text\n\nCarey AF, Carlson JR: Insect olfaction from model systems to disease control. Proc Natl Acad Sci U S A. 2011; 108(32): 12987–12995. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRamdya P, Benton R: Evolving olfactory systems on the fly. Trends Genet. 2010; 26(7): 307–316. PubMed Abstract | Publisher Full Text\n\nChen S, Luetje CW: Identification of new agonists and antagonists of the insect odorant receptor co-receptor subunit. PLoS One. 2012; 7(5): e36784. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJones PL, Pask GM, Romaine IM, et al.: Allosteric antagonism of insect odorant receptor ion channels. PLoS One. 2012; 7(1): e30304. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChen S, Luetje CW: Phenylthiophenecarboxamide antagonists of the olfactory receptor co-receptor subunit from a mosquito. PLoS One. 2013; 8(12): e84575. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJones WD, Nguyen TA, Kloss B, et al.: Functional conservation of an insect odorant receptor gene across 250 million years of evolution. Curr Biol. 2005; 15(4): R119–121. PubMed Abstract | Publisher Full Text\n\nBohbot JD, Dickens JC: Odorant receptor modulation: ternary paradigm for mode of action of insect repellents. Neuropharmacology. 2012; 62(5-6): 2086–2095. PubMed Abstract | Publisher Full Text\n\nBlenau W, Thamm M: Distribution of serotonin (5-HT) and its receptors in the insect brain with focus on the mushroom bodies: lessons from Drosophila melanogaster and Apis mellifera. Arthropod Struct Dev. 2011; 40(5): 381–394. PubMed Abstract | Publisher Full Text\n\nCazzamali G, Klaerke DA, Grimmelikhuijzen CJ: A new family of insect tyramine receptors. Biochem Biophys Res Commun. 2005; 338(2): 1189–1196. PubMed Abstract | Publisher Full Text\n\nNassel DR: Histamine in the brain of insects: a review. 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PubMed Abstract | Publisher Full Text\n\nMitchell RF, Hughes DT, Luetje CW, et al.: Sequencing and characterizing odorant receptors of the cerambycid beetle Megacyllene caryae. Insect Biochem Mol Biol. 2012; 42(7): 499–505. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWanner KW, Nichols AS, Allen JE, et al.: Sex pheromone receptor specificity in the European corn borer moth, Ostrinia nubilalis. PLoS One. 2010; 5(1): e8685. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHill CA, Fox AN, Pitts RJ, et al.: G protein-coupled receptors in Anopheles gambiae. Science. 2002; 298(5591): 176–178. PubMed Abstract | Publisher Full Text\n\nZucchi R, Chiellini G, Scanlan TS, et al.: Trace amine-associated receptors and their ligands. Br J Pharmacol. 2006; 149(8): 967–978. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBirmingham JT, Tauck DL: Neuromodulation in invertebrate sensory systems: from biophysics to behavior. J Exp Biol. 2003; 206(Pt 20): 3541–3546. PubMed Abstract | Publisher Full Text\n\nGrosmaitre X, Marion-Poll F, Renou M: Biogenic amines modulate olfactory receptor neurons firing activity in Mamestra brassicae. Chem Senses. 2001; 26(6): 653–661. PubMed Abstract | Publisher Full Text\n\nPerry CJ, Barron AB: Neural mechanisms of reward in insects. Annu Rev Entomol. 2013; 58: 543–562. PubMed Abstract | Publisher Full Text\n\nCole SH, Carney GE, McClung CA, et al.: Two functional but noncomplementing Drosophila tyrosine decarboxylase genes: distinct roles for neural tyramine and octopamine in female fertility. J Biol Chem. 2005; 280(15): 14948–14955. PubMed Abstract | Publisher Full Text\n\nMonastirioti M: Biogenic amine systems in the fruit fly Drosophila melanogaster. Microsc Res Tech. 1999; 45(2): 106–121. PubMed Abstract | Publisher Full Text\n\nRoeder T: Tyramine and octopamine: ruling behavior and metabolism. Annu Rev Entomol. 2005; 50: 447–477. PubMed Abstract | Publisher Full Text\n\nBembenek J, Sehadova H, Ichihara N, et al.: Day/night fluctuations in melatonin content, arylalkylamine N-acetyltransferase activity and NAT mRNA expression in the CNS peripheral tissues and hemolymph of the cockroach, Periplaneta americana. Comp Biochem Physiol B Biochem Mol Biol. 2005; 140(1): 27–36. PubMed Abstract | Publisher Full Text\n\nRichter K, Peschke E, Peschke D: Effect of melatonin on the release of prothoracicotropic hormone in the brain of Periplaneta americana (Blottodea: Blattidae). Eur J Entomol. 1999; 96: 341–345. Reference Source\n\nDacks AM, Riffell JA, Martin JP, et al.: Olfactory modulation by dopamine in the context of aversive learning. J Neurophysiol. 2012; 108(2): 539–550. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuser A, Rohwedder A, Apostolopoulou AA, et al.: The serotonergic central nervous system of the Drosophila larva: anatomy and behavioral function. PLoS One. 2012; 7(10): e47518. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhukovskaya MI: Modulation by octopamine of olfactory responses to nonpheromone odorants in the cockroach, Periplaneta americana L. Chem Senses. 2012; 37(5): 421–429. PubMed Abstract | Publisher Full Text\n\nSun XQ, Zhang MX, Yu JY, et al.: Glutathione S-transferase of brown planthoppers (Nilaparvata lugens) is essential for their adaptation to gramine-containing host plants. PLoS One. 2013; 8(5): e64026. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOliveira RR, Brito TB, Nepel A, et al.: Synthesis, Activity, and QSAR Studies of Tryptamine Derivatives on Third-instar Larvae of Aedes aegypti Linn. Med Chem. 2013. Epub ahead of print. PubMed Abstract | Publisher Full Text\n\nMcKenna DJ: Plant hallucinogens: springboards for psychotherapeutic drug discovery. Behav Brain Res. 1996; 73(1-2): 109–116. PubMed Abstract\n\nShulgin A, Shulgin A: Pihkal: A chemical love story. (Transform Press, Berkeley, California). 1991; p 978. Reference Source\n\nShulgin A, Shulgin A: Tihkal: The continuation. (Transform Press, Berkeley, California). 1997; p 804. Reference Source\n\nAdkins EM, Barker EL, Blakely RD: Interactions of tryptamine derivatives with serotonin transporter species variants implicate transmembrane domain I in substrate recognition. Mol Pharmacol. 2001; 59(3): 514–523. PubMed Abstract | Publisher Full Text\n\nChen S, Luetje CW: Inhibition of odorant and Orco agonist initiated current responses of oocytes expressing insect odorant receptors by various amines. Figshare. 2014. Data Source"
}
|
[
{
"id": "4395",
"date": "07 Apr 2014",
"name": "Joseph C. Dickens",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well-written paper that documents the discovery of modulatory effects of biogenic amines on the responses of odorant receptor assemblages (OR = odorant receptor + Orco = odorant receptor co-receptor) in insects. Both plant- and insect-produced amines are shown to antagonize responses of OR/Orco complexes to known agonists through interactions suggested to be with Orco. The potential role of the amines in regulating insect chemosensory behavior is suggested, and Orco is proposed as an important target for development of novel insect repellents.",
"responses": []
},
{
"id": "4357",
"date": "06 May 2014",
"name": "Coral Warr",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this study the authors report the identification of new antagonists for the Orco protein, a co-receptor for the ligand binding Olfactory receptor (Or) family in insects. This is of interest as they suggest Orco antagonists might be a useful approach to modifying insect behaviour. The authors screened a panel of biogenic amines and found several that functioned as Orco antagonists, of which tryptamine, naturally produced in plants, was the most effective. They further showed that this activity was conserved against Orco from a number of insect species. The paper is well written, the data is solid and well presented, and is appropriately analysed and interpreted. The caveat to the use of currently identified Orco antagonists in biocontrol, namely that they appear to affect Orco in all insects and are not species-specific, is appropriately acknowledged. My one feedback comment is that the justification for screening just amines was not really clear, and I wondered if the authors had also in fact screened other types of compounds? If so it would be valuable to other researchers to also mention any screened compounds that did not have any effect on Orco function.",
"responses": []
}
] | 1
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https://f1000research.com/articles/3-84
|
https://f1000research.com/articles/3-4/v1
|
06 Jan 14
|
{
"type": "Case Report",
"title": "Two fatal cases of melioidosis on the Thai-Myanmar border",
"authors": [
"Cindy S. Chu",
"Stuart Winearls",
"Clare Ling",
"Aung Phae Phyo",
"Warat Haohankunnathum",
"Paul Turner",
"Vanaporn Wuthiekanun",
"François Nosten",
"Cindy S. Chu",
"Stuart Winearls",
"Clare Ling",
"Aung Phae Phyo",
"Warat Haohankunnathum",
"Paul Turner",
"Vanaporn Wuthiekanun"
],
"abstract": "Melioidosis is endemic in areas of Southeast Asia, however, there are no published reports from the Thai-Myanmar border. We report the first two cases of fatal melioidosis in this region. This is of great public health importance and highlights the need to increase clinical awareness of melioidosis on the Thai-Myanmar border and to assess the true burden of disease in the area through improved case detection and Burkholderia pseudomallei prevalence studies.",
"keywords": [
"Melioidosis",
"caused by Burkholderia pseudomallei",
"is endemic in areas of Southeast Asia. Published case reports from the Thai-Myanmar border are absent and the general belief is that meliodosis does not occur in this region. Presented below are two fatal case reports suggesting that meliodosis does in fact occur and may be under-recognized and not reported."
],
"content": "Presentation\n\nMelioidosis, caused by Burkholderia pseudomallei, is endemic in areas of Southeast Asia. Published case reports from the Thai-Myanmar border are absent and the general belief is that meliodosis does not occur in this region. Presented below are two fatal case reports suggesting that meliodosis does in fact occur and may be under-recognized and not reported.\n\nThe first case of this series was a 17-year old female who presented with an intermittent fever for two months, more than 10% weight loss and confusion. She had been previously healthy. Her parents were farmers and the family lived in close proximity to the farm in eastern Myanmar along the Thailand border. On physical examination, her temperature was 40.1°C, pulse 130 bpm, respiration 28/min, and blood pressure 80/50 mmHg. In general, she was prostrate with conjunctival pallor. Her Glasgow Coma Score was 10/15. The cardiovascular and pulmonary exams were normal aside from tachycardia and tachypnea. Abdominal examination revealed a 1 cm liver and spleen. There were no other significant findings on clinical exam. Evidence from a random dextrose level test (432 mg/dL) and a urine dipstick was consistent with anorexia. A malaria smear showed Plasmodium vivax at a low parasitemia. Because of decreased consciousness, a lumbar puncture was performed and clear cerebrospinal fluid (CSF) was obtained. The CSF cell counts were normal and no organisms were grown after 48 hours of culture. Blood cultures were also sent. The patient was resuscitated with normal saline, given ceftriaxone 1 g intravenous BID and stabilized. Over the following two days, further laboratory results were obtained. A complete blood count (CBC) showed an elevated white blood count (WBC) at 13.5 103/µL with a neutrophilia, hemoglobin (Hgb) 14.1 g/dL and a normal platelet count. Chest X-ray was abnormal with a left sided consolidation, cavitation on the right side and bilateral hilar lymphadenopathy. Sputum tested for acid fast bacilli was negative. Despite the addition of gentamicin 280 mg intravenous daily and oxygen, the patient deteriorated and died more than 8 weeks after the onset of symptoms. Admission blood cultures grew Burkholderia pseudomallei after one day of incubation.\n\nThe second case was a 45-year-old male who presented with fever and weakness for two days. He also complained of chills, rigors, cough and generalized body aches. He had a history of gastric ulcer and hypertension, neither of which were currently being treated. He was a rice farmer on the eastern border of Myanmar and Thailand and had continued to work until he fell ill. He was treated with ceftriaxone 1 g intravenous daily and metronidazole 500 mg oral TID by a village health worker. At this time a malaria smear was negative and other diagnostic testing was not performed. On the sixth day of illness he had still not improved and was sent to our clinic. At initial consultation, tympanic temperature was 36°C, pulse 76 bpm, respiration 20/min, and blood pressure 100/70 mmHg. His general appearance was weak but he was ambulatory with a Glasgow Coma Score of 15/15. He had icteric sclera and pale conjunctivae. The cardiovascular, pulmonary and abdominal exams were normal. On skin examination, a 3 cm lesion that appeared like a superficial abscess was noted on the right leg. Additional history revealed that this lesion had intermittently appeared and resolved over more than two years.\n\nDuring his hospitalization, the patient developed congestive heart failure and pulmonary edema that improved with initial management. A random venous dextrose level was 110 mg/dL and field hematocrit 27%. A Urine dipstick result was abnormal with protein 2+ and blood 4+. Urine sediment was normal. Dengue rapid test was NS-1 negative, IgM and IgG positive (Standard Diagnostics, Inc, Kyonggi province, Korea). A scrub typhus rapid test for total antibody was positive (Standard Diagnostics, Inc). Stool test microscopy was negative and the stool sample was noted to be of black color. His CBC showed a Hgb 8.1 g/dL, WBC 26.2 103/µL with a neutrophilia and normal platelets. Blood culture was negative. Biochemistry was sent to the district hospital, however, the results were not available immediately. His treatment regimen included cloxacillin 1 g intravenous QID, ceftriaxone 1 g intravenous daily, doxycycline 100 mg oral BID and furosemide 40 mg oral BID. Six days after admission the biochemistry results were returned and showed markedly abnormal renal function; blood urea nitrogen (BUN) 112.5 mg% and creatinine 15.4 mg%. He was diagnosed with chronic renal failure exacerbated by dengue and scrub typhus infections. After two weeks of hospitalization, the patient’s clinical condition deteriorated and he died despite prolonged broad antibiotic coverage. A second blood culture obtained one day prior to his death grew Burkholderia pseudomallei after one day of incubation.\n\n\nDiagnosis\n\nB. pseudomallei isolates from both cases were obtained using the BacT/Alert® 3D automatic blood culture system (bioMérieux, Marcy L’Etoile, France). The isolate from case one was identified using the API 20NE test kit (bioMérieux) and confirmed with a B. pseudomallei latex agglutination test. The B. pseudomallei latex test, produced and provided by Mahidol Oxford Tropical Medicine Research Unit (MORU), is based on an anti-exopolysaccharide monoclonal antibody1. The B. pseudomallei isolate from case two was identified using the B. pseudomallei latex test and then sent to MORU for confirmation. Susceptibility testing by disc diffusion was performed at SMRU for the first isolate and at MORU for the second (Table 1). Two colony morphologies were found for case 2; one, which was susceptible to meropenem, and one with intermediate susceptibility to meropenem (bioMérieux Etest minimum inhibitory concentration of 8 μg/mL).\n\nR=resistant, S=sensitive, I=intermediate, NT=not tested\n\n\nDiscussion\n\nAt the beginning of the 20th century in Rangoon, Burma, Dr. Alfred Whitmore and his assistant Krishnaswami identified a new bacteria causing a “glanders-like illness” in humans. It was originally named Bacillus pseudomallei2,3 and renamed as Burkholderia pseudomallei in 19924. They proceeded to document over a hundred cases and the bacterium was isolated in over 5% of Rangoon’s autopsies at that time5. The disease, now termed melioidosis, was named by Stanton and Fletcher in 19326 and is characterised by severe sepsis, pneumonia, abscess formation and is associated with a high mortality7. It has become an important public health concern across Southeast Asia and northern Australia. In this region, incidence rates vary between 1.1/100000 and 412.7/1000008, and Lao PDR has recorded 400 cases between 1999 and 20109,10. This variability may be explained by the geographical preference of B. pseudomallei for specific soils11,12, the locations of the necessary containment level three laboratories for culture diagnosis7 and population risk factors that include occupation8 or medical conditions such as diabetes, renal failure and immunosuppressed states7. Little is known about the current disease burden in Myanmar and along its borders. Indeed the last published case report from within Myanmar was in 19457. A recent serological survey of new Burmese migrants to Thailand revealed that 78% were seropositive for antibodies to B. pseudomallei13. This high seropositivity rate is supported by Aung et al who found that 2% of pus specimens obtained from abscesses from patients examined in Yangon, also known as Rangoon, Myanmar, contained organisms consistent with B. pseudomallei14.\n\nThe first case described here was a healthy young female and her only risk factor was environmental exposure. The elevated dextrose on admission was attributed to glycemic dysregulation rather than from new onset diabetes. Concurrent vivax malaria infection may have caused immunosuppression enabling sepsis with B. pseudomallei. The second case was a male with environmental exposure as well as undiagnosed chronic renal disease. The first blood culture was negative, however, concurrent infection with dengue and possibly scrub typhus (the rapid test used detects IgG, IgM and IgA as total Ab and cannot differentiate acute or resolved infection) may have caused worsening immunosuppression resulting in B. pseudomallei sepsis. Both cases had risk factors and concomitant infections.\n\n\nManagement\n\nAntimicrobial therapeutic options for melioidosis are limited due to the intrinsic resistance of the organism. Current guidelines recommend parenteral ceftazidime 50 mg/kg up to 2 g every 6 hours or meropenem 25 mg/kg up to 1 g every 8 hours for at least 10–14 days (longer in severe or complicated cases) followed by oral co-trimoxazole plus doxycycline (first-line) or co-amoxiclavulanate alone (second-line) for 20 weeks. In these cases, ceftazidime or meropenem was not given due to the low suspicion for B. pseudomallei sepsis. New data supports the use of monotherapy with oral trimethoprim-sulfamethoxazole for the oral phase of treatment15. This simplifies the treatment regimen and should have a positive impact on prescribing patterns in suspected cases. Melioidosis has not been reported to be endemic in eastern Myanmar along the Thailand border, therefore, health providers may not consider it as a diagnosis in persons with sepsis. The detection of B. pseudomallei in this case series highlights the need for further epidemiologic study and case diagnosis in this region.\n\n\nConsent\n\nWritten informed consent to report these cases was obtained from the families of the patients.",
"appendix": "Author contributions\n\n\n\nCSC, SW, AAP managed the patients, CL, WH, PT, VW performed the laboratory work, all authors contributed to the discussion and writing of the report.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nAcknowledgements\n\nWe would like to acknowledge the contribution of all SMRU staff involved in the management of these two cases including clinic and laboratory staff.\n\n\nReferences\n\nWuthiekanun V, Anuntagool N, White NJ, et al.: Short report: a rapid method for the differentiation of Burkholderia pseudomallei and Burkholderia thailandensis Am J Trop Med Hyg. 2002; 66(6): 759–61. PubMed Abstract\n\nWhitmore A, Krishnaswami CS: An account of the discovery of a hitherto undescribed infective disease occurring among the population of Rangoon. Indian Med Gaz. 1912; 47: 262–7.\n\nWhitmore A: An Account of a Glanders-like Disease occurring in Rangoon. J Hyg (Lond). 1913; 13(1): 1–34.1. PubMed Abstract | Free Full Text\n\nYabuuchi E, Kosako Y, Oyaizu H, et al.: Proposal of Burkholderia gen. nov. and transfer of seven species of the genus Pseudomonas homology group II to the new genus, with the type species Burkholderia cepacia. (Palleroni and Holmes 1981) comb. nov. Microbiol Immunol. 1992; 36(12): 1251–75. PubMed Abstract | Publisher Full Text\n\nKrishnaswami CS: Morphia injectors’ septicaemia. Indian Med Gaz. 1917; 52: 296–9.\n\nStanton AT, Fletcher W: Melioidosis: Notes on a culture of B. whitmori from Saigon. Danielsson B editor. J Hyg (Lond). Cambridge University Press; 1932.\n\nWhite NJ: Meliodosis. Lancet. 2003; 361(9370): 1715–22. PubMed Abstract | Publisher Full Text\n\nLimmathurotsakul D, Kanoksil M, Wuthiekanun V, et al.: Activities of daily living associated with acquisition of melioidosis in northeast Thailand: a matched case-control study. Small PLC editor. PLoS Negl Trop Dis. Public Library of Science; 2013; 7(2): e2072. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPhetsouvanh R, Phongmany S, Newton P, et al.: Meliodosis and Pandora’s box in the Lao People's Democratic Republic. Clin Infect Dis. 2001; 32(4): 653–4. PubMed Abstract | Publisher Full Text\n\nCheng AC, Currie BJ: Melioidosis: epidemiology, pathophysiology, and management. Clin Microbiol Rev. 2005; 18(2): 383–416. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRattanavong S, Wuthiekanun V, Langla S, et al.: Randomized soil survey of the distribution of Burkholderia pseudomallei in rice fields in Laos. Appl Environ Microbiol. 2011; 77(2): 532–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVuddhakul V, Tharavichitkul P, Na-Ngam N, et al.: Epidemiology of Burkholderia pseudomallei in Thailand. Am J Trop Med Hyg. 1999; 60(3): 458–61. PubMed Abstract\n\nWuthiekanun V, Langa S, Swaddiwudhipong W, et al.: Short report: Melioidosis in Myanmar: forgotten but not gone? Am J Trop Med Hyg. 2006; 75(5): 945–6. PubMed Abstract\n\nAung MK, Mar TT: Re-emergence of melioidosis in Myanmar. Trans R Soc Trop Med Hyg. Elsevier Ltd; 2008; 102(Suppl 1): S10–S11. PubMed Abstract | Publisher Full Text\n\nChetchotisakd P, Chierakul W, Chaowagul W, et al.: Trimethoprim-sulfamethoxazole versus trimethoprim-sulfamethoxazole plus doxycycline as oral eradicative treatment for melioidosis (MERTH): a multicentre, double-blind, non-inferiority, randomised controlled trial. Lancet. 2013. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "2977",
"date": "22 Jan 2014",
"name": "Kavitha Saravu",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article certainly raises awareness about the presence of melioidosis in Thai Myanmar border and the expanding map of melioidosis globally. The two cases also reiterate the importance of timely diagnosis and appropriate chemotherapy for successful outcome. This report also underscores the fact that there may be many undiagnosed cases in the region, because of low index of suspicion by the clinicians and lack of facility for diagnosis.However, a few points could have been included as follows: Evidence from a random dextrose level test (432 mg/dL) and a urine dipstick was consistent with anorexia. This statement is not clear. It would have been better if a glycated hemoglobin value was available to rule out diabetes. Additionally, further values of plasma glucose would have been helpful. Also, the finding of the urinary dipstick is not mentioned. It is not clear if the patient was treated for vivax malaria. The course and events of the patient's hospitalization of 8 weeks has not been mentioned.",
"responses": [
{
"c_id": "681",
"date": "28 Jan 2014",
"name": "Cindy Chu",
"role": "Reader Comment",
"response": "Thank you for your useful comments. Our reply to your points are as follows:We agree that it would have been better had diabetes been ruled out. A blood sample for glycated hemoglobin was not sent to the sub-district hospitals, and was not available at our hospital. In this case, repeated random dextrose levels remained elevated with the highest measure being 520 mg/dL. The urine dipstick results were WBC 1+, ketones 3+, glucose 1+. Serum biochemistry for K+ was 3.14 mmol/L and for Na+ was 129.5 mmol/L, not consistent with dehydration. Oral chloroquine 500 mg daily was given for 2 days. The patient expired before the final dose could be given. The patient's symptoms had started approximately 8 weeks before admission. She was admitted 3 days prior to her death and the case report describes the events over those 3 days."
}
]
},
{
"id": "3595",
"date": "17 Feb 2014",
"name": "Blandine Rammaert",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nF. Nosten and colleagues report two cases of fatal melioidosis in the Myanmar border and remind the international community that this disease was discovered in Myanmar. Melioidosis is probably a forgotten public health issue in Myanmar and this article has the advantage of emphasizing it. However a description of the Myanmar environment, healthcare system and available antibiotics could help to better understand the general background around these two cases.I have a few comments on the cases: The first case illustrates a chronic pulmonary melioidosis with a fatal septic choc due to B. pseudomallei.“lived in close proximity to the farm” - Could you please reinterate the context of the Myanmar border; are there irrigated rice fields in the area? Is the patient in close contact with wet soil or any kind of water sources? “Abdominal examination revealed a 1 cm liver and spleen” - This statement is not really clear. Do you mean that there are an hepatomegaly and a splenomegaly? “died more than 8 weeks after the onset of symptoms” - It would be more helpful to have the time from hospitalization. The second case is about a fatal disseminated melioidosis from cutaneous inoculation. What is less clear is the global clinical presentation. The patient had initial pulmonary oedema. Maybe it would be helpful to discuss pulmonary melioidosis and to have the result of the chest X-ray (if it could have been done in this setting).“stool sample was noted to be of black color” - This statement doesn’t bring out any new evidence.\n\n“A second blood culture obtained one day prior to his death grew Burkholderia pseudomallei after one day of incubation.” - Does that mean that you got the result after his death? How long did it take to get the result? Do the authors think that earlier result of blood cultures could have had a positive impact on the outcome of their two patients? It could be interesting to discuss it. Do the authors have any comments on the Ashdown media, which is a cheap, specific and easy-to-make medium. Could it be implemented in the Myanmar border to help diagnosing cases? Is ceftazidime available in Myanmar?",
"responses": [
{
"c_id": "715",
"date": "24 Feb 2014",
"name": "Cindy Chu",
"role": "Author Response",
"response": "Thank you for reviewing this case series. Our reply to your comments are as follows:Response to comments on the first case:Land use along the Thailand Myanmar border is primarily agricultural. Crops that are grown are wide ranging, but most importantly, rice farming is common. All rice farms have some form of irrigation. As is custom, this patient and her family lived in a wooden home situated beside the fields of the farm where they worked and would certainly have been exposed to wet soil and natural water sources on a daily basis. “Abdominal examination revealed a 1cm liver and spleen” would mean that both the liver and spleen were enlarged and both measured 1cm. The patient's symptoms had started approximately 8 weeks before admission. She was admitted 3 days prior to her death and the case report describes the events over those 3 days.Response to comments on the second case:At our clinics, situated in a resource-limited setting, we are not able to test stool for occult blood, therefore we rely on the visual examination to determine if there is gastrointestinal bleeding. In this particular case, the presence of melanic stool would indicate either the severity of his sepsis or an underlying gastrointestinal disorder contributing to his poor clinical condition. The second blood culture result grew B. pseudomallei within 24 hours. Results were called immediately to the clinical doctor, however, the patient had already expired the day prior. Gram stain, blood culture results, and sensitivities when available are called immediately to the clinical doctor. Usually, microbiological identification is available within 24 hours of blood culture positivity. If the first blood culture had been positive then the course of events may have been different. We agree that that early recognition and confirmation of B. pseudomallei by blood culture would save lives. Ashdown media would be a suitable selective media for testing a variety of samples for B. pseudomallei, particularly from sites with normal flora and if incubated for 48 hrs. However, it must be noted that when handling B. pseudomallei cultures appropriate biosafety level three precautions must be taken, which may be a limiting factor in some areas of Myanmar. Ceftazidime is available in hospitals and pharmacies in Myanmar, however, the fee for service medical system currently present in Myanmar may limit patient access to medical care and treatment."
}
]
},
{
"id": "4028",
"date": "18 Mar 2014",
"name": "Nicholas Feasey",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have reported two cases meliodosis in an area in which this disease was not believed to occur and make an important point about paucity of diagnostic technology in the region.Abstract:“We report the first two cases of fatal melioidosis in this region...” The phrasing of this sentence implies that these are the first actual cases of fatal meliod in the region, rather than the first formally diagnosed cases. It currently implies that Meliod is a new/emerging disease to this region rather than a new diagnosis of a condition which is likely to have been present, but unrecognised. Presentation:“Presented below are two fatal case reports...” The case reports were not fatal. \"Presented below are two case reports of fatal...” would be better. The cases are a little brief on detail and if possible, it would be nice to make the corrections/additions suggested by the other two reviewers. Having said that, the weakness of the case descriptions do not detract from the message regarding the description of Meliod in this setting.Diagnosis:This is a challenging condition to diagnose and it is important to have a little more detail here – reassuring readers that this really was B. pseudomallei is the most important part of this paper. The authors should have described the colonial morphology of the pathogen as B. pseudomallei has a characteristic appearance (although I would discourage them from re-culturing it simply to answer this point unless there is a safe laboratory environment to do so). It would also have been useful to record the API20NE score (both the 7 digit number and the percentage ID).What does SMRU stand for? Lastly, what confirmatory test did MORU perform?Management:It is a little unusual that management follows the discussion. In particular, the last 2 sentences are conclusions and they should be separated and labelled as such.",
"responses": [
{
"c_id": "743",
"date": "27 Mar 2014",
"name": "Francois Nosten",
"role": "Author Response",
"response": "\"“We report the first two cases of fatal melioidosis in this region...” The phrasing of this sentence implies that these are the first actual cases of fatal meliod in the region, rather than the first formally diagnosed cases. It currently implies that Meliod is a new/emerging disease to this region rather than a new diagnosis of a condition which is likely to have been present, but unrecognised.\"It is true that there are multiple interpretations of this phrase. This report did clarify, and further stated that melioidosis is endemic in areas of Southeast Asia and points out that there are no published reports from the Thai-Myanmar border. Prior to these two cases it was thought not to occur in the region. The report also highlights the need to assess the true burden of disease in the area, which we feel will help shed light on whether this is a new, emerging or unrecognised infection in the area. \"“Presented below are two fatal case reports...” The case reports were not fatal. \"Presented below are two case reports of fatal...” would be better.\" We agree that this should be corrected. \"The cases are a little brief on detail and if possible, it would be nice to make the corrections/additions suggested by the other two reviewers. Having said that, the weakness of the case descriptions do not detract from the message regarding the description of Meliod in this setting.\"Details to the cases have been added to the updated version of the article. \"This is a challenging condition to diagnose and it is important to have a little more detail here – reassuring readers that this really was B. pseudomallei is the most important part of this paper. The authors should have described the colonial morphology of the pathogen as B. pseudomallei has a characteristic appearance (although I would discourage them from re-culturing it simply to answer this point unless there is a safe laboratory environment to do so). It would also have been useful to record the API20NE score (both the 7 digit number and the percentage ID).What does SMRU stand for? Lastly, what confirmatory test did MORU perform?\"We agree with the reviewer that it would be useful to add more detail to this section and have made changes to the diagnosis section of the manuscript. \"It is a little unusual that management follows the discussion. In particular, the last 2 sentences are conclusions and they should be separated and labelled as such.\"The initial manuscript followed an example from the journal. We agree with this comment, and will amend the manuscript to follow a more traditional layout."
}
]
}
] | 1
|
https://f1000research.com/articles/3-4
|
https://f1000research.com/articles/3-80/v1
|
31 Mar 14
|
{
"type": "Commentary",
"title": "On genomics, kin, and privacy",
"authors": [
"Amalio Telenti",
"Erman Ayday",
"Jean Pierre Hubaux",
"Erman Ayday"
],
"abstract": "The storage of greater numbers of exomes or genomes raises the question of loss of privacy for the individual and for families if genomic data are not properly protected. Access to genome data may result from a personal decision to disclose, or from gaps in protection. In either case, revealing genome data has consequences beyond the individual, as it compromises the privacy of family members. Increasing availability of genome data linked or linkable to metadata through online social networks and services adds one additional layer of complexity to the protection of genome privacy. The field of computer science and information technology offers solutions to secure genomic data so that individuals, medical personnel or researchers can access only the subset of genomic information required for healthcare or dedicated studies.",
"keywords": [
"The recent authorization of a sequencing platform for clinical use by the Food and Drug Administration will expand and accelerate the use of genetic information in medical care1. Progress is particularly impressive in the deployment of sequencing tools for neonatal diagnostics2. Commoditization of genome-wide genotyping and sequencing is happening as rapidly outside of the medical setting – prominently through companies offering “direct to consumer” (DTC) services. There is full awareness of the need to protect these data1 – while simultaneously supporting their use in research3. Here",
"we discuss how protection of genome data from medical and non-medical sources needs to be reframed considering the mutual implications of personal decision",
"online social networks and consequences to relatives."
],
"content": "Introduction\n\nThe recent authorization of a sequencing platform for clinical use by the Food and Drug Administration will expand and accelerate the use of genetic information in medical care1. Progress is particularly impressive in the deployment of sequencing tools for neonatal diagnostics2. Commoditization of genome-wide genotyping and sequencing is happening as rapidly outside of the medical setting – prominently through companies offering “direct to consumer” (DTC) services. There is full awareness of the need to protect these data1 – while simultaneously supporting their use in research3. Here, we discuss how protection of genome data from medical and non-medical sources needs to be reframed considering the mutual implications of personal decision, online social networks and consequences to relatives.\n\nParadoxically, genomics is an attractive field for individual or collective altruism – many people are willing to place their genome data in the public domain, and to actively engage in genomic research. The academic community is also calling for definitive actions to support global data-sharing3. Many research participants count on the protection of their identity. However, current strategies have proven insufficient to stop sophisticated attacks on genetic data. A recent study4 demonstrated the feasibility of re-identifying DNA donors from a public research database by using information available from popular genealogy websites. Attackers can also take advantage of gaps in the protection of other sources of data, for example census and voter lists, hospital insurance reports, and increasingly, from online social networks (see below). Genome data in the wrong hands could have undesirable consequences: from discrimination, or release of paternity, ancestry or other data that the participant did not intend to be public, to more prosaic usages such as targeted advertisements based on genome information.\n\nOnline social platforms are convenient sites for posting data but they are susceptible to “multilayer attacks”: the possibility to simultaneously aggregate data from online social networks (e.g., Facebook), health related websites (e.g., patientslikeme.com), platforms for sharing genome data (e.g., OpenSNP.org), family history resources (e.g., ancestry.com), research datasets (e.g., 1000 Genomes Project), and public records (e.g., voter registration forms) can help an attacker de-anonymize the owner of an anonymized genome and/or infer the genomic data of his/her family members. We illustrate in Figure 1A the feasibility and ease of cross-identification of a given individual across various genetic and non-genetic platforms, including the reconstitution of parts of the family pedigree.\n\n(A) Multilayer attacks using data from genomic and non-genomic platforms. An attacker can obtain the anonymized genomic data of an individual from one of the genome data websites (e.g., openSNP.org). Then, the attacker can de-anonymize the owner of the genome (i.e., learn his/her identity) by matching his/her phenotypic, demographic and administrative information (e.g., profile picture, age, gender, ZIP code) across the individual’s online social network profile. Once the individual is de-identified, the attacker can also determine his/her family members from a family history resource (e.g., ancestry.com) and infer the genomic data of family members from the individual’s retrieved genome. For example, owners of some genomes uploaded to openSNP can be de-anonymised using their Facebook profiles. For 6 individuals who publicly revealed the names of some of their relatives on Facebook, 29 familial relationships could be identified9. (B) Decrease in genomic privacy of the target person (circled in red) when the genomes of his family members are gradually revealed. The health privacy of family members can be quantified. For example, two single nucleotide polymorphisms (rs7412 and rs429358) of the Apolipoprotein E (ApoE) gene are associated with increased risk for Alzheimer's disease. The identification in several members of the pedigree of a carrier status for those risk alleles can reveal the ApoE4 status of the target person to the attacker.\n\nKin aspects of genomics were well publicized by the recent controversy regarding the public release of the genome of Henrietta Lacks (August 1, 1920 – October 4, 1951). HeLa, a cell line established from Lacks, has been used for decades in research laboratories world-wide. Recently, HeLa cells were sequenced and the genome data posted online without the consent of her relatives, who subsequently complained that this accounted to revealing private information about the family. The multilayer attacks mentioned above can reconstruct phylogenies from revealed genomes and open the door to genetic prediction of family members. The amount of kin privacy lost from such attacks can be precisely estimated (Figure 1B). As more individuals will have their genome sequenced or genotyped in coming years, the loss of privacy of family members through multilayer attacks will increase if no action is taken.\n\nThere is little doubt that genome privacy will be challenged – in particular if the medical establishment relies solely on legal deterrents and conventional protection of stored data, or if it resorts to ineffective deidentification and anonymization of genome data shared for the purpose of research. However, personal genetic tests and genomic research are possible without jeopardizing the genomic privacy of the individual or of family members. In particular, IT security provides a trove of solutions. These include using efficient cryptographic techniques for privacy-preserving personalized medicine5,6, and for genomic research7. With such approaches, genomic data are always stored in encrypted form and medical personnel or researchers can access only the subset of genomic information required for healthcare or dedicated studies. Similarly there are obfuscation-based solutions8 to use genomic data in research settings in a privacy-preserving way.\n\nSome genome researchers may be tempted to belittle the threat raised by the possible leakage of genomic data. This is a mistake, because progress in genetics is likely to make these data more and more meaningful. In addition, if it appears that genomic data are not properly protected, people could start distrusting genetics, with negative consequences for the progress of medicine. Protection needs to consider both the interest of the individual and of relatives. It is important to learn from errors in Internet security over the last decades. In that field, tools and solutions are often lagging behind threats.\n\nThe first meeting exclusively dedicated to genomic privacy took place in October 2013 at the Leibniz Center for Informatics in Dagstuhl, Germany (http://www.dagstuhl.de/13412). As one of the outcomes, the community set up a web site reporting the efforts and progress on this topic: https://genomeprivacy.org/. Notably, this site contains the list of research groups active in this field, as well as basic information to facilitate the understanding of this novel field. It is our conviction that by pooling together the skills of geneticists, law scholars, ethicists and computer scientists, we are still in time to strike an appropriate balance between accessibility to genome data and their protection.",
"appendix": "Author contributions\n\n\n\nA.T., E.A. and H.-P. H. conceived the content of the commentary and wrote the paper.\n\n\nCompeting interests\n\n\n\nThe authors hold a patent on protecting the privacy of genomic data in medical tests using cryptographic techniques.\n\n\nGrant information\n\nA.T. is funded by the Swiss National Science Foundation (SNF #141234 and CRSII3_147665).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nCollins FS, Hamburg MA: First FDA authorization for next-generation sequencer. N Engl J Med. 2013; 369(25): 2369–2371. PubMed Abstract | Publisher Full Text\n\nYang Y, Muzny DM, Reid JG, et al.: Clinical whole-exome sequencing for the diagnosis of mendelian disorders. N Engl J Med. 2013; 369(16): 1502–1511. PubMed Abstract | Publisher Full Text\n\nHayden EC: Geneticists push for global data-sharing. Nature. 2013; 498(7452): 16–17. PubMed Abstract | Publisher Full Text\n\nGymrek M, McGuire AL, Golan D, et al.: Identifying personal genomes by surname inference. Science. 2013; 339(6117): 321–324. PubMed Abstract | Publisher Full Text\n\nAyday E, Raisaro JL, Rougemont J, et al.: Protecting and evaluating genomic privacy in medical tests and personalized medicine. ACM Workshop on Privacy in the Electronic Society (WPES 2013), Berlin, Germany, 2013. Publisher Full Text\n\nBaldi P, Baronio R, De Cristofaro E, et al.: Countering GATTACA: Efficient and secure testing of fully-sequenced human genomes. ACM Conference on Computer and Communications Security (CCS), 2011. Publisher Full Text\n\nKantarcioglu M, Jiang W, Liu Y, et al.: A cryptographic approach to securely share and query genomic sequences. IEEE Trans Inf Technol Biomed. 2008; 12(5): 606–617. PubMed Abstract | Publisher Full Text\n\nJohnson A, Shmatikov V: Privacy-preserving data exploration in genome-wide association studies. ACM SIGKDD Conference on Knowledge Discovery and Data Mining (KDD), 2013. Publisher Full Text\n\nHumbert M, Ayday E, Hubaux JP, et al.: Addressing the concerns of the lacks family: quantification of kin genomic privacy. ACM Conference on Computer and Communications Security (CCS), 2013. Publisher Full Text"
}
|
[
{
"id": "4304",
"date": "14 Apr 2014",
"name": "XiaoFeng Wang",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper discusses the challenge of protecting human genome data, particularly its unique feature in that one’s DNA data can be used to infer the private health information of those genetically related to them. The authors talk about the conflict between the perception that the decision on releasing one’s DNA materials is personal, and how it can actually impact on the privacy of their kin. They further sketch a technique for quantifying such an information leak, and demonstrate that the threat is realistic, given the de-anonymization attack that can happen through the booming online social networks. I feel that this article provides useful information for raising the awareness of the uniqueness and significance of genome privacy. This, hopefully, will lead to a broad, in-depth conversation among genomics researchers, security and privacy researchers, bioethics experts, genomics industry, policy makers and the public on how to effectively regulate the dissemination of human DNA data to facilitate scientific research, without undermining DNA donors’ privacy and well-being.",
"responses": []
},
{
"id": "5889",
"date": "29 Aug 2014",
"name": "Xiaoqian Jiang",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a timely commentary on privacy, kin, and genomics. Today, many gene donators are still ignorant of the potential impact of information leakage to the family when their genome data are made public. This problem is becoming more critical as the younger generation reveals themselves and family members on online social networks and open family history resources made it possible to link individuals. I found this topic to be extremely important.",
"responses": []
},
{
"id": "5895",
"date": "29 Aug 2014",
"name": "Florian Kerschbaum",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article raises a very important issue: the difficulty of providing privacy for genetic information in the light of inheritance. I cannot stress enough how important this aspect is, since it requires data protection measures, such as the mentioned cryptographic or obfuscation-based approaches, to be utterly restrictive. The article gives clear examples where -- intentionally or unintentionally -- leaked information allowed harmful inferences. The authors combine information from public genomes and social networks to infer information about people who have not released any information about their genes. These examples should be taken very, very seriously, since they are raised only at the very beginning of the scientific development. As the paper argues we can expect the use of genomics to significantly grow. Genetic information can prove at least as harmful as location information provided by cell phones, but it is impossible (for now) to change it. It is therefore a scientific and societal challenge to protect genomic information much better than we protect our information in current telecommunication networks. The article references excellent works from the computer security community that can certainly provide a guiding direction, but even these mechanisms need to necessarily leak some information about the genomes. I hope that medical and computer science researchers will take the challenge described by the article seriously and look for mechanisms that control the entire information in all medical or non-medical information system based on direct or indirect genomic data.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-80
|
https://f1000research.com/articles/2-220/v1
|
18 Oct 13
|
{
"type": "Research Article",
"title": "The Human dsRNA binding protein PACT is unable to functionally substitute for the Drosophila dsRNA binding protein R2D2",
"authors": [
"Benjamin K Dickerman",
"Jocelyn A McDonald",
"Ganes C Sen",
"Benjamin K Dickerman",
"Jocelyn A McDonald"
],
"abstract": "The primary function of the dsRNA binding protein (dsRBP) PACT/RAX is to activate the dsRNA dependent protein kinase PKR in response to stress signals. Additionally, it has been identified as a component of the small RNA processing pathway. A role for PACT/RAX in this pathway represents an important interplay between two modes of post-transcriptional gene regulation. The function of PACT/RAX in this context is poorly understood. Thus, additional models are required to clarify the mechanism by which PACT/RAX functions. In this study, Drosophila melanogaster was employed to identify functionally orthologous dsRNA-binding proteins. Transgenic Drosophila expressing human PACT were generated to determine whether PACT is capable of functionally substituting for the Drosophila dsRBP R2D2, which has a well-defined role in small RNA biogenesis. Results presented here indicate that PACT is unable to substitute for R2D2 at the whole organism level.",
"keywords": [
"The dsRNA binding protein (dsRBP) PACT (referred to as RAX in the mouse) activates the dsRNA dependent protein kinase PKR in response to various stress signals1–5. This in turn results in repression of translation through phosphorylation of the eukaryotic translation initiation factor eIF2α6. Although the biochemical function of PACT/RAX as an activator of PKR is well established",
"the physiological role of this protein in vivo is unclear. Indeed",
"generation of mice lacking RAX revealed a role for this protein in craniofacial7",
"8 and anterior pituitary development9 that is not readily explained by current knowledge of its biochemical function. More recently",
"PACT/RAX has been identified as a component of the RNA silencing (siRNA/miRNA) pathway10",
"11",
"although a detailed understanding of its role in this pathway is limited. Thus",
"PACT/RAX functions in two independent pathways that govern post-transcriptional gene regulation and as such it is important to understand the overall physiological function of this protein."
],
"content": "Introduction\n\nThe dsRNA binding protein (dsRBP) PACT (referred to as RAX in the mouse) activates the dsRNA dependent protein kinase PKR in response to various stress signals1–5. This in turn results in repression of translation through phosphorylation of the eukaryotic translation initiation factor eIF2α6. Although the biochemical function of PACT/RAX as an activator of PKR is well established, the physiological role of this protein in vivo is unclear. Indeed, generation of mice lacking RAX revealed a role for this protein in craniofacial7,8 and anterior pituitary development9 that is not readily explained by current knowledge of its biochemical function. More recently, PACT/RAX has been identified as a component of the RNA silencing (siRNA/miRNA) pathway10,11, although a detailed understanding of its role in this pathway is limited. Thus, PACT/RAX functions in two independent pathways that govern post-transcriptional gene regulation and as such it is important to understand the overall physiological function of this protein.\n\nRNA silencing through the micro-RNA (miRNA) and short-interfering RNA (siRNA) pathways regulates nearly every aspect of cellular biology (reviewed by van Kouwenhove et al.12,13). Both siRNA and miRNA pathways require cytoplasmic processing of precursor molecules (either dsRNA or ~70bp pre-miRNAs) to generate mature guide RNAs (reviewed by van Kouwenhove et al.12). In both cases, this cytoplasmic processing step is accomplished by the endonuclease Dicer in complex with one or more dsRNA binding proteins, which cleaves precursor RNA molecules into ~20–25bp dsRNA. One of the two strands of the Dicer-processed mature RNA molecule (known as the guide strand) is then loaded into the effector complex, the RNA-induced silencing complex (RISC) (reviewed by Czech and Hannon13). Although the precise composition of the RISC is unclear, various studies have demonstrated the presence of a mature guide RNA, Dicer, one or more accessory dsRBPs, and one of the four members of the Argonaute protein family (Ago1–4) which function as the effector enzymes in the terminal silencing step14.\n\nThe RNA silencing pathways of metazoans have largely been investigated in the model organisms Caenorhabditis elegans and Drosophila melanogaster. In both systems, it was discovered that the endonucleases required for cleavage of pri-miRNAs (Drosha) and pre-miRNAs/siRNA precursors (Dicer) form complexes with specific dsRBPs. These proteins, like PACT/RAX, are comprised of tandem dsRBDs with no other identifiable domains (Drosha complexes with Pasha/DGCR815, C. elegans Dicer binds RDE-416, D. melanogaster Dicer-1 binds Loquacious (LOQS)17 and D. melanogaster Dicer-2 binds R2D218). Furthermore, these dsRBPs are required for the processing functions of the complexes. In mammalian systems, processing of both siRNA and miRNA is performed by a common Dicer enzyme. Both PACT/RAX and a similar dsRBP Tat Trans-activation element RNA binding protein (TRBP) in human/Protamine-1 RNA binding protein (PRBP) in mouse have been demonstrated to bind Dicer through either their N-terminal dsRNA binding motifs or their C-terminal Merlin-Dicer-PACT Liaison (Medipal) domain (which contains the PKR activation domain of PACT/RAX)10,11,19. The binding of PACT, TRBP or both with Dicer enhances the ability of Dicer to process dsRNA into siRNAs in vitro10. These findings suggest a functional significance to the PACT/TRBP/Dicer interactions, although the mechanism by which this occurs is unclear. These reports suggest that in addition to the regulating PKR activation, PACT/RAX may affect post-transcriptional control via small RNA pathways.\n\nIn Drosophila, processing of small RNA precursors is accomplished by separate enzymes, Dicer-1 for miRNA and Dicer-2 for siRNA20. The dsRNA binding proteins LOQS17 and R2D218 bind Dicer-1 to Dicer-2 respectively. LOQS encodes four isoforms; LOQS-PA and LOQS-PB contain three tandem dsRBDs whereas both LOQS-PC and LOQS-PD21,22 lack the third C-terminal dsRBD. R2D2 encodes a single isoform that is comprised of two dsRBDs18 (Figure 1). LOQS and R2D2 sort precursor RNA molecules based on the presence or absence of mismatches in the stemloop of the hairpin or dsRNA precursor to the appropriate Dicer processing pathway23 and facilitate RISC loading24,25. Within dsRBDs there is a high degree of sequence similarity (71–78% similarity, see Table 1) between PACT and LOQS PA and PB (which have all three dsRBDs in common). This sequence similarity coupled with the tandem dsRBD architecture (see Figure 1 and Table 1) indicates potential functional orthology between PACT and LOQS. Although R2D2 is significantly less similar to PACT (46% sequence similarity within dsRBD2, with no detectable similarity within dsRBD1), it has nonetheless been demonstrated to bind Dicer-218 and possesses a tandem dsRBD architecture similar to PACT and LOQS and thus may also function orthologously to PACT/RAX.\n\nAmino acid sequence identity and similarity (positives) between dsRNA binding domains of PACT and the Drosophila dsRNA binding proteins determined by BLAST (blastp) alignment; ~ indicates no detectable similarity. The three dsRBDs shown for LOQS correspond to the shared dsRBDs of PA and PB isoforms; the PC and PD isoforms share the first and second but lack the third dsRBD.\n\nSchematic diagram of the PACT, LOQS and R2D2 proteins depicting the organization of their dsRBD domains. Dashed lines designate dsRBD1, 2 or 3.\n\nPrevious studies have produced mutant D. melanogaster lines lacking either of the dsRBPs that interact with Dicer-1 and Dicer-2. Flies deficient in all four isoforms of LOQS display developmental lethality, and do not survive past the pupal stage26. In contrast, flies homozygous mutant for a null allele of r2d2 are semi-lethal but the surviving flies have severe fertility defects27. The fertility phenotype results from defective oogenesis. Specifically, loss of R2D2 disrupts formation of the stalk cells, which normally separate individual follicles from each other28. This phenotype is enhanced by a dicer-1 mutant, indicating an unexpected interaction between R2D2 and Dicer-1 rather than Dicer-227.\n\nWe hypothesized that one or more dsRNA binding proteins with known function in D. melanogaster are functionally orthologous to PACT/RAX, and that identification of the functional ortholog(s) would provide insight into the small RNA processing function of PACT/RAX. We sought to identify proteins orthologous to PACT/RAX by transgenically expressing human PACT in the well-characterized Drosophila genetic model system. We report here our efforts to determine the degree to which human PACT is a functional ortholog of Drosophila R2D2.\n\n\nMaterials and methods\n\nHeLa cells (ATCC CCL-2) were maintained in Dulbecco's modified Eagle's medium (DMEM) containing glucose (4.5g/L), penicillin (50U/ml), streptomycin (50µg/ml), L-glutamine (2mM) and sodium pyruvate (1mM) (Cleveland Clinic Lerner Research Institute Media Preparation Core) supplemented with 10% fetal bovine serum (Atlanta Biologicals) at 37°C in 5% CO2. The pUASP vector was obtained from Drosophila Genomics Resource Center. A polyclonal rabbit antibody raised against full length PACT (produced in-house)29 was used at 1:8000 and was detected using 1:10000 horseradish peroxidase (HRP) conjugated polyclonal goat anti-rabbit secondary (Rockland Immunochemicals # 611-103-122).\n\nTo obtain PACT cDNA, total RNA was extracted from HeLa cells using Trizol (Invitrogen) according to the manufacturer’s instructions, followed by DNAse treatment using the DNA-free kit (Ambion). Extracted, DNase-treated RNA was then reverse transcribed with the Superscript III system (Invitrogen) using random hexamers. The resulting cDNA was used to amplify the coding sequence of PACT by PCR using the primers 5´BamHI-PACT(UAS) (AAG GAT CCA AAC ATG TCC CAG AGC AGG CAC C) and 3´XbaI-PACT (GGC GGA TCC TTA CTT TCT TTC TGC TAT TAT CTT TAA ATA C). The resulting product was digested with BamHI (New England Biolabs Inc. #R0136) and XbaI (New England Biolabs Inc. #R0145) and ligated into pUASP to generate pUASP-PACT. The full-length Drosophila r2d2 cDNA was amplified by PCR from a plasmid obtained from Drosophila Genome Research Center (cDNA #LD06392) using 5´BamHI-R2D2(UAS) (AAG GAT CCA AAC ATG GAT AAC AAG TCA GCC GTA TC) and 3´XbaI-R2D2 (AAA TCT AGA TTA AAT CAA CAT GGT GCG AAA ATA GTC TAT TAT ATG G). The resulting product was digested with BamHI and XbaI and ligated into pUASP to generate pUASP-R2D2. Final cloned plasmids were sequence verified using the primers listed above by the Cleveland Clinic Lerner Research Institute Genomics Core.\n\nProtein was isolated by homogenizing individual adult flies in Triton X-100 lysis buffer (20mM Tris-HCl pH 7.5, 150mM NaCl, 1% Triton X-100, 1mM EDTA, 5mM 2-mercaptoethanol, 10% glycerol, supplemented with Complete protease inhibitor and PhoSTOP (Roche)). Protein was separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane (Immobilon, Millpore) for western blot analysis. Western blots were visualized using enhanced chemiluminescence (ECL) detection reagents (GE Healthcare Lifesciences #RPN2106).\n\nFlies were maintained and crossed at 25°C according to standard protocols30. Fly food was prepared according to the standard recipe from Bloomington Drosophila Stock Center with minor modifications (83.3ml/L molasses, 33.75g/L yeast, 97.58g/L cornmeal, 31g/L agar, 15.67ml/L Tegosept, all purchased from Genesee Scientific). Transgenic UAS-PACT and UAS-r2d2 flies were generated31 by Model Systems Genomics (Duke University, Durham, NC) using the plasmids pUASP-PACT and pUASP-R2D2 described above. The following fly strains were obtained from the Bloomington Drosophila Stock Center: w1118, r2d21/CyO (a null allele;27) and Df(2L)BSC142/CyO. Tubulin (tub)-GAL4 (insert on X) was a gift of Dr. A. Page-McCaw. Homozygous viable r2d21 flies were only obtained infrequently, suggesting that the stock acquired a second-site lethal mutation. Therefore, all experiments were performed by outcrossing r2d21 to the deficiency Df(2L)BSC142 (deletion of 28C3; 28D3, including the r2d2 gene at 28C4). Standard genetic methods were used to construct the following genotypes:1 tub-Gal4; r2d21/CyO;2 Df(2L)BSC142/CyO; UAS-PACT; and3 Df(2L)BSC142/CyO; UAS-r2d2.\n\nFemales of the genotype tub-GAL4; r2d21/Df(2L)BSC142; +/UAS-x (where x is PACT or r2d2) were outcrossed with w1118 males for fertility tests. Crosses to test fertility were established in vials of food for 24 hours prior to transferring flies to an egg collection chamber. Females were allowed to lay eggs on grape juice agar plates (Genesee Scientific, #47–102) for 1 hr prior to collecting the plate, and incubating at 25°C for 24 hours to allow eggs to hatch. The total number of eggs laid was determined by counting the number of larvae and the number of unhatched eggs. Hatch rate was calculated as the number of larvae divided by the number of total eggs laid. Student’s t-test of two independent experiments (defining P < 0.05 as statistically significant) was performed using Graphpad Prism version 6.02 software package (Graphpad Software).\n\n\nResults\n\nWe reasoned that the genetically tractable Drosophila model system could be used to study the in vivo functions of PACT. Because there is no known PKR ortholog in Drosophila, this also allows the role of PACT in small RNA processing to be investigated independent of PKR activation. Moreover, the well-characterized siRNA and miRNA processing pathways and availability of loqs and r2d2 mutant alleles make this an attractive system to study the role of PACT in the small RNA pathways. Various human proteins have been used to rescue Drosophila mutant phenotypes in order to identify homologous and orthologous proteins32–34, therefore to identify proteins orthologous to PACT, a human PACT transgene was constructed and introduced into Drosophila. This transgenic line was used to determine whether PACT can substitute for Drosophila dsRNA binding proteins that regulate small RNA processing (see below).\n\nHuman PACT-expressing D. melanogaster were created using the Gal4-UAS bipartite expression system35. This system allows the generation of flies containing transgenes of interest under the transcriptional control of the UAS element. These lines then can be crossed to a number of Gal4-driver flies to tailor transgene expression to the needs of a specific experiment. The full-length coding region of the Human PACT gene was cloned into the pUASP vector under the transcriptional control of the UASp element, a modified form of UAS that permits expression in both somatic and germline cells36. Several UAS-PACT transformants were obtained and their chromosomal insertions mapped. A stock that carried an insert on chromosome 3 was retained and crossed to the ubiquitous tub-GAL4 driver.\n\nWe next tested whether Human PACT was expressed at detectable levels in vivo. Flies expressing tub-Gal4 alone, containing the UAS-PACT transgene alone, and flies with tub-Gal4 driving UAS-PACT were analyzed by western blot for PACT expression in whole flies. Using an antibody that recognizes Human PACT29, we detected a protein band of the expected size (~34 kDa) in tub-GAL4; UAS-PACT flies but not in the individual parental lines (Figure 2). Importantly, because we obtained viable tub-GAL4; UAS-PACT adult flies, this experiment also demonstrates that ubiquitous transgenic expression of PACT does not impair Drosophila development.\n\nWestern blot analysis of total protein isolated from whole flies of the indicated genotypes. Flies carrying both the tub-Gal4 and UAS-PACT elements show a protein band of expected size for PACT that is not present in either stock alone; a non-specific band is indicated (*). Arrow denotes predicted molecular weight of Human PACT (34 kDa). Additional bands of smaller size likely represent degradation products.\n\nThe focus of the work presented here is on PACT rescue of the r2d2 loss-of-function mutant phenotype. Investigation of the functional substitution between human PACT and Drosophila LOQS was investigated in a separate project in our lab and will be reported elsewhere.\n\nFlies mutant for r2d2 have reduced fertility27. Therefore, we sought to determine the extent to which PACT can suppress r2d2 mutant fertility defects. We performed fertility tests using a standard egg-laying and hatching assay. Crosses were performed to obtain female progeny that carried the r2d21 allele over a deficiency that removes the r2d2 genetic locus (i.e. r2d2–/–), and simultaneously carried tub-GAL4 driving either UAS-PACT or UAS-r2d2 (Figure 3A; see Materials and methods for details). These female F1 progeny were then outcrossed to w1118 males to test for fertility; a cross of w1118 females to w1118 males was used as the wild-type control. Eggs resulting from these outcrosses were collected on grape juice agar plates, and allowed to hatch for 24 hours at 25°C. The total number of eggs produced and the number of hatched larva were counted for each cross and hatch rate was calculated as the number of hatched larva divided by the total number of eggs (Figure 3B & 3C). Flies heterozygous for r2d2 (either r2d21/+ or Df(2L)BSC142/+) produced eggs and hatch rates equivalent to the wild-type control. Notably, expression of either UAS-PACT or UAS-r2d2 in r2d2 heterozygous flies did not influence fertility or hatch rates. Consistent with a previous report27, flies homozygous mutant for r2d2 had a strong reduction in both the total number of eggs laid and the hatch rate compared to wild-type (Figure 3). We observed a rescue of both total number of eggs (p=0.0512) and hatch rate (p=0.0671) in r2d2 mutant flies by ubiquitous (tub-GAL4) transgenic expression of UAS-r2d2 that is approaching statistical significance and consistent with previous reports27. In contrast, ubiquitous expression of UAS-PACT did not suppress the homozygous r2d2 mutant fertility defects; the hatch rate and number of eggs laid was equivalent to the r2d2 mutants alone (Figure 3). Despite a documented interaction between PACT and the mammalian small RNA processing machinery and similar domain architecture between these proteins, these results demonstrate that Human PACT is not sufficient to rescue the R2D2 deficient phenotype.\n\n(A) Schematic representation of the experimental cross used to test fertility. UAS-x refers to the transgene shown in (B) and (C), for control crosses with no transgene the genotype Df(2L)BSC142/CyO; UAS-x was replaced with DF(2L)BSC142/CyO. (B) Calculated hatch rate of eggs produced by wild-type (w1118) flies, flies with one copy of r2d2 (+/-; either r2d21/CyO or Df(2L)BSC142/CyO) and flies lacking both copies of r2d2 (-/-, r2d21/Df(2L)BSC142) and either expressing no UAS-transgene or expressing UAS-PACT or UAS-r2d2; these flies were outcrossed to w1118 males for the fertility tests. (C) Total number of eggs laid from the crosses depicted in (B). Graphs show mean ± SEM of two independent experiments, p-values were calculated by Student’s t-test.\n\n\nDiscussion\n\nIdentification of PACT/RAX as a component of the small RNA processing pathway implicates this protein in multiple pathways of post-transcriptional gene regulation through general inhibition of translation by PKR and target-specific regulation by siRNA/miRNA. As such it is important to determine the precise nature of PACT/RAX’s involvement in this pathway in order to understand the overall function of PACT/RAX in post-transcriptional gene regulation, and the physiological effects of the protein. Here, we investigated the role of PACT/RAX in RNA silencing by attempting to identify orthologous proteins in D. melanogaster. Human PACT was introduced into Drosophila to determine whether it could substitute for R2D2, which has a well characterized function in RNA silencing. Expression of PACT was unable to rescue the phenotypes of flies lacking R2D2. These results suggest that the Dicer-associated dsRBPs PACT and R2D2 are not functionally orthologous.\n\nWhile our study demonstrates that Human PACT cannot substitute for R2D2 function in flies, there are a number of important caveats to the results described here. Despite the lack of rescue, it is nonetheless possible that PACT plays the same (or a similar) role in mammalian cells as R2D2 plays in Drosophila cells. One possibility could be that PACT protein is sufficiently different from R2D2 that it cannot interact in vivo with Drosophila partner proteins that are necessary for R2D2 function. Alternatively, R2D2 may be required to bind specific RNA precursor molecules that PACT is unable to bind (or has a lower affinity for). Investigation of biochemical interactions between PACT and Drosophila RNA silencing proteins such as Dicer-1 and Dicer-2 and a biochemical comparison of the specific RNA binding functions of these proteins would begin to address these issues.\n\nIn addition to functional discrepancies between these proteins, there may be other potential explanations for these results that would not rule out orthologous function between PACT and R2D2. Although less likely, the lack of PACT rescue of the r2d2 mutant may be due to inherent difficulty in controlling the tissue-specificity and relative levels of transgene expression in vivo. In the case of R2D2, expression in the stalk and follicle cells of the ovary is required for egg development27. Expression of UAS-PACT by tub-GAL4 in whole flies was validated by western blot; however, this does not provide information about transgenic expression of PACT in the appropriate tissues. Thus, even though PACT expression was driven by a ubiquitous GAL4 driver we cannot rule out insufficiently high expression of PACT in the relevant cell types. Expression of UAS-R2D2 using the same tub-GAL4 driver however rescued the r2d2 loss-of-function fertility defect. Although this result provides some degree of confidence in the expression levels, the genomic context of the P-element insertion and stability of transgenically-produced RNA and protein can all contribute to expression variability in vivo. As such, it still remains a possibility that PACT was not expressed as highly as R2D2 using the same transgenic expression system, and thus PACT may not have rescued fertility due to insufficient expression rather than a lack of orthologous function. Additional experiments investigating tissue-specific expression levels of these proteins as well as using tissue-specific GAL4 drivers would begin to address these concerns.\n\nWhile the complementation experiments described here indicate that PACT is unable to functionally substitute for R2D2, there are complications to the interpretation of these experiments. Further technical refinements will be necessary to definitively demonstrate or rule out functional orthology between PACT/RAX and R2D2. It also remains a distinct possibility that PACT/RAX is instead orthologous to LOQS, a topic that we are interested in investigating further in the future.",
"appendix": "Author contributions\n\n\n\nBKD contributed to the conception, design, execution, analysis and interpretation of the experiments detailed in this manuscript, as well as drafting and revising the manuscript. JAM contributed to the conception, design, analysis and interpretation of the experiments detailed in this manuscript, as well as revising the manuscript. GCS contributed to the conception, design, analysis and interpretation of the experiments detailed in this manuscript, as well as revising the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by NIH grant CA062220, awarded to G. Sen.\n\n\nAcknowledgments\n\nWe wish to thank George Aranjuez for technical assistance with Drosophila work.\n\n\nSupplementary materials\n\n\n\n\nReferences\n\nPatel RC, Sen GC: PACT, a protein activator of the interferon-induced protein kinase, PKR. EMBO J. 1998; 17(15): 4379–4390. 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}
|
[
{
"id": "2676",
"date": "06 Dec 2013",
"name": "Anthony Sadler",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a useful study that is well executed and that makes a valuable contribution. The results are clearly described. An attempt to address the acknowledged uncertainties in this study, by measuring the levels of PACT in the critical tissues, comparison of the levels of expression of PACT to the transgenic R2D2, detecting an association between human PACT and Drosophila Dicer-2, and/or otherwise confirming function of the PACT transgene would have increased confidence in the conclusion. These measures would be less germane if the parallel experiment to rescue the function of Drosophila LOQS, which is the more likely homolog for PACT, had demonstrated the functionality of the PACT transgene. Perhaps a sentence could be introduced into the discussion that these separate experiments validated the PACT transgene functioned in Drosophila - if this was the case. I have a number of very minor criticisms that might improve the manuscript. The statement in the abstract that the primary function of PACT is to activate PKR is not accurate. As the authors themselves state the relative physiological consequence of PACT, in small RNA biogenesis or protein translation, is uncertain. This sentence might be better expressed by, for instance, writing PACT was first described to function by activating PKR. The sentence in the abstract stating that additional models are required to clarify the function of PACT does not best justify the study approach. Alternatively, describing the utility of Drosophila to decipher small RNA biogenesis would seem more appropriate. It might improve the clarity of the text if the authors selected a simpler abbreviation, rather than PACT/RAX, such as either PACT or RAX.",
"responses": [
{
"c_id": "749",
"date": "03 Apr 2014",
"name": "Benjamin Dickerman",
"role": "Author Response",
"response": "We agree that inclusion of LOQS rescue data would strengthen the arguments presented in this manuscript. Despite repeated efforts, and likely due to technical limitations, Drosophila LOQS (UAS-LOQS-PB) was unable to rescue loqs loss-of-function mutations in our hands. This important control has been previously reported (Park et al., 2007) to be sufficient to rescue the loqs loss-of-function mutation that was used in this study. Because these rescue studies were inconclusive, we are currently unable to confirm that LOQS and PACT are functional orthologous.We have addressed the remaining comments presented here in a revised manuscript. The abstract has been revised to clarify stress-induced PKR activation as the first described rather than the primary function of PACT, as well as to highlight the genetic utility of Drosophila as a model system, rather than merely suggesting that additional model systems are required. To address the final comment regarding PACT nomenclature, the manuscript has been revised for clarity by changing PACT/RAX to PACT unless specifically referring to the mouse protein RAX."
}
]
},
{
"id": "2387",
"date": "18 Feb 2014",
"name": "Anita Corbett",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript by Dickerman et al. describes an attempt to study the functional conservation of RNA binding proteins implicated in processing of small RNAs and activation of dsRNA-dependent protein kinase (PKR). The work exploits a Drosophila model where PKR is not relevant thus allowing focus on the small RNA processing pathways. The approach is logical to try to rescue a fertility phenotype observed for the Drosophila protein (R2D2) with transgenic expression of a related human protein, PACT. Unfortunately, the authors were not able to demonstrate rescue and thus are left with a negative result. The data presented do support the conclusions drawn, that no rescue could be detected. There are a number of caveats for this result as the authors describe in the Discussion. This is a reasonable contribution to the literature in that it would prevent other researchers from wasting time undertaking the identical experiment. The data presented is robust and convincing. I would suggest some modifications to the text for clarification: The authors begin by describing both related human proteins, PACT and the isoforms of LOQS. While it is logical to describe the family of proteins, this study actually only analyzes the PACT protein. This point should be clearly communicated earlier in the Introduction to the study not just as the final sentence of the Introduction. The section in the Introduction describing Figure 1 is very convoluted and really could be re-written to be clearer. An example with grammatical errors is the second sentence of this paragraph, which reads: “The dsRNA binding proteins LOQS and R2D2 bind Dicer-1 to Dicer-2 respectively.” I think what the authors mean to say is: “The dsRNA binding proteins, LOQS and R2D2, bind TO Dicer-1 AND Dicer-2, respectively.” The description of the proteins really needs to be rewritten for clarification. If the authors could perform an additional experiment to address one of the caveats that they mention, ideally they could compare the expression levels for the transgenic human and fly proteins. Such a direct comparison would really only be possible if the proteins are tagged with an epitope tag. Given the number of caveats for the study presented here, the real value as described above is simply to communicate that these experiments have been attempted using this approach.",
"responses": [
{
"c_id": "750",
"date": "03 Apr 2014",
"name": "Benjamin Dickerman",
"role": "Author Response",
"response": "We have addressed the comments presented in a revised manuscript. A sentence reading “To this end, we investigated the functional orthology between PACT and the Drosophila melanogaster dsRBP R2D2.” has been added to the end of the first paragraph of the introduction, to highlight the focus of this manuscript before describing the family of dsRBPs. The paragraph describing figure 1 has been reordered and edited for clarity, and to address the specific grammatical error mentioned by the reviewer. A sentence reading “This issue could be addressed in future experiments by expressing epitope-tagged R2D2 or PACT in r2d2 mutant flies to correlate rescue of the r2d2 deficient phenotype with a direct comparison of protein expression level.” has been added to the relevant paragraph of the discussion section suggesting this experiment as part of future studies. This has been referenced as a comment by the reviewer to credit the suggestion."
}
]
}
] | 1
|
https://f1000research.com/articles/2-220
|
https://f1000research.com/articles/3-77/v1
|
26 Mar 14
|
{
"type": "Short Research Article",
"title": "Prevalence of primary outcome changes in clinical trials registered on ClinicalTrials.gov: a cross-sectional study",
"authors": [
"Sreeram V. Ramagopalan",
"Andrew P. Skingsley",
"Lahiru Handunnetthi",
"Michelle Klingel",
"Daniel Magnus",
"Julia Pakpoor",
"Ben Goldacre",
"Andrew P. Skingsley",
"Lahiru Handunnetthi",
"Michelle Klingel",
"Daniel Magnus",
"Julia Pakpoor"
],
"abstract": "Background: An important principle in the good conduct of clinical trials is that a summary of the trial protocol, with a pre-defined primary outcome, should be freely available before the study commences. The clinical trials registry ClinicalTrials.gov provides one method of doing this, and once the trial is registered, any changes made to the primary outcome are documented. The objectives of this study were: to assess the proportion of registered trials on ClinicalTrials.gov that had the primary outcome changed; to assess when the primary outcome was changed in relation to the listed study start and end dates and to assess whether the primary outcome change had any relation to the study sponsor.Methods: A cross-sectional analysis of all interventional clinical trials registered on ClinicalTrials.gov as of 25 October 2012 was performed. The main outcome was any change made to the initially listed primary outcome and the time of the change in relation to the trial start and end date.Findings: Our analysis showed that 28229 of 89204 (31.7%) registered studies had their primary outcome changed. Industry funding was associated with all primary outcome changes, odds ratio (OR)= 1.36, 95% confidence interval (CI)=1.31-1.41, p<0.001; with primary outcome changes after study start date OR=1.37, 95% CI=1.32-1.42, p<0.001; with primary outcome changes after primary completion date OR=1.84, 95% CI=1.75-1.94, p<0.001 and with primary outcome changes after study completion date OR=1.82, 95% CI=1.73-1.91, p<0.001. Conclusions A significant proportion of interventional trials registered on ClinicalTrials.gov have their primary outcomes altered after the listed study start and completion dates. These changes are associated with funding source.",
"keywords": [
"Clinical trials provide the principal method with which to assess the effectiveness of therapeutic strategies1. An important principle in the good conduct of clinical trials is that a summary of the trial protocol",
"with a pre-defined primary outcome",
"should be freely available before the study commences1. In February 2000",
"the United States (US) Food and Drug Administration (FDA) created an online clinical trials registry named ClinicalTrials.gov2. From 2005",
"the International Committee of Medical Journal Editors (ICMJE) required that clinical trials should be listed in a clinical trial registry to qualify for publication3. The registration of a clinical trial usually involves reporting informationon 20 items proposed by the World Health Organization (WHO) registration advisory group",
"including the primary outcome of the study4. One reason for the creation of this registry was to help to reduce the risk of selective reporting of outcomes that had been previously identified. For example",
"a cohort study using protocols and published reports of randomized trials approved by the Scientific-Ethical Committees for Copenhagen and Frederiksberg in Denmark in 1994–1995 found that 62% of trials had at least one primary outcome that was changed",
"introduced",
"or omitted5. A more recent study looking at trials that were registered on trial websites (such as ClinicalTrials.gov)",
"found that 31% of trials displayed some evidence of discrepancies between the outcomes registered and the outcomes published6. Therefore",
"even trial registration may not be a complete barrier to selective outcome reporting."
],
"content": "Introduction\n\nClinical trials provide the principal method with which to assess the effectiveness of therapeutic strategies1. An important principle in the good conduct of clinical trials is that a summary of the trial protocol, with a pre-defined primary outcome, should be freely available before the study commences1. In February 2000, the United States (US) Food and Drug Administration (FDA) created an online clinical trials registry named ClinicalTrials.gov2. From 2005, the International Committee of Medical Journal Editors (ICMJE) required that clinical trials should be listed in a clinical trial registry to qualify for publication3. The registration of a clinical trial usually involves reporting informationon 20 items proposed by the World Health Organization (WHO) registration advisory group, including the primary outcome of the study4. One reason for the creation of this registry was to help to reduce the risk of selective reporting of outcomes that had been previously identified. For example, a cohort study using protocols and published reports of randomized trials approved by the Scientific-Ethical Committees for Copenhagen and Frederiksberg in Denmark in 1994–1995 found that 62% of trials had at least one primary outcome that was changed, introduced, or omitted5. A more recent study looking at trials that were registered on trial websites (such as ClinicalTrials.gov), found that 31% of trials displayed some evidence of discrepancies between the outcomes registered and the outcomes published6. Therefore, even trial registration may not be a complete barrier to selective outcome reporting.\n\nClinicalTrials.gov tracks all changes made to registered protocols. The objectives of this study were: to assess the proportion of registered trials on ClinicalTrials.gov that had the primary outcome changed; to assess when the primary outcome was changed in relation to the listed study start and end dates and to assess whether the primary outcome change had any relation to the study sponsor.\n\n\nMethods\n\nClinicalTrials.gov is a publicly available trial registry and results database developed and maintained by the US National Library of Medicine on behalf of the US National Institutes of Health.\n\nWe wrote scripts in R to download all interventional clinical studies registered with ClinicalTrials.gov as of 25 October 2012. Data from the ‘tabular view’ for all studies (e.g. http://clinicaltrials.gov/ct2/show/record/NCT00548405?term=alemtuzumab+multiple+sclerosis&rank=2) were downloaded, and the information was automatically extracted from each field and used to populate a spreadsheet for analysis. This spreadsheet is available in figshare (doi: 10.6084/m9.figshare.967827). We downloaded data from all interventional studies registered with ClinicalTrials.gov as of 25 October 2012 preventing any bias in study selection (please contact the corresponding author for details on the scripts).\n\nThe following information was collected from each study: study registration date (the date the study is registered with ClinicalTrials.gov); the study start date (defined as the date that enrollment to the protocol begins); the primary completion date (defined as the anticipated or actual date the final subject was examined or received an intervention for the purposes of final collection of data for the primary outcome, whether the clinical trial concluded according to the pre-specified protocol or was terminated); study completion date (defined as the final date on which data was collected); original primary outcome (defined as a specific key measurement(s) or observation(s) used to measure the effect of experimental variables in a study) and date submitted; current primary outcome and date submitted; study phase (phase of investigation as defined by the US FDA), data monitoring committee (whether an independent group of scientists has been appointed to monitor the safety and the scientific integrity of a human research intervention, and to make recommendations to the sponsor regarding the termination of the trial for efficacy, for harm or for futility); study sponsor (defined as the primary organization that oversees the implementation of the study and is responsible for data analysis) and collaborators (defined as other organizations (if any) providing support, including funding, design, implementation, data analysis and reporting).\n\nFor studies to be included in this analysis a primary outcome had to be registered. A study was classified as not having a primary outcome changed if the original primary outcome was listed as ‘same as current’. For a subset of interventional trials, specifically those that had ‘multiple sclerosis’ or ‘diabetes’ in the title, we looked for primary outcomes that had a significant change, as the authors have experience in these fields. We defined a discrepancy between primary outcomes if the two were clearly different (e.g. in one study the primary outcome was changed from ‘expanded disability status scores assessed every 12 weeks’ to ‘annualized relapse rate’). Two authors (SVR and JP) independently assessed whether or not the studies had significant changes; then they met to compare results. For any discrepancies they met with another author (LH) and a consensus was reached. The kappa statistic for agreement between the first two observers was 0.87.\n\nClinicalTrials.gov stores funding organization information using two data elements: lead sponsor (defined as the organization or person who oversees the clinical study and is responsible for analyzing the study data) and collaborator (defined as an organization other than the sponsor that provides support for a clinical study). We derived probable funding source from the lead sponsor and collaborator fields using the following algorithm: if the lead sponsor and any collaborators were from industry and no non-industry sponsors or collaborators were listed, then the study was categorized as industry funded; if the lead sponsor and/or collaborator included industry and non-industry sources, then the study was categorized as mixed; finally if the lead sponsor and any collaborators were not from industry and no industry sponsors or collaborators were listed, then the study was categorized as non-industry funded. Two authors (SVR and JP) independently appraised the funding status of all studies; then they met to compare results. For any discrepancies they consulted another author (LH) and a final consensus was reached. The kappa statistic for agreement between the first two observers was 0.76.\n\nStatistical analysis. We used logistic regression to calculate odds ratios (ORs) and 95% confidence intervals (95% CI) for comparisons between outcome changed and non-outcome changed groups, using registration date and funding source as explanatory variables. In a full model, we included study phase and data monitoring committee as additional explanatory variables. The p-values <0.05 were interpreted as significant. Statistical analyses were conducted using the STATA 12.0.0 software.\n\n\nResults\n\nAs of 25 October 2012, 97,668 interventional trials were registered with ClinicalTrials.gov. These trials were registered between 1999 and 2012; the distribution of registrations by year is shown in Table 1. There were 46,803 (52.5%) trials classed as non-industry funded, 11,986 (13.4%) as mixed and 30,415 (34.1%) as industry funded (Table 1). There were 39,992 studies registered as completed.\n\nWe found that 28,129 studies (31.7% of 89,204) had their primary outcome changed. Funding source and registration year were associated with trials that had their primary outcome changed (Table 2). Restricting analyses to completed trials (39,992 trials) did not materially change these associations (mixed funding odds ratio (OR)=1.02; 95% confidence interval (CI)=0.95–1.10, p=0.63; industry funding OR=1.30, 95% CI=1.24–1.36, p<0.001).\n\nOR=odds ratio.\n\nThe dates of primary outcome changes were first compared in relation to the registered study start date. This analysis showed that 26,457 studies (30.3% of 87384 studies with a registered start date) had their primary outcome changed after the study had supposedly started. Funding source and registration year were associated with trials that had their primary outcome changed after the study start date (Table 2). Restricting analyses to completed trials (39236 trials) did not affect the associations with funding (mixed funding OR=1.01; 95% CI=0.94–1.09, p=0.70; industry funding OR=1.29, 95% CI=1.23–1.35, p<0.001).\n\nThe dates of primary outcome changes were then compared to the listed study end dates. The results showed that 11,834 studies (30.4% of 38,974 studies with a registered primary completion date) had their primary outcome changed after the primary completion date and 10,623 (26.2% of 40,615 studies with a registered study completion date) studies had their primary outcome changed after the study completion date. Funding source and registration year were associated with trials that had their primary outcome changed after either study completion dates (Table 2). Restricting analyses to completed trials (32,124 trials with a primary completion date and 35,719 trials with a study completion date) rendered the associations with mixed funding non-significant (primary completed trials mixed funding OR=1.10; 95% CI=0.99–1.19, p=0.057; completed trials mixed funding OR=1.08, 95% CI=0.99–1.17, p=0.085); the associations with industry funding remained (primary completed trials industry funding OR=1.94; 95% CI=1.83–2.05, p<0.001; completed trials industry funding OR=1.81, 95% CI=1.72–1.91, p<0.001).\n\nIn a full model we included study phase and the use of a data monitoring committee (DMC) as additional explanatory variables. There were 48,471 trials that provided information for all four variables, 47,748 trials that also had a study start date, 23,080 that also had a primary completion date and 21,747 that also had a study completion date. Funding source was associated to all primary outcome changes, primary outcome changes after listed study start date and primary outcome changes after listed primary completion date following adjustment for study phase and presence of a DMC (Table 3). Study phase and use of a DMC were also associated to primary outcome changes. All associations remained similar when restricting to completed trials, although the associations of mixed funding with all primary outcome changes and primary outcome changes after study start date became non-significant.\n\nOR=odds ratio.\n\nA subset of interventional trials in multiple sclerosis was investigated to assess the actual change in primary outcome. Fifty out of 422 (11.9%) registered trials were deemed to have a significant change in the primary outcome, 49 out of 416 (11.8%) were changed after the study start date, 32 out of 187 (17.1%) were changed after the primary completion date and 26 out of 191 (13.6%) were changed after the study completion date. After adjusting for study registration year, industry funding was associated with all primary outcome changes (OR=4.92, 95% CI 2.12–11.41, p<0.001), primary outcome changes after listed study start date (OR=4.75, 95% CI 2.04–11.05, p<0.001), primary outcome changes after listed primary completion date (OR=14.3, 95% CI 1.87–109.3, p=0.01), and primary outcome changes after listed study completion date (OR=14.52, 95% CI 1.90–111.06, p=0.01). These associations remained also when adjusting for presence of DMC and study phase and when looking at completed trials.\n\nA subset of interventional trials in diabetes was also investigated to assess the actual change in primary outcome. Two hundred and forty eight out of 2836 (8.7%) registered trials were deemed to have a significant change in the primary outcome, 225 out of 2786 (8.1%) were changed after the study start date, 158 out of 1475 (10.7%) were changed after the primary completion date and 147 out of 1561 (9.4%) were changed after the study completion date. After adjusting for study registration year, industry funding was associated to all primary outcome changes (OR=1.76, 95% CI 1.31–2.36, p<0.001), primary outcome changes after listed study start date (OR=1.75, 95% CI 1.28–2.38, p<0.001), primary outcome changes after listed primary completion date (OR=1.83, 95% CI 1.20–2.77, p=0.005), and primary outcome changes after listed study completion date (OR=2.63, 95% CI 1.68–4.11, p<0.001). These associations remained also when adjusting for presence of DMC and study phase and when looking at completed trials.\n\n\nDiscussion\n\nWe assessed the proportion of primary outcome changes for interventional trials registered on ClinicalTrials.gov from 1999 to 2012, when these changes occurred and whether these changes related to funding source. Industry funding was associated with primary outcome changes. These associations remained even after adjusting for the phase of the study and the presence of a DMC. The changes appeared to peak in the period between 2004 and 2007. This may suggest that a lower number of trials were registered before this time period and perhaps that there was not enough time to accrue for changes to be made in more recently registered trials (or that the changes made to the protocol decrease over time). When looking at significant primary outcome changes, the proportion of trials with their outcome changed was much less than all changes; nevertheless, industry funding was still associated with significant primary outcome changes.\n\nThere are many reasons for departures from the initial study protocol. Authors should identify and explain any such changes, however no such information is given on ClinicalTrials.gov and thus we have no explanation for our results. The results obtained here may suggest that industry funded trials are more diligent in reporting changes to protocols. Indeed, the primary outcome measure data element was not available in ClinicalTrials.gov until late 2004 and it was considered an optional data element until December 2012. There are implications of the data reported here for medical journals, reviewers and drug approval agencies.\n\nA limitation for our study is the scope of the data. We used data from only one trial website, and thus the generalisability to other registration sites is unclear. We have also assumed the data entered regarding study start and completion dates were accurate. We have no information on the reasons for the changes being made. We also did not assess the significance of the primary outcome change for the vast majority of changes; the sub-studies in multiple sclerosis and diabetes suggested that significant changes may only occur in approximately 8% of all registered trials, but this may or may not be applicable to trials for other disorders. Some primary outcome changes may be typographical/semantic and may not reflect actual changes to the nature of the outcome (although one would expect these to occur equally regardless of funding source).\n\nPrevious reports have not investigated changes to a primary outcome as entered on a registration website, but they have compared trial protocols to published studies. Four previous studies have shown that in 47–74% of studies the primary outcome stated in a protocol was the same as in a subsequent publication; between 13 and 31% of primary outcomes specified in the protocol were omitted in the publication and between 10 and 18% of reports introduced a primary outcome in the publication that was not specified in the protocol7. The proportion of primary outcome changes that we found was perhaps lower than that found previously, but this may be due to the different methodology used in this study. The relationship between protocol changes and funding has not been thoroughly investigated. Chan and colleagues found that 61% of the 51 trials with major discrepancies between the study protocol and publication were funded solely by industry sources compared to 49% of the 51 trials without discrepancies5.\n\n\nConclusions\n\nPrimary outcome changes are made to study protocols registered on ClinicalTrials.gov and these changes are associated with funding source.\n\n\nData availability\n\nfigshare: Data set of primary outcome changes in interventional clinical trials registered on ClinicalTrials.org8. http://dx.doi.org/10.6084/m9.figshare.967827",
"appendix": "Author contributions\n\n\n\nSVR and BG conceived and designed the study. SVR, JP, LH, APS, MK and DM analysed the data. SVR and BG interpreted the data. SVR drafted the article. All authors revised the article and gave final approval for publication.\n\n\nCompeting interests\n\n\n\nThe authors declare no competing interests.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nPocock SJ: Clinical trials: a practical approach. Wiley. 1983. Reference Source\n\nZarin DA, Tse T, Williams RJ, et al.: The ClinicalTrials.gov results database--update and key issues. N Engl J Med. 2011; 364(9): 852–860. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDeAngelis CD, Drazen JM, Frizelle FA, et al.: Clinical trial registration: a statement from the International Committee of Medical Journal Editors. JAMA. 2004; 292(11): 1363–1364. PubMed Abstract | Publisher Full Text\n\nZarin DA, Keselman A: Registering a clinical trial in ClinicalTrials.gov. Chest. 2007; 131(3): 909–912. PubMed Abstract | Publisher Full Text\n\nChan AW, Hróbjartsson A, Haahr MT, et al.: Empirical evidence for selective reporting of outcomes in randomized trials: comparison of protocols to published articles. JAMA. 2004; 291(20): 2457–2465. PubMed Abstract | Publisher Full Text\n\nMathieu S, Boutron I, Moher D, et al.: Comparison of registered and published primary outcomes in randomized controlled trials. JAMA. 2009; 302(9): 977–984. PubMed Abstract | Publisher Full Text\n\nDwan K, Altman DG, Arnaiz JA, et al.: Systematic review of the empirical evidence of study publication bias and outcome reporting bias. PLoS One. 2008; 3(8): e3081. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRamagopalan SV, Skingsley A, Handunnetthi L, et al.: Data set of primary outcome changes in interventional clinical trials registered on ClinicalTrials.org. Figshare. 2014. Data Source"
}
|
[
{
"id": "4267",
"date": "07 Apr 2014",
"name": "Janet Wale",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well written, competently undertaken, cross-sectional analysis. The study finds that changes to primary outcomes listed in a trials register, at any stage of the trial, are clearly associated with industry funding. Studying the extent of the changes in two disease areas is a practical way used by the authors to assess the changes and their significance (extensive in some 8%).The authors point out that there are many reasons for changes from the initial study protocol. No information is given on ClinicalTrials.gov on the reasons for changes and thus they have no explanation for their results. The authors suggest that industry funded trials are more diligent in reporting changes to protocols.This is a possible area for change which has been provided in this article.",
"responses": []
},
{
"id": "4266",
"date": "23 Apr 2014",
"name": "Deborah Korenstein",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis cross-sectional study of trials registered on Clinicaltrials.gov found that changes in primary outcome are common and are associated with industry funding. The study was appropriately done and reported. The reasons for the changes in primary outcomes and the explanation for the association with funding source are not clear, but it seems likely that changes to the primary outcome might be important for the sound interpretation of results.",
"responses": []
},
{
"id": "4262",
"date": "09 May 2014",
"name": "Steven Julious",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting report with some interesting results. Upon reading the report it did lead to the thinking of possible confounding factors which could explain some of the differences if not all. Some of the confounding factors could not be easily investigated in the current data set and I will consider these first.The first is whether the treatment being investigated in the trial is a licensed or unlicensed therapy. Though in theory \"do-able\" the authors can be forgiven for not investigating this. However, could it be proposed that a trial of a new unlicensed treatment where knowledge is emerging - and being published as the trial is ongoing - is more likely to have an endpoint changed than a licensed treatment with known properties?The other consideration is therapeutic area. The authors to a degree looked into this by investigating two sub populations of MS and diabetes trials but it could be that certain therapeutic areas predominate in certain sectors?One final consideration is if the endpoint change was pre-planned in some way in some form of adaptive design. With respect to confounders which could be investigated the main consideration is study duration. Might a study that has gone on longer be more likely to have an endpoint change? This could be legitimate if, as mentioned previously this change is due to published work becoming available. It could also be (maybe) legitimate if, for completed studies, it has dragged on for an age with no chance to reach the target sample size. So to a degree there is a case of fitting tailoring the study to what can be answered with the sample size. The interaction between study duration and funder would also be interesting (as well as just allowing in the model)There are two markers in the analysis which do suggest study duration may impact on endpoint change. The first is study registration year and the other is phase of development. In the case of phase of development the effect seems to be the reverse, it could be argued, to what one would expect. Early phase trials are learning trials not confirming trials. A consequence is the endpoints may change to reflect the properties of a treatment an investigator is learning about in an ongoing manner. It would be interesting with study duration in the model how it would effect the comparison of phase of developmentFollowing on from the point in the previous paragraph therefore an analysis of just trials which are Phase III would be of interest. These trials should have everything set and pre-specified in terms of analysisOne final point about the work is that I could not see mention of a protocol for the audit itself undertaken for the paper. In particular what the pre-specified primary endpoint and analysis population was. It is not the convention for audits to do this but it would have been optimal given the subject matter.In summary therefore it would be optimal to comment on the potential limitations of the work and to undertake the suggested analyses. However, the work itself is of interest and will almost certainly feed debate.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-77
|
https://f1000research.com/articles/3-40/v1
|
06 Feb 14
|
{
"type": "Commentary",
"title": "Drugs acting on central nervous system (CNS) targets as leads for non-CNS targets",
"authors": [
"Prashant S. Kharkar"
],
"abstract": "Out-of-the-box approaches are currently needed to replenish the souring pipelines of pharmaceutical companies across the globe. Here a theme is presented – the use of central nervous system (CNS) drugs as leads for non-CNS targets. The approach is related to the use of existing drugs for new indications. Suitable chemical modifications of the CNS drugs abolish their CNS penetration. These novel analogs may then be screened for activity against non-CNS targets. Careful selection of the appropriate structural modifications remains the key to success.",
"keywords": [
"The pharmaceutical industry worldwide is suffering from ‘productivity crisis’1. The number of new molecular entities (NMEs) launched annually has decreased significantly despite rising discovery and developmental expenses2. Drug discovery researchers have started seeking approaches with higher probabilities of success. Drug repositioning",
"drug rescue and related strategies such as selective optimization of side activities (SOSA) are the front-runners3. In May 2012",
"the US National Institutes of Health (NIH) launched ‘Discovering New Therapeutic Uses for Existing Molecules’",
"a collaborative program administered by the National Center for Advancing Translational Sciences (NCATS). Such an initiative emphasized the importance of lead discovery approaches based on existing drugs."
],
"content": "Commentary\n\nThe pharmaceutical industry worldwide is suffering from ‘productivity crisis’1. The number of new molecular entities (NMEs) launched annually has decreased significantly despite rising discovery and developmental expenses2. Drug discovery researchers have started seeking approaches with higher probabilities of success. Drug repositioning, drug rescue and related strategies such as selective optimization of side activities (SOSA) are the front-runners3. In May 2012, the US National Institutes of Health (NIH) launched ‘Discovering New Therapeutic Uses for Existing Molecules’, a collaborative program administered by the National Center for Advancing Translational Sciences (NCATS). Such an initiative emphasized the importance of lead discovery approaches based on existing drugs.\n\nThe design and development of drugs that cross the blood-brain barrier (BBB) and act at some target site(s) in the central nervous system (CNS) is a formidable task. In contrast, for the drugs to act on peripheral targets, it is important to restrict their passage through the BBB in order to avoid unwanted CNS side effects. Several physicochemical and molecular properties of CNS drugs differ from peripherally acting drugs; the former have lower molecular weights, are more lipophilic, have a smaller polar surface area (PSA), a fewer H-bond acceptors and donors and fewer rotatable bond4. There is a fine balance between the physicochemical properties of CNS and non-CNS drugs.\n\nDuring typical lead optimization cycles in the discovery phase, the lead molecules undergo several chemical modifications in order to improve their potency and pharmacokinetic properties, which usually lead to increased a) molecular weight, b) lipophilicity, c) molecular complexity, d) number of rotatable bonds, e) number of H-bond donors and acceptors, etc5. In general, the drugs are more complex than the leads and exhibit higher values for the majority of the associated molecular properties listed above. Based on these findings, it can be hypothesized that ‘CNS drugs which are smaller and lower ranges of the aforementioned molecular properties make excellent starting points (as leads) for the development of non-CNS drugs’. Several aspects of this hypothesis are outlined in the discussion given below.\n\nThe majority of the CNS drugs are basic in nature. The presence of an ionizable functional group (mostly cationic) favors BBB penetration. Strong acids (pKa < 4) and strong bases (pKa > 10) are prohibited from crossing the BBB4. Chemical modifications of the basic functional group (primary and secondary) to a neutral species (e.g., conversion of primary amine to a substituted urea or amide) may impede the entry of the NME into the CNS. Several physicochemical and molecular properties can then be tailor-made once suitable potency against a non-CNS target is found.\n\nAnother molecular property, PSA, is crucial for BBB penetration. A PSA cutoff of 90 Å2 has been suggested for CNS drugs6. Increased PSA is likely to create hurdles in the passage of NMEs across the BBB. An increased PSA can be achieved through the introduction of polar functional groups such as sulfonamide, carboxylic acid, substituted amides, etc., on the aromatic rings present in majority of the CNS drugs. Structural modifications leading to a higher PSA will ultimately lead to an increased number of H-bond donors and acceptors and reduced lipophilicity. The cumulative effect is reduced CNS penetration.\n\nCNS drugs tend to have less molecular flexibility, lighter molecular weights and less molecular volume4. Significant increases in these molecular properties may create obstacles in absorption following oral administration leading to reduced bioavailability, e.g., an increased number of rotatable bonds can result in increased hepatic metabolism of the drug7. Nonetheless, the overall effects of an increase in the molecular weight and/or molecular flexibility on BBB penetration may depend on alterations in other properties such as lipophilicity and PSA.\n\nIn terms of toxicity, inhibition of the hERG channel by several CNS drugs (e.g., haloperidol) is a major concern. Many CNS drugs contain the hERG pharmacophore (aromatic rings and suitably placed cationic N)8. Suitable chemical modifications of the CNS drugs such as attenuating the basicity of the cationic N and suitably placed aromatic substituents may lead to abolished hERG binding and associated adverse effects. Thus, conversion of a CNS drug into its non-CNS counterpart, according to the theme of this commentary, may lead to diminished hERG toxicity.\n\nFrom the above discussion, it appears convincing that the CNS drugs can serve as suitable leads for non-CNS targets after appropriate structural modifications leading to considerable alterations in their property space. This leads to the question: what are the potential applications of such a strategy?\n\nCombinatorial libraries starting with CNS drugs can be designed in silico and then synthesized after selecting desirable substituents to introduce structural novelty (see Figure 1). These libraries may be unique in terms of structural and property space due to their origin from a known drug and thus may serve as a novel compound collection for high-throughput screening (HTS) campaigns. Once suitable hits are identified, development time may be reduced because of prior knowledge of the original drug. The pharmacokinetics (PK), pharmacodynamics (PD), adverse reactions, toxicity and the clinical trials data obtained from the original drug may guide the design of such experiments for the non-CNS analogs. Similarly, the synthetic routes, bulk scale-up and the analytical methods may also be used for the novel non-CNS derivatives. The utility of any and every piece of information about the CNS drug may vary from case to case. Since the original drug was never intended for a peripheral target, the intellectual property issues (novelty, non-obviousness) between the peripheral analogs and the original drug analogs for the CNS target may be minimal.\n\nFluoxetine, an antidepressant agent, is used as a template. The synthesis of the analogs can be achieved by modifying the synthetic route of fluoxetine itself. The major structural modification includes abolishing the basicity of the secondary N in fluoxetine by converting it to urea or sulfonamide. Similarly, the 4-CF3Ph ring is replaced with an acetic acid side chain. The resultant structural modifications lead to increased PSA, decreased cLogP (calculated logarithm of partition coefficient) and an increased number of H-bond donors and acceptors.\n\nSome functional groups convey particular therapeutic effects, e.g., acetic acid or related aliphatic acids as analgesic and anti-inflammatory agents (diclofenac, ibuprofen), sulfonamides as carbonic anhydrase (CA) inhibitors and as anti-bacterials (sulfisoxazole). As such introduction of such functional groups during chemical modifications of CNS drugs may lead to a gain in potency for non-CNS targets such as CA. Careful selection of the chemical modifications aimed at the non-CNS target coupled to virtual screening of the designed analogs may potentially increase the rate of success of this approach.\n\nIn summary, the commentary outlines a novel approach for generating ‘interesting’ compound collections for lead discovery. Such an idea is of potential interest in times of pharmaceutical ‘productivity crisis’. The designed libraries can be tailor-made to suit the target requirements for potency and/or selectivity, in addition to pharmacokinetic and toxicity properties. Compared to the traditional lead discovery method based on HTS (with or without virtual screening) of in-house or commercially available compound collections, the ‘intelligent’ (virtual or physical) libraries developed using established CNS drugs may yield higher success rate (% of hits).\n\n\nConclusion\n\nThe use of CNS drugs as a starting point for developing non-CNS leads seems interesting with reference to potentially altered molecular, pharmacokinetic, pharmacodynamic and/or toxicity properties. The combinatorial libraries based on the CNS drug scaffolds may perturb novel chemical space not accessed previously by the non-CNS small molecule drugs. Curious researchers interested in the above strategy may help in demonstrating its potential utility.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nThe author acknowledges Dr. R. S. Gaud, Dean, SPP School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai, India, for his help during preparation of this commentary.\n\n\nReferences\n\nPaul SM, Mytelka DS, Dunwiddie CT, et al.: How to improve R&D productivity: the pharmaceutical industry’s grand challenge. Nat Rev Drug Discov. 2010; 9(3): 203–214. PubMed Abstract | Publisher Full Text\n\nMullard A: 2011 FDA drug approvals. Nat Rev Drug Discov. 2012; 11(2): 91–94. PubMed Abstract | Publisher Full Text\n\nMullard A: Could pharma open its drug freezers? Nat Rev Drug Discov. 2011; 10(6): 399–400. PubMed Abstract | Publisher Full Text\n\nPajouhesh H, Lenz GR: Medicinal chemical properties of successful central nervous system drugs. NeuroRx. 2005; 2(4): 541–553. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOprea TI, Davis AM, Teague SJ, et al.: Is there a difference between leads and drugs? A historical perspective. J Chem Inf Comput Sci. 2001; 41(5): 1308–1315. PubMed Abstract | Publisher Full Text\n\nHitchcock SA, Pennington LD: Structure-brain exposure relationships. J Med Chem. 2006; 49(26): 7559–7583. PubMed Abstract | Publisher Full Text\n\nVeber DF, Johnson SR, Cheng HY, et al.: Molecular properties that influence the oral bioavailability of drug candidates. J Med Chem. 2002; 45(12): 2615–2623. PubMed Abstract | Publisher Full Text\n\nPerry M, Stansfeld PJ, Leaney J, et al.: Drug binding interactions in the inner cavity of hERG channels: molecular insights from structure-activity relationships of clofilium and ibutilide analogs. Mol Pharmacol. 2006; 69(2): 509–519. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "3543",
"date": "10 Feb 2014",
"name": "Vijay Gokhale",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis commentary describes a general approach for using CNS-active drugs as leads for non-CNS targets. As physicochemical properties for CNS-active drugs are more restrictive than for non-CNS drugs, it would appear that the CNS-active drugs could serve as a good leads. It is a well-written commentary discussing general approaches to modify properties of these lead molecules. The approach is also exemplified by use of fluoxetine as a lead and some suggested modifications of it. One main problem with this approach is the challenge of removing or minimizing CNS activity of these leads for non-CNS targets since the activity on CNS will lead to side effects. It would be useful to include a table comparing desired value ranges for different physicochemical properties (molecular weight, log D, polar surface area, hydrogen bond donor) for CNS and non-CNS drugs. I am not aware if this approach has been used in the medicinal chemistry literature. But if such reports exist, it would be good to include those references in the commentary.",
"responses": []
},
{
"id": "3540",
"date": "26 Feb 2014",
"name": "Evans Coutinho",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe author has proposed a method of using CNS drugs as leads and developing them into candidates for non-CNS target. The logic behind the approach is to tailor the attributes of a) molecular weight, b) lipophilicity, c) molecular complexity, d) number of rotatable bonds, e) number of H-bond donors and acceptors and f) polar surface area that are in consonance with values appropriate for non-CNS agents. He has illustrated his point with fluoxetine as an example. This is an idea is worthwhile looking at, but the final test would be to see some real applications of the approach on some established CNS drugs and their activity on some non-CNS targets experimentally established.",
"responses": []
},
{
"id": "3539",
"date": "07 Mar 2014",
"name": "Hariprasad Vankayalapati",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis F1000Research article on “Drugs acting on central nervous system (CNS) targets as leads for non-CNS targets\" submitted by Kharkar is on a subject not uncommon to the CNS research community.The following comments need to be addressed:The author should state that several CNS agents, particularly multifunctional targets, are currently in clinical trial or have been approved for the treatment of non-CNS diseases despite the fact that they can cause inflammation and cancer. However, recently reported targeted CNS agents can bring an opportunity for repurposing some of the non-CNS targets as well. (Reference: http://www.ncats.nih.gov/research/reengineering/rescue-repurpose/rescue-repurpose.html). Abstract: \"Out-of-the-box approaches are currently needed to replenish the souring pipelines of pharmaceutical companies across the globe\" this statement is arguable and needs to be revised. Page 2 paragraph 9: this may not be true and needs to be revised completely to something like - \"Even the slightest modification to the physicochemical and physiological properties of the entity proposed as the non-CNS target of interest may alter its medicinal chemistry and tractability. One therefore needs to conduct every phase of non-clinical/IND work and may not reduce the development time due to the different PK and PD profile of the newly designed entity. However this is true only for repositioned drugs for new indications\". Figure 1: The introduction of just a -COOH functional group as a strategy may not abolish the CNS activity completely (-COOH is negatively charged at the physiological pH). There is no evidence to support Figure 1 because reports show that at least 3−4% of CNS drugs have a carboxylic acid group. The author should therefore emphasize that this proposal is just theoretical as yet. The author also missed some important references that I feel need to be included to support this article:a) Gosh et al., (2012) Knowledge-Based, Central Nervous System (CNS) Lead Selection and Lead Optimization for CNS Drug Discovery. ACS Chem. Neurosci. 2012, 3, 50−68b) Palmer and Alavijeh (2012) Translational CNS medicines research. in Drug Discov Today.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-40
|
https://f1000research.com/articles/2-280/v1
|
19 Dec 13
|
{
"type": "Review",
"title": "Thermoregulation strategies in ants in comparison to other social insects, with a focus on Formica rufa",
"authors": [
"Štěpánka Kadochová",
"Jan Frouz",
"Jan Frouz"
],
"abstract": "Temperature influences every aspect of ant biology, especially metabolic rate, growth and development. Maintenance of high inner nest temperature increases the rate of sexual brood development and thereby increases the colony fitness. Insect societies can achieve better thermoregulation than solitary insects due to the former’s ability to build large and elaborated nests and display complex behaviour. In ants and termites the upper part of the nest, the mound, often works as a solar collector and can also have an efficient ventilation system. Two thermoregulatory strategies could be applied. Firstly the ants use an increased thermal gradient available in the mound for brood relocation. Nurse workers move the brood according to the thermal gradients to ensure the ideal conditions for development. A precise perception of temperature and evolution of temperature preferences are needed to make the correct choices. A second thermoregulatory strategy used by mound nesting ants is keeping a high temperature inside large nests. The unique thermal and insulation properties of the nest material help to maintain stable conditions, which is the case of the Wood ant genus Formica. Ants can regulate thermal loss by moving nest aggregation and alternating nest ventilation. Metabolic heat produced by ant workers or associated micro organisms is an important additional source of heat which helps to maintain thermal homeostasis in the nest.",
"keywords": [
"review",
"wood ants",
"Formicinae",
"thermoregulation",
"metabolic heat production"
],
"content": "Introduction\n\nTemperature is an important factor for all ectothermic organisms, including ants. Their rate of development is accelerated with high temperatures1, the movement rate speeds up2 and the rate of food and oxygen consumption also increases3. Higher temperatures can be advantageous for colony fitness as it can increase reproduction rate though at the same time can be disadvantageous due to higher energy expenditure4.\n\nMost insect species have a solitary mode of life in adulthood; these individuals are able to regulate their body temperature through behavioural reactions such as sunning or seeking cool shelter5. More sophisticated forms of thermoregulation can be found among social insects. They are able not only of regulating their own body temperature but also the temperature of the immediate surroundings. This ability is due to the large number of individuals in the society, their behavioural interactions and nest construction6. The nest protects the whole colony and serves as a shelter for adults and, more importantly, as an incubator for the brood. Improved colony homeostasis could even be one of the reasons why insect sociality evolved5. Thermoregulation, however, requires the expenditure of energy and so comes with costs as well as benefits.\n\nThe level of nest thermoregulation depends on many other factors, e.g. nest size, population size, and the moisture and thermal conductivity of the nest material. This paper’s objective is to provide an overview of nest thermoregulation strategies occurring among ant societies, with a focus on thermoregulation in the Wood Ant genus Formica, in comparison to other social insects.\n\nAnts nest in wide range of materials; in soil, under stones, in leaf litter and even in living trees (see Table 1). Some of them build above-ground nests, usually from soil or organic material, called ant hills or mounds. These nests show an advanced ability in regulating inner temperature.\n\nThe red imported fire ant Solenopsis invicta and members of the genus Pogonomyrmex build soil mounds from excavated soil7,8. These nests gain heat through solar radiation and the brood is moved along an increased thermal gradient1,9. Weaver ants from the genus Oecophylla construct their nest from living leaves, with the help of their own larvae, which produce a special “glue” from their salivary glands10.\n\nWood ants from the genus Formica build large nests from organic material that is based on a mixture of soil, twigs, coniferous needles and pebbles3,11–13. In these nests a stable heat core can be maintained thanks to the good insulation properties of these materials and the metabolic heat produced by the ants or their associated microflora14,15.\n\nThe composition of the organic material is not the same throughout the whole nest volume3,16 and the mound structure is not rigid either. The ants loosen and renovate the nest structure and the organic material is continuously moved from the inside to the outer layers3. Nest structure and architecture plays a vital role in nest thermoregulation. The tunnels and passages build by ants are important mainly for ventilation and humidity control, which affect the nest temperature. Pieces of tree resin are often incorporated into the Formica nest material because of its antimicrobial effect17. The resin inhibits the growth of potentially pathogenic bacteria and fungi in the nest.\n\nThatch ants, Formica obscuripes18 and Acromyrmex heyeri19, use plant fragments as a building material and arrange them in a thick compact surface layer called “thatch” which has a lower thermal diffusivity than the surrounding soil. The thatch prevents nest overheating by the incoming solar radiation and avoids losses of the accumulated heat into the cold air during night19.\n\nNest moisture can have two different and opposite thermoregulatory effects: 1) moisture can support microbial heat production (i.e. increase the temperature)15; 2) it can decrease the insulating properties of nest material (i.e. decrease the temperature)12. A study of the relationship between daily temperature regime and moisture in Formica polyctena nests revealed two possible thermoregulation strategies which differ between dry nests and wet nests15. Dry nests are usually located in sunny open places. Solar radiation heats up the nest and keeps the nest material dry, with low heat capacity and conductivity. The thermal losses of dry nests are estimated to be 0.15–4.3 W per nest12. The temperatures of dry nests are usually the highest in the evening and they drop during the night. Thermoregulation in dry nests is based on a combination of metabolic heating from the ants, the insulating properties of the nest and solar heating. The dry nest thermoregulatory strategy is more common than the wet nest strategy in F. polyctena15.\n\nWet nests are mostly found in the shade and thus solar heating is limited. In the evening the temperature in these nests is low and it increases during the night. The high night temperature at the nest surface indicates substantial heat loss, about 24–30 W per nest. The wet nests have a high thermal capacity; increasing the temperature by 1°C requires a thermal input of 35 W12. This means that maintaining a sufficient temperature in a wet nest requires a heat source beyond the metabolic heat produced by ants and the heat obtained from solar radiation. The additional heat source in wet nests is provided by microbial activity3,15.\n\nThe microbial community in F. polyctena nests differs from that in the surrounding soil in part because of differences in pH and food availability and quality13. Thermoregulation via microbial heating was first proposed in 1915 by Wasmann3. In 1980 the existence of microbial heating in F. polyctena mounds was confirmed in an experiment showing that in the absence of ants non-sterilized nest material generated a substantial amount of heat but sterilized nest material generated almost no heat3.\n\nMicrobial activity can be estimated by calculating the respiration rate of the nest material, which is used as a proxy for the respiration rate of the microbes living in it3,15. There are detectable seasonal changes in the respiration rates of nest material with the highest rates found in summer. The mass-specific heat production of ants is higher than that of the nest material but, when considering the total mound volume, microbial heat production is more than seven times higher than the heat evolved by ants3. Ants can affect microbial activity via nest material aeration, supply of fresh plant material and their own metabolic heat production. Since the microbial activity of wet nest material depends strongly on temperature3, a temperature increase in some small parts of the nest (due to ant metabolism or sun radiation) results in an increase in microbial activity and consequently in a subsequent overall increase of nest temperature.\n\nAs first mentioned by Forel in the early 1920s the ant mound often serves as a solar collector20. Solar energy can both increase the metabolism of ants and help heat the nest mound21. Compared to underground nests, mounds absorb heat more quickly both in the direct sun and in the shade9. Ants in the Northern Hemisphere usually remove shading grass from the south side of the mound so that the temperature increases quickly on that side. This creates a temperature gradient that many species use for brood displacement9. Other species decorate the mound surface with small pebbles or dead vegetation, which can work as heat collectors or as radiation reflectors22.\n\nMounds of some Formica, Solenopsis and Lasius species are asymmetric, with the main axis oriented in a south-north direction23,24. In S. invicta the angle of the south slope of the mound is negatively correlated with the maximal sun angle22. Sun-influenced thermoregulation in termites has also been documented, for example in fungus-growing members of the genus Macrotermes25. The amount of intercepted sunlight influences the shape of termite mounds and leads to great structural differences in nests in forests vs. savannahs25. But the most admirable sun-induced nest shape differences can be seen in Australian “magnetic termites” Amitermes meridionalis26. The nests are wedge-shaped with apparent north-south orientation which prevents overheating at noon and enables maintaining the warmth of nest in the evening.\n\nEffective ventilation takes place as part of nest thermoregulation in many ant species, being regulated by the opening and closing of nest entrances (see below). A ventilation system in nests of the leaf-cutting ant genus Atta was described by Kleineidam et al.27. There is not a thermal gradient big enough to generate thermal convection flow; rather the ventilation in Attini nests is driven by the wind. There are many openings on the nest surface, which are functionally divided into entry and exit tunnels. Wind flowing over the nest from any direction causes air to exit from the central tunnel and to enter tunnels at the nest periphery27. This ensures optimal thermal conditions for symbiotic fungi which are damaged by temperatures higher than 30°C28.\n\nBehavioural reactions of ants are based on sensing temperature and temperature preferences5. To react to these gradients, ants and termites have evolved inherent temperature preferences, which are the key element in thermoregulatory behaviour4,29. Nurse workers are able to choose the optimal temperature for pupae production and sexual brood incubation and to move the brood along temperature and humidity gradients to achieve the best conditions for its development5,29,30.\n\nThe optimal temperature range is variable for different groups of social insects, for example the brood of the honey bee Apis mellifera develops fastest at 35°C31. In Formica polyctena a temperature 29°C is preferred for pupal development14, whilst colonies of Solenopsis invicta can grow only between 24 and 36°C1. In contrast the genus Myrmica is adapted to cold climates, M. rubra4 and M. punctiventris32 prefer temperatures between 19 and 21°C, about 8°C lower than the temperature preferred by other ants.\n\nTemperature preferences can be affected by many factors including age and sex33, working caste or feeding condition1, or prior acclimation34. Ant queens in Formica polyctena35 and Solenopsis invicta1 prefer slightly higher temperatures than workers, especially during the egg-laying phase; inactive queens may prefer cooler temperatures. Workers generally prefer lower temperatures, which decrease their metabolic rate and increase their lifespan1,36. A decrease of 2°C can lengthen the worker lifespan in S. invicta by 14%37. Preferences of nurse workers tend to be shifted towards the higher temperatures that favour brood development1,4,21,29. Similar patterns have also been found in Apis mellifera5,31.\n\nBrood translocation along temperature gradients has characteristic time rhythms in many ant species. Camponotus mus follow a photoperiodic circadian rhythm. In the presence of a temperature gradient, nurse workers move the brood twice each day29. The first displacement starts at 2 pm, when the brood is transported from the colder night location to a warmer day location. This movement occurs 6 h after sunrise, i.e., in the middle of photophase. At 10 pm (8 h after the first displacement and 2 h after nightfall), the brood is transported back to the night location. Under artificial light/dark day cycles the brood translocation rhythm changes according to the new photophase length29. In Solenopsis invicta moving the brood up and down along temperature gradients does not seem to depend on the time of day or photoperiod9.\n\nIn response to temperature gradients, leaf-cutting ants from the genus Acromyrmex move not only the brood but also the symbiotic fungi which provide their food. The fungus requires high humidity and temperatures between 25 and 30°C28. Acromyrmex ambiguus workers move the fungus garden according to humidity conditions, but they are also capable of nest humidity regulation by changing nest architecture. The flow of dry air into the colony is a signal for workers to plug ventilation tunnels to prevent nest from drying out38. Similar behaviour has been proposed for termites39.\n\nWhen the nest interior becomes too hot, workers can reduce the inner temperature in several ways. In ants and termites nest cooling is usually achieved by changes in building behaviour. Workers of leaf-cutting ants in the genera Atta and Acromyrmex open tunnels to allow air circulation27,30,38; this behaviour could be limited by a trade off for humidity control40. Ants of the genus Formica can also partly remove the nest material, which reduces the wall thickness and increases heat dissipation16. In the genus Eciton, loosening of bivouacs´ structure (temporary nests similar to a honeybee swarm) is an effective way of cooling41. Even more efficient ventilation systems can be found in nests of the termite Mactorermes bellicose. These nests can have either externally or internally driven ventilation, depending on the habitat, nest shape (cathedral vs. dome shaped nest) or the time of day42. Cathedral shaped nests in open habitats are warmed by the sun, which creates a steep thermal gradient leading to convention currents in peripheral air tunnels. Dome shaped nests located in the forest rely more on internally driven ventilation. The same is true for cathedral nests at night42.\n\nIn contrast, bees, wasps and bumblebees cool their nests by wing-fanning and regurgitating water droplets. Water is spread over the brood comb surface enabling cooling through evaporation5. Cooling by water evaporation is very effective. Lindauer43 placed a bee hive onto a lava plain where the surface temperature reached 70°C. Taking water ad libitum, the bees were able to maintain the hive temperature at the favoured 35°C.\n\nIf the nest becomes too cold, heating strategies are applied. Bees, bumblebees and wasps are capable of direct incubation of selected pupae. Specialized workers sit on the surface of the brood cell and maintain their thoracic temperature over 35°C. Bees sitting inside empty brood cells can heat six brood cells at one time44. Metabolic heat can also be used for protection, as shown in the interaction between the predatory hornet Vespa mandarina japonica and Japanese honeybees Apis cerana japonica45. If the hornet attacks a honeybee nest it is surrounded by bee workers who increase their body temperature to a level which is lethal for the hornet but not for the honeybees45. Although ants cannot actively produce heat they are able to use indirect metabolic heat (i.e. heat produced as a by-product of metabolism) for ensuring brood development. This ability has so far been documented in the Formica rufa group3,15,46 and the army ant genus Eciton41,47,48.\n\nThe Neotropical army ants Eciton hamatum and Eciton burchelli form temporary swarms called bivouacs, which can regulate temperature very precisely to ensure optimal conditions for developing brood41. According to Franks48 bivouacs have a similar construction to a bee swarm; they can be divided into an outer mantel and inner core, which together maintain a stable temperature between 27.5 and 29.5°C regardless of the ambient temperature. On cold days the bivouacs change shape: they become more hemispherical to reduce the surface to volume ratio48. Franks48 postulates that all the heat required by the bivouac can be produced by ant metabolism.\n\nWood ants of the Palearctic genus Formica use metabolic heat to maintain a heat core, an area with high and stable temperatures, in their nests. The heat core’s position moves according to nest shape and size3,15. From April to October the inner nest temperatures are significantly higher than air temperatures49. High temperatures in spring are required for sexual brood development; nests producing sexual offspring always have higher temperatures than those producing only workers46. In winter the mound temperature changes with falling ambient temperatures but the temperature in the hibernation chambers remains stable at 1–2°C46.\n\nThe temperature inside a nest of wood ants (Formica rufa group) begins to increase very early in the spring, even when the nest surface is covered by ice and snow46. At this time some nests can contain larvae, pupae, and even some winged individuals, indicating that the inner heating must have started much earlier, because larval development cannot start in a cold nest and requires some weeks of constant warm temperature21. It is supposed that in large nests of F. rufa containing over 1 million workers, spring nest heating can start as an autocatalytic process46 that relies on utilizing lipid reserves in young workers50.\n\nAnother factor contributing to spring thermoregulation in red wood ants is temperature intake by ant bodies. The ants are dark colored so they heat up quickly when exposed to the sun during their outside-nest activities. In the spring ants are observed to create clusters on the mound surface as they bask in the sun51. Their bodies contain a substantial amount of water which has high thermal capacity making ant bodies an ideal medium for heat transfer. After getting hot enough the ants move inside the nest where the accumulated heat is released. This principle works throughout the year but in spring it is most obvious and supported by ant clustering on the nest surface15,46.\n\nDaily temperature fluctuations in the nest seem to be correlated with temperature-dependent changes in ant density and ant aggregations in the nest. The highest nest temperatures usually occur in the afternoon or in the evening which corresponds with the return of foragers15,46. This apparently results from the heat brought into the nest by returning workers (heat coming from absorbed solar energy) as well as the heat generated by worker metabolic heat production within the nest. In some nests the temperature drops slightly in the morning when ants leave the nest15,16. Heat coming from the metabolism of foragers clustered in the nest centre on cool days, when ambient temperature limits outdoor activities, could also explain a negative correlation between the inner nest temperature and the ambient temperature found occasionally in spring46.\n\nThe seasonal fluctuations in the thermoregulation behaviour of Formica polyctena along a geographic gradient were studied by Frouz & Finer49. Both in Finland and the Czech Republic the ant colonies maintained a high nest temperature (over 20°C) in spring and summer, for about 65–129 days. A rapid increase in the inner nest temperature in early spring was observed, mostly at the beginning of April. Annual nest temperature peaked in June in both locations and decreased gradually from August to November. F. polyctena nest temperatures were affected by location, season, nest shading, ambient temperature, nest size, soil moisture, and rainfall. Nest temperatures fluctuated more in the Czech Republic than in Finland, possibly because of greater differences between day and night ambient temperatures49.\n\nAn interesting question is why F. polyctena maintain stable nest temperatures for the same period in Finland and the Czech Republic even though the length of the vegetation season and the ambient temperatures in Finland and Czech Republic are different. This might be explained by the regular shifts between reproduction and diapauses in F. polyctena queens, which are driven by endogenous factors. The queen enters diapauses after 100 days of reproduction even at a constant temperature and photoperiod35,52. It has been postulated that ants maintain high temperatures only during the queen’s reproduction phase49. After that the nest temperature drops, despite the fact that the outside temperature usually does not limit foraging and the ants are still active.\n\nMaintenance of a high inner nest temperature has been observed in ants, especially in species which build above-ground nests or inhabit tree hollows15,46,53. Heating of these structures is much easier than heating underground nests, because the surrounding soil has a large heat capacity and conductivity.\n\nIn moderate climates most ants build nests in the soil where the temperature is quite stable (Table 1) or on the soil surface under a layer of leaf litter where the temperature can be buffered by the insulating properties of the nest material. Many species in the Northern Hemisphere also nest under rocks or stones which serve as heat collectors (Table 1). In the tropics only a few species nest in soil and the majority of species inhabit small pieces of rotting wood5. More precise microclimate regulation is achieved in the mound-building species of the genera Atta, Acromyrmex, Myrmicaria, Pogonomyrmex, Solenopsis, Iridomyrmex, Formica, and Lasius (Table 1).\n\nThe maintenance of a stable temperature in Wood Ant genus Formica nests during spring and summer is widely known and has been the subject of many studies12,16,46,49,53. These ants are able to maintain thermal homeostasis in the nest because of the insulation and heat storage provided by the nest material20. Mound size is generally correlated with the number of inhabitants54,55. In small nests located in sunny areas the solar radiation and the insulation properties of the nest material are thought to be the key elements in nest thermoregulation11,12. In larger nests there are internal sources of heat production, such as ant and microbial metabolic heat, that enable the maintenance of high inner temperatures even in a permanently cold environment46,49.\n\nMany other ant species (see Table 1) build hill-shaped nests from soil. These nests show large spatiotemporal variations in temperature and ants select the optimal temperature for brood development by brood displacement1. These nests, however, do not have a stable heat core. A heat core where the temperature is stable and higher than ambient temperature for an extended period of time (several months) can be found only in a minority of ant species. Why is this the case?\n\nThe heat core exists in the nests/hives of winged insects that are capable of active heating, i.e. bees, wasps, bumblebees (thermogenesis in flying muscles), and some ants, especially the genus Formica3,15,46, the army ant genus Eciton41,47,48 and probably in fungus-growing termites42. But there is no evidence of a stable heat core in Solenopsis invicta or in Acromyrmex heyeri, which also build upper-ground hills from materials with suitable insulation properties such as soil or dead vegetation. As discussed earlier, thermoregulation can also be the result of nest architecture, and only those species that build nests with a low thermal heat capacity and a low thermal conductivity are likely to maintain a heat core. In summary, the use of metabolic heat production for maintenance of a heat core depends on the insulation properties of the nest and the size of the individual workers and of the entire colony.\n\nThe latest review about nest thermoregulation in social insects6 distinguished three types of ant thermoregulation strategies: “First, like many social bees, some ant species rely on protection from a cavity, such as a tree stump or underground burrows56. Second some migrate their nest frequently, varying the amount of cover they select, depending on the temperature and season57,58. Third some others move their brood to areas of optimal temperature within the same nest”29. We suggest dividing the last category in the following way: a) ants that move the brood in daily cycles to places with optimal temperatures within the whole nest structure, for example Solenopsis invicta9 or Camponotus mus29 b) ants that keep a stable heat core inside their nest and do not move the brood from the nest interior. An example of this strategy is seen in the Formica fuga ant group3,15.\n\n\nConclusion\n\nDespite a wide variety of thermoregulatory strategies observed among ant societies some general trends can be found. Two opposite thermoregulatory strategies in mound building ants could be distinguished. Firstly nests with low insulative properties which work as solar collectors and thus increase the available thermal gradient for brood development. Secondly nests which steadily maintain higher inner temperature thanks to low thermal conductivity of the nest material, good insulative properties and metabolic heat produced by ants or associated micro organisms.",
"appendix": "Author contributions\n\n\n\nBoth authors contributed equally and agreed the final manuscript for publication.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nReferences\n\nPorter SD, Tschinkel WR: Fire ant thermal preferences: behavioral control of growth and metabolism. Behav Ecol Sociobiol. 1993; 32(5): 321–329. Publisher Full Text\n\nChallet M, Jost C, Grimall A, et al.: How temperature influences displacements and corpse aggregation behaviors in the ant Messor sancta. Insectes Sociaux. 2005; 42(4): 309–315. Publisher Full Text\n\nCoenen-Stass D, Schaarschmidt B, Lamprecht I: Temperature distribution and calorimetric determination of heat production in the nest of the wood ants Formica polyctena (Hymenoptera Formici-dae). Ecology. 1980; 61: 238–244. Publisher Full Text\n\nBrian MV: Temperature choice and its relevance to brood survival and caste determination in the ant Myrmica rubra L. Physiol Zool. 1973; 46(4): 245–252. Reference Source\n\nWilson EO: The insect societies. Belknap Press of Harvard University Press, Massachusetts. 1971; 548. Reference Source\n\nJones JC, Oldroyd BP: Nest Thermoregulation in Social Insects. Adv in Insect Phys. 2006; 33: 153–191. Publisher Full Text\n\nPorter SD: Impact of temperature on colony growth and developmental rates of the ant Solenopsis invicta. J Insect Phys. 1988; 34(12): 1127–1133. Publisher Full Text\n\nCarlson SR, Whitford WD: Ant mound influence on vegetation and soils in semiarid mountain ecosystem. American Midland Naturalist. 1991; 126(1): 125–139. Reference Source\n\nPenick CA, Tschinkel WR: Thermoregulatory brood transport in the fire ant Solenopsis invicta. Insec Soc. 2008; 55(2): 176–182. Publisher Full Text\n\nHölldobler B: Territorial Behaviour in the Green Tree Ant (Oecophylla smaragdina). Biotropica. 1983; 15(4): 241–250.Reference Source\n\nBrandt CJ: The thermal diffusivity of the organic material of a mound of Formica polyctena Foerst. in relation to the thermoregulation of the brood (Hymenoptera, Formicidae). Neth J Zool. 1980; 30(2): 326–344. Publisher Full Text\n\nDlusskij GM: [The ants of genus Formica]. Nauka, Moskva. 1967; 231 pp. Reference Source\n\nFrouz J, Kalčík J, Cudlín P: Accumulation of phosphorus in nest of red wood ants Formicas. Str. Ann Zool Fennici. 2005; 42(3): 269–275. Reference Source\n\nCoenen-Stass D: Zum Verhalten der roten Waldameise, Formica polyctena. (Hymenoptera, Formicidae) im Klimagradient während der Brutpflege. Verhandlungen der Deutschen Zoologischen Gesellschaft. 1985; 78: 204–112. Reference Source\n\nFrouz J: The effect of nest moisture on daily temperature regime in the nests of Formica polyctena. wood ants. Insec Soc. 2000; 47(3): 229–235. Publisher Full Text\n\nHorstmann K, Schmid H: Temperature regulation in nests of the wood ant Formica polyctena (Hymenoptera: Formicidae). Entomologia Generalis. 1986; 11(3–4): 229–236. Publisher Full Text\n\nCastella G, Chapuisat M, Christe P: Prophylaxis with resin in wood ants. Anim Behav. 2008; 75(4): 1591–1596. Publisher Full Text\n\nMciver JD, Torgersen TR, Cimon NJ: A supercolony of the thatch ant Formica obscuripes Forel (Hymenoptera: Formicidae) from the Blue Mountains of Oregon. Northwest Sci. 1997; 71(1): 18–29. Reference Source\n\nBollazzi M, Roces F: The thermoregulatory function of thatched nests in the South American grass-cutting ant Acromyrmex heyeri. J Insect Sci. 2010; 10(137): 1–17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSeeley TD, Heinrich B: Regulation of temperature in the nest of social insects. In: Insect Thermoregulation (HEINRICH, B. Ed.). John Wiley and Sons, Inc,. 1981; pp. 160–234. Reference Source\n\nKneitz G: Versuche zur Wärmeorien-tierung von Arbeiterinhen der Waldameisenart Formica polyctena. Foerst. (Hymenoptera, Formicidae). Insect Soc. 1966; 13: 285–296. Publisher Full Text\n\nVogt JT: Quantifying imported Fire ant (Hymenoptera: Formicidae) mounds with airborne digital imagery. Environ Entomol. 2004; 33(4): 1045–1051. Publisher Full Text\n\nHöLldobler B, Wilson EO: The Ants. The Belknap Press of Harvard University Press, Cambridge, 1990; 732 pp. Reference Source\n\nVogt JT, Wallet B, Freeland TB Jr: Imported fire ant (Hymenoptera: Formicidae) mound shape characteristics along a north-south gradient. Environ Entomol. 2008; 37(1): 198–205. PubMed Abstract | Publisher Full Text\n\nKorb J, Linsenmair KE: The effect of temperature on the architecture and distribution of Macrotermes bellicosus. (Isoptera, Macrotermitinae) mounds in different habitats of West African Guinea savanna. Insec Soc. 1998; 45(1): 51–65. Publisher Full Text\n\nGrigg GC: Some consequences of the shape and orientation of 'magnetic' termite mounds. Aust J Zool. 1973; 21(2): 231–237. Publisher Full Text\n\nKleineidam C, Ernst R, Roces F: Wind induced ventilation of the giant nests of the leaf-cutting ant Atta vollenweideri. Naturwissenschaften. 2001; 88(7): 301–305. PubMed Abstract | Publisher Full Text\n\nPowell RJ, Stradling DJ: Factors influencing the growth of the Attamyces bromatificus, a symbiont of Attine ants. Trans Br Mycol Soc. 1986; 87(2): 205–213. Publisher Full Text\n\nRoces F, Núñez JA: Brood translocation and circadian variation of temperature preference in the ant Campotonus mus. Oecologia. 1989; 81(1): 33–37. Publisher Full Text\n\nBollazzi M, Roces F: To build or not to build: circulating dry air organizes collective building for climate control in the leaf-cutting ant Acromyrmex ambiguus. Anim Behav. 2007; 74(5): 1349–1355. Publisher Full Text\n\nJones JC, Myerscough MR, Graham S, et al.: Honey bee nest thermo-regulation: diversity promotes stability. Science. 2004; 305(5682): 402–404. PubMed Abstract | Publisher Full Text\n\nBanschbach VS, Levit N, Herbers JM: Nest temperatures and thermal preferences of a forest ant species: is seasonal polydomy a thermoregulatory mechanism? Insec Soc. 1997; 44(2): 109–122. Publisher Full Text\n\nCokendolhper JC, Francke OF: Temperature preferences of four species of the fire ants (Hymenoptera: Formicidae: Solenopsis). Psyche. 1985; 92(1): 91–101. Publisher Full Text\n\nCoenen-Stass D: Untersuchungen fiber die jahreszeitlichen Klimaprfiferenz der roten Waldameise Formica polyctena. (Hymeno-ptera, Formicidae). Mitteilungen der Deutschen Gesellschaft für Allgemeine und Angewandte Entomologie. 1987; 5. : 44–48.\n\nKipyatkov VE, Schederova SS: Seasonal changes in behavior patterns of the ant Formica polyctena. in an artificial nest with temperature gradient. Zool Zhurnal. 1985; 65(12): 1847–1857. Reference Source\n\nCeusters R: Social homeostasis in colonies of Formica polyctena. Foerst. (Hymenoptera, Formicidae): nestform and temperature preferences. Proc. 8th Int. Cong, International Union for the Study of Social Insects. Wageningen, 1977; pp. 111–112. Reference Source\n\nCalabi P, Porter SD: Worker longevity in the fire ant Solenopsis invicta, ergonomic considerations of correlations between temperature size and metabolic rates. J Insect Physiol. 1989; 35(8): 643–649. Publisher Full Text\n\nBollazzi M, Roces F: Thermal preference for fungus culturing and brood location by workers of the thatching grass-cutting ant Acromyrmex heyeri. Insectes Soc. 2002; 49: 153–157. Publisher Full Text\n\nWeir J: Air flow, evaporation and mineral accumulation in mounds of Macrotermes subhyalinus. (rambur). J Anim Ecol. 1973; 42(3): 509–520. 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Publisher Full Text\n\nRosengren R, Fortelius W, Lindström K, et al.: Phenology and causation of nest heating and thermoregulation in red wood ants of the Formica rufa. group studied in coniferous forest habitats in southern Finland. Ann Zool Fennici. 1987; 24: 147–155. Reference Source\n\nHölldobler B, Wilson EO: The Ants. The Belknap Press of Harvard University Press, Cambridge, 1990; 732 pp. Reference Source\n\nFranks NR: Thermoregulation in army ant bivouacs. Physiol Entomol. 1989; 14(4): 397–404. Publisher Full Text\n\nFrouz J, Finer L: Diurnal and seasonal fluctuations in wood ant (Formica polyctena) nest temperature in two geographically distant populations along a south-north gradient. Insect Soc. 2007; 54(3): 251–259. Publisher Full Text\n\nMartin SJ: Thermoregulation in Vespa simillima xanthoptera. (Hymenoptera, Vespidae). Kontyu. 1988; 56(3): 674–677. Reference Source\n\nZahn M: Temperatursinn, Wärmehaushat und Bauweise der role Waldameisen (Formica rufa. L.). 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Insect Soc. 1970; 17(1): 49–81. Publisher Full Text\n\nMiyata H, Shimamura T, Hirosawa H, et al.: Morphology and phenology of the primitive ponerine army ant Onychomyrmex hedleyi. (Hymenoptera: Formicidae: Ponerinae) in a highland rainforest of Australia. J Nat Hist. 2003; 37(1): 115–125. Publisher Full Text\n\nSanada-Morimura S, Satoh T, Obara Y: Territorial behavior and temperature preference for nesting sites in a pavement ant Tetramorium tsushimae. Insect Soc. 2006; 53(2): 141–148. Publisher Full Text\n\nCole BJ: Nest architecture in the Western harvester ant, Pogonomyrmx occidentalis (Cresson). Insect Soc. 1994; 41(4): 401–410. Publisher Full Text\n\nFletcher DJC, Crewe RM: Nest structure and thermoregulation in the stingless bee Trigona (Plebeina) denoiti Vachal (Hymenoptera: Apidae). J Entomol Soc South Afr. 1981; 44(2): 183–196. Reference Source\n\nLüscher M: Air-conditioned termite nests. Sci Am. 1961; 205: 138–145. Publisher Full Text\n\nIshay JS: Thermoregulation by social wasps: behavior and pheromones. Trans N Y Acad Sci. 1973; 35(6): 447–462. PubMed Abstract\n\nIshay JS, Barenholz-Paniry V: Thermoelectric effect in hornet (Vespa orientalis) silk and thermoregulation in a hornet’s nest. J Insect Physiol. 1995; 41(9): 753–759. Publisher Full Text\n\nGreves T: Temperature studies of termite colonies in living trees. Aust J Zool. 1964; 12(2): 250–262. Publisher Full Text\n\nMorse RA, Laigo FM: Apis dorsata in the Philippines. Monogr. Philipp Assoc Entomol. 1969; 1: 1–96. Reference Source\n\nNielsen MG: An Attempt to Estimate Energy Flow Through a Population of Workers of Lasius Alienus. (Forst), (Hymenoptera: Formicidae). Natura Jutlandica. 1972; 16: 99–107. Reference Source\n\nO´donnell S, Foster RL: Thresholds of response in nest thermoregulation by worker bumble bees, Bombus bifarius nearcticus (Hymenoptera: Apidae). Ethology. 2001; 107(5): 387–399. Publisher Full Text\n\nCassill DL, Tschinkel WR, Vinson SB: Nest complexity, group size and brood rearing in the fire ant, Solenopsis invicta. Insectes Soc. 2002; 49(2): 158–163. Publisher Full Text"
}
|
[
{
"id": "2858",
"date": "22 Jan 2014",
"name": "Jouni Sorvari",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting review on nest thermoregulation strategies in social insects. I think the work is sound and I agree with most parts of the text. However, I think that the authors give too much weight to an assumption that the “dry nest strategy” is a typical strategy for red wood ants. At least in Finland, the large and populous nests are usually located in shaded forests and seem to perform better than the dry nests located in open areas. This is however, overall a good work and deserves to be approved after some fine-tuning. Title and Abstract:The title expects focus on Formica rufa, but in fact the species focused on is Formica polyctena, a member of the Formica rufa species group. I recommend changing the title so that the focus is on Formica rufa group (or “red wood ants”). The Abstract is ok. Article content:The temperature-humidity regulation of red wood ant nest mounds is a very complex process. Because young small nests of Formica polyctena need direct solar radiation to increase the inner temperature of nest, the new nests need to be built in open spots in or close to forests123. Later, during natural succession those spots will turn into forests, i.e., more shaded habitat but richer in food. The nests will become more shaded but due to the “wet nest strategy” they are no longer dependent on direct sun light. Therefore, both of the two types of thermoregulation strategies are applied in natural populations, but in different stages of nest the development. The situation would be different if the forest succession did not advance and the nest grows in a spot which stays open for a longer period. This is a typical situation in habitats with anthropogenic influence, where the natural succession and penetration of forest towards agricultural or urban field is prevented. Then the same strategy that is used in shaded forest is not optimal for more permanently open areas. Therefore, the statement that “The dry nest thermoregulatory strategy is more common than the wet nest strategy in F. polyctena” may be true due to anthropogenic pressure, but would possibly not be so in natural habitats. In addition, the statement seems to be based on data studied in two locations in Czech Republic without mention of the naturalness of the habitats. The more fragmented the forests are the higher the proportion of nests that are located in, or close to forest edges and other open areas, possibly causing an impression that the ant prefers open areas. I recommend using term “moist nest” instead of “wet nest”, because “wet” sounds too wet. The nest architecture and building method of Oecophylla weaver ants is mentioned in the text, but their function in thermoregulation is not mentioned. The authors should either describe how the architecture (or building method) affects thermoregulation of such a nest, or remove the sentence. Although Acromyrmex heyeri builds thatched nests this species does not belong to the ‘thatch ants’ which is in fact an American term reserved for red wood ants of the Formica rufa group. The table:Large and most populous nests of the red wood ants, especially the polygynous ones, e.g., Formica aquilonia and F. polyctena, are often located in shaded forests where direct sun light does not cause high oscillations in outer layers of the nest. It is possible that the description does not fit mature nests in natural forest environment, but young and smaller nests, or nests in anthropogenic fragmented forests. References list:The reference list should be proofread because of some mistakes e.g., in reference #13 Formicas. Str. -> Formica s. str.; in some references the species name is not in italics where it probably should be; and the journal abbreviation for Insectes Sociaux should always be Insect Soc.",
"responses": [
{
"c_id": "736",
"date": "19 Mar 2014",
"name": "Štěpánka Kadochová",
"role": "Author Response",
"response": "We thank Dr. Jouni Sovari very much for his review and valuable comments concerning our review paper. All typos and mistakes have been corrected; suggested comments and changes have been included in the latest version of the paper including the change of title. For comments which need more explanation please see authors replies bellow.We hope that their explanation and changes made are sufficient enough for the paper version 2 to be accepted. JS: I recommend using term “moist nest” instead of “wet nest”, because “wet” sounds too wet. Reply: The major reason for using wet nests was to be consistent with previous literature. Term „wet nests“ was adopted because it is how the thermoregulation strategy was described in the previous literature namely Frouz 1996, 2000, Frouz and Finner 2007 . Moreover the moisture of nest material (measured gravimetrically) should reach up to 72%. We believe such level of moisture justify using term wet (15. Frouz J: The effect of nest moisture on daily temperature regime in the nests of Formica polyctena. wood ants. Insec Soc. 2000; 47(3): 229–235.) JS: “… Oecophylla weaver ants is mentioned in the text, but their function in thermoregulation is not mentioned …” Reply: This is very good note, however the thermoregulatory function of Oecophylla nests is not described in any paper so far. We double check that text do not imply any thermoregulation function in these nest. The nest of Oecophylla ants were mentioned only to emphasis the great variability of nesting strategies occurring in ants. JS – Table 1 „. It is possible that the description does not fit mature nests in natural forest environment, but young and smaller nests, or nests in anthropogenic fragmented forests.“ Reply: Authors agree with the referee, the thermoreegulatory strategy description is bassed mainly on limited number of studies performed in Czech republic and in Finland (49. Frouz J, Finer L: Diurnal and seasonal fluctuations in wood ant (Formica polyctena) nest temperature in two geographically distant populations along a south-north gradient. Insect Soc. 2007; 54(3): 251–259). These studies include large variety of nest sizes from nest with volume several litres to volume many thousand of liters. We agree that role of solar radiation is bigger in smaller nests and inner heating is more important in bigger nest and we note this in Table 1Moreover to make it clear authors mention the possible of different temperation fluctuation in the text as follow: „The seasonal temperature fluctuations ... studied by Frouz & Finer49... both in Finland and the Czech Republic the ant colonies maintained a high nest temperature. Nest temperatures fluctuated more in the Czech Republic than in Finland, possibly because of greater differences between day and night ambient temperatures.“"
}
]
},
{
"id": "2859",
"date": "04 Feb 2014",
"name": "Haitao Wu",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis review manuscript represents an interesting study into the various thermoregulation strategies used in ant nests and mounds. A particular strength of the manuscript is the conclusion that there are two main types of thermoregulation strategies used: ‘thermal gradient’ in nests or a ‘stable temperature’ with a heat core. The review concentrates mainly on three aspects to reveal these strategies: nest/mound material and architecture properties, ant behavioral reaction and ant metabolic heat. The manuscript is well written and the discussion seems to be sound. I believe it is a good paper on ant nest ecology, but there are several issues that should be considered. Upon reading this paper it is clear that the two different thermoregulatory strategies are its core point, which is explained from three aspects. However these two thermoregulatory strategies are an existing phenomena which are simply being discussed and explored in more detail and should therefore not be the ‘conclusion’ of this article. Instead I think it would be better to state the existence of these two strategies in the Introduction section and then discuses the mechanisms of the phenomena in the rest of the article. Some of the content is rather long and would benefit from being condensed a little to make the overall message clearer. For example: the termites in Table 1 and the details of strategies used by bees, bumblebees and wasps. As this paper focuses on ant thermoregulation characteristics, the content on other insects should deleted as they are not really necessary. The explanations of \"ant metabolic heat\" for ant nest thermoregulation in \"Use of metabolic heat for nest thermoregulation in ants\" are not clear. From the explanations given I still cannot understand how ant metabolic heat impacts on ant mound temperatures or how the ants adopt an ant mound temperature. This content needs to be further explained. The conclusion could do with a rewrite. It should not only reiterate the two opposite thermoregulatory strategies but also condense the messages from other sections, namely: nest/mound material and architecture properties, ant behavioral reaction and ant metabolic heat.",
"responses": [
{
"c_id": "735",
"date": "19 Mar 2014",
"name": "Štěpánka Kadochová",
"role": "Author Response",
"response": "We thank Haitao Wu very much for his review and approval of our review paper. Suggested comments and changes have been included in the latest version of the paper. For cases of authors disagreement with the referee please see comments bellow. Point 2 – details of thermoregulatory strategies in other insectsReply: The strategies of other social insects species are used as comparism, especialy the bees thermoregulation which is very well studied. Authrs would like to point out certan paralel between the thermoregulation in Formica polyctena mound to that in bee hive, even though achieved by different mechanisms Point 3 – ant metabolic heatingReply: The concept of „metabolic heating“ has been described using all known information so far. The metabolic heat production of Formica rufa ants has not been studied since the Coenen-Strass study in 1980 so the exact physiological mechanism of metabolic heating has not been clarified yet."
}
]
},
{
"id": "3521",
"date": "03 Mar 2014",
"name": "Clint Penick",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have provided a clear update to the literature on thermoregulation of ant nests and also include interesting anecdotes from the literature, such as Lindauer’s experiment with bees on lava plains. The arrangement of the article focuses on species that regulate temperature by moving brood across a temperature gradient (e.g. Solenopsis) vs. species that modify the thermal environment to maintain a core nest temperature (e.g. Formica polyctena). This distinction is perhaps the most interesting part of the paper, but in its current form the organization is not clear. I have the following comments:The authors make their strongest argument for distinguishing between these two strategies in the second to last paragraph. This paragraph should instead be the last paragraph of the Introduction, which would help establish their argument earlier. In addition, several other sections throughout the manuscript could make this distinction more apparent (especially in “Behavioural reactions of ant workers”). It may help to identify which species fit your definitions. Pogonomyrmex, Solenopsis invicta, Camponotus, and Acromyrmex fit into brood/fungus movers; Formica, Atta, Eciton, termites, and bees seem to fit more into core nest temperature maintainers. Likewise, the table does not clearly fit within the papers main theme. It may help to add a column that specifies whether each type of nest is associated with brood translocation, maintaining a stable core temperature, or possibly neither. One question that is not addressed in this paper is why these two strategies exist? There is some discussion about wet vs. dry thatch nests in open and forested areas, but I think it would make sense to describe the ecological conditions where each thermoregulation strategy is commonly used in more detail. The discussion about nest ventilation in Atta species is not quite accurate. Currently, there is little evidence that nest ventilation strongly impacts temperature; rather, nest ventilation facilitates CO2 exchange. This has also been argued with respect to termite nests, where termite nest chimneys do not seem to affect nest temperature.",
"responses": [
{
"c_id": "734",
"date": "19 Mar 2014",
"name": "Štěpánka Kadochová",
"role": "Author Response",
"response": "We thank Clint Penick very much for his review and approval of our review paper. Suggested comments and changes have been included in the latest version of the paper. For cases of authors disagreement with the referee please see comments bellow. Point 4 – why those strategies exist? Reply: More discussion concerning this topic could be found in part „Use of metabolic heat for nest thermoregulation in ants“ Point 5 – ventilation in Atta speciesReply: Authors used information quoted in references, which suppose the ventilation to affect nest temperature.(Kleineidam et. all 2001; Powell and Stradling 1986, Bollazzi and Roces 2010). However to make situation clear the sentences about ventilation have been changed to „This ensures exchange of respiratory gasses and optimal thermal conditions for symbiotic fungi which are damaged by temperatures higher than 30°C“ and „Even more efficient ventilation systems, providing both temperature regulation and respiratory gasses exchange, can be found in nests of the termite Mactorermes bellicose.“"
}
]
}
] | 1
|
https://f1000research.com/articles/2-280
|
https://f1000research.com/articles/2-125/v1
|
10 May 13
|
{
"type": "Data Article",
"title": "A novel variable delay Go/No-Go task to study attention, motivation and working memory in the head-fixed rodent",
"authors": [
"Samuel D Dolzani",
"Shinya Nakamura",
"Donald C Cooper",
"Samuel D Dolzani",
"Shinya Nakamura"
],
"abstract": "In order to parse the causal elements underlying complex behaviors and decision-making processes, appropriate behavioral methods must be developed and used in concurrence with molecular, pharmacological, and electrophysiological approaches. Presented is a protocol for a novel Go/No-Go behavioral paradigm to study the brain attention and motivation/reward circuitry in awake, head-restrained rodents. This experimental setup allows: (1) Pharmacological and viral manipulation of various brain regions via targeted guide cannula; (2) Optogenetic cell-type specific activation and silencing with simultaneous electrophysiological recording and; (3) Repeated electrophysiological single and multiple unit recordings during ongoing behavior. The task consists of three components. The subject first makes an observing response by initiating a trial by lever pressing in response to distinctive Go or No-Go tones. Then, after a variable delay period, the subject is presented with a challenge period cued by white noise during which they must respond with a lever press for the Go condition or withhold from lever pressing for the duration of the cue in the No-Go condition. After correctly responding during the challenge period (Challenge) and a brief delay, a final reward tone of the same frequency as the initiation tone is presented and sucrose reward delivery is available and contingent upon lever pressing. Here, we provide a novel procedure and validating data set that allows researchers to study and manipulate components of behavior such as attention, motivation, impulsivity, and reward-related working memory during an ongoing operant behavioral task while limiting interference from non task-related behaviors.",
"keywords": [
"Go/No-Go",
"attention",
"motivation",
"working memory",
"head-fixed rodent"
],
"content": "Introduction\n\nThe use of operant behavioral tasks that utilize the head-immobilized condition in rodents have some advantages over tasks that occur under freely moving conditions by limiting the range of possible non task-relevant behaviors and distractors. While the head-restrained behaving rodent has been examined for many years, the relevant literature is minimal in comparison to similar behavioral tasks involving freely moving animals. Head immobilization is common in acute electrophysiological recordings and has been extended to study cardiovascular function, neuroimaging, reflex adaptation, and licking movements1. To date, very few experiments have assessed psychological and physiological processes simultaneously occurring during operant behavioral tasks in the awake, head-restrained rodent1.\n\nThe Go/No-Go task, a canonical paradigm in animal behavior and psychology, requires the subject to initiate an operant conditioned response during one stimulus (CS+) and withhold from such a response during the presence of the opposite type of stimulus (CS-)1,2. This type of behavioral task has a high level of construct validity and clinical utility when studying the brain motivation/reward pathways and behaviors such as impulsivity. Impulsivity and motivational deficits are complex behavioral phenotypes implicated in a number of psychiatric disorders including attention deficit hyperactivity disorder (ADHD), mania and depression associated with bipolar disorder, schizophrenia, pathological gambling, borderline personality disorder, and substance abuse2. Utilization of an appropriate variant of the rodent Go/No-Go task allows for these complex, clinically relevant behaviors to be interrogated and manipulated. Data obtained from these studies provide the opportunity for development of novel future pharmacotherapies and behavioral interventions2.\n\nThe head-immobilized rodent Go/No-Go task we have developed is a procedure that provides the framework for performing repeated electrophysiological recordings and simultaneous molecular and pharmacologic interventions during ongoing operant behavioral tasks. In addition, this technique allows for accurate stereotaxic localization during electrophysiological recordings, daily anesthesia-free recordings in various brain regions, rapid electrode repositioning during chronic recording sessions, and high quality stabilized recordings3. With the advent of optogenetics, the head-restrained procedure allows for these recordings to be coupled with cell-type specific silencing and activation, free from electrophysiological artifacts4,5.\n\n\nMethods\n\nFour male Sprague Dawley rats and 28 male Long Evans rats (8 weeks and 200–300 g at time of surgery), bred at the University of Colorado Boulder, were used for all experiments. Animals were maintained on a 12 h reverse light/dark cycle (lights off at 07:00). The behavioral experiments were conducted during the dark period. Animals were singly housed and water-restricted, while food was available ad libitum for the duration of the experiment. All procedures were in compliance with animal care standards set forth by the Institute for Animal Care and Use Committee at the University of Colorado Boulder.\n\nAnimals were stereotaxically affixed with stainless steel head-caps prior to any behavioral training. Rats were deeply anesthetized with an intraperitoneal injection of ketamine-xylazine (Sigma-Aldrich; 80 mg/ml ketamine/6 mg/ml xylazine) and restrained on a custom stereotaxic device (Old School Industries, CO, USA). Head fur was shaved and a rostro-caudal incision was made from the region roughly 10 mm anterior to bregma, extending 5–7 mm posterior to lamba. Connective tissue on the skull was carefully removed and temporal muscles were retracted from the dorsolateral region of the skull. Vasculature was cauterized using 30% hydrogen peroxide (Sigma-Aldrich, USA) and quickly rinsed with sterilized saline. Eight self-tapping screws (Small Parts, IN, USA) were implanted on the dorsal surface of the skull and four screws were implanted on the lateral region of the skull to confer head-cap stability and strength. After screws were implanted, the skull was sterilized using povidone-iodine (9%) (Sigma-Aldrich, USA) then desiccated with sterile cotton swabs. A thin layer of radiopaque glass ionomer restorative cement (GC America Inc., USA) was applied to the skull and around the base of the reinforcing screws to increase adhesion strength between the skull and head holder cap. After allowing the radiopaque cement to set, two horizontal stainless steel head restraint bars were stereotaxically positioned directly above the exposed dorsal region of the skull (5 mm anterior to bregma and 3 mm posterior to lamda). Bars were positioned approximately 5 mm above the dorsal surface of the skull and acrylic dental cement (Stoelting, USA) was applied between the head restraint bars and the skull. After implantation of screws and head restraint bars, surrounding areas of the skull were disinfected using povidone-iodine and antibiotic ointment (Sigma-Aldrich, USA) was applied around the site of incision. Animals were given 7 days to recover with food and water available ad libitum.\n\nAll behavioral training was conducted using a custom head-restraint system (Old School Industries, CO, USA) in a red acrylic box. A reward-delivery nozzle, lever, sensor systems for detecting licking movements and lever-presses, and a speaker were placed around the head-restraint stage. All computer software used for the experiments was customized using LabVIEW (National Instruments, USA) and a digital input/output interface (USB-6008, National Instruments, USA) was used for controlling all devices.\n\n\nBehavioral conditioning procedure\n\nAfter animals recovered from surgery, they were handled for 3–5 days and allowed to explore (20 min/day) the operant conditioning boxes that they were restrained within for the remaining duration of the experiment. The purpose of this was to familiarize the animal with the experimental environment and reduce context-related stress and excitability. Subjects were head restrained on an ergonomically designed custom head-restraint system (Old School Industries, CO, USA) for 60 minutes/day during the habituation period of the training procedure. During this time period, subjects were administered 0.3 M sucrose (Sigma-Aldrich, USA) non-contingently through a reward delivery nozzle positioned near the mouth. Approximately 40 µl sucrose was delivered every 10 seconds (10 s inter-trial interval (ITI)) over the course of the 60 minute habituation session, yielding a total reward delivery of 15 ml/60 minute session.\n\nFollowing successful sucrose consumption under non-contingent reward delivery conditions, subjects were trained to press a right forepaw lever to receive sucrose reinforcement (Figure 1). This stage of operant behavioral training was intended to facilitate the contingent relationship between lever pressing and reward delivery. This phase of training persisted until the subject responded with >1000 lever presses for three 2–3 consecutive days. The next phase of training was the Lever/Tone association phase during which subjects learned to lever press in the presence of an audible tone. The two tones randomly presented were either 3000 Hz or 9000 Hz, which corresponded to the same frequencies used in the Go and No-Go constituents of the later stages of the training procedure. This stage of training began with a 5 s tone presentation with 8 µl sucrose delivered for every lever press occurring during the tone (15 s ITI). Subjects were switched to the next stage of training after >70% correct responding during the presence of the tone. The next phase of training was a reduction in the duration of the tone presentation (5 s to 3 s). After achieving >70% correct responding using these parameters, tone duration was finally decreased to 2 s with a single lever press contingent sucrose delivery of 40 µl. The final phase persisted until subjects achieved >80% correct responding for 2 consecutive sessions.\n\nLever presses, initiation tone, challenge period, reward tone, and sucrose reward delivery components are shown for both trials. The black boxes represent the relative timing of the rats response (lever press) in response to the auditory cues. ITI: inter-trial interval.\n\nFirst, subjects were trained to perform the No-Go constituent of the behavioral task after successfully completing the Lever/Tone association phase (Figure 1). During No-Go trials, the subject was required to initiate a trial by lever pressing in response to 3000-Hz, 2-s tone (initiation tone). Correct trial initiation yielded an 8 µl sucrose delivery. Following the initiation tone response, a variable delay (1–3 s) was followed by a 2-s white-noise presentation (challenge period). This was the specific epoch in which the subject must withhold from lever pressing (No-Go). Following successful suppression of lever pressing during the challenge period, the 2-s 3000-Hz tone was presented again (reward tone). A second lever press was required during the presence of the final reward tone and a larger 40 µl sucrose was delivered in response to this lever press. Following a 15-s ITI, the next initiation tone was presented. If the subject failed to initiate a trial or withhold from lever pressing during the challenge period, the trial was terminated and a 15-s ITI followed. No-Go acquisition training proceeded until the subject responded at >80% correct challenge period responding for two consecutive days before moving to the Go condition training.\n\nAfter successful acquisition of the No-Go constituent of the training procedure, subjects began daily 90 minute Go training sessions (Figure 1). In a Go trial, subjects must lever press to initiate a trial during the presence of a 9000-Hz, 2-s initiation tone. 8 µl of sucrose was delivered in response to the lever press. Following initiation of a trial, a 2-s white noise (challenge period) was presented after a variable delay (1–3 s). The subject must lever press (Go) during the challenge period. Lever pressing during the challenge period terminated the white noise and was followed by a 2-s final reward tone (9000 Hz). The larger 40 µl sucrose reward was only delivered if the subject correctly lever pressed during the reward tone. In the early stage of Go training, a white noise signal of indefinite duration was used for the challenge period. As subjects learned the association between the 9000-Hz initiation tone and lever pressing during the challenge period their reaction time decreased and the challenge period duration was decreased to 2 s. Subjects were trained using these parameters until they achieved >80% correct responding during the 2-s challenge period for 2 consecutive days before a randomize within session Go/No Go condition began (data not included).\n\n\nResults\n\nSubjects rapidly acquired the ability to consume sucrose delivered non-contingently through a reward delivery nozzle. Following acquisition of sucrose consummatory behavior, subjects quickly learned (<3 days) an operant response by lever pressing (Lever) to receive sucrose reinforcement (Figure 2). On average, subjects reached the specified criteria of >1000 presses/90 minute training session in <3 consecutive daily training sessions (n=24). Next, subjects reliably acquired operant lever pressing behavior during the presence of a tone (Lever/Tone association) to receive sucrose reinforcement (Figure 2). The Lever/Tone association stage of training required an average of 8 training sessions to achieve the criteria of >70% correct responding for two consecutive days (n=20).This requisite stage of training was intended to facilitate the association between tone presentation, lever pressing, and concomitant primary reinforcement. No-Go training (No-Go) followed the Lever/Tone association training (Figure 2). The No-Go constituent of the behavioral task required an average of 22 daily training sessions to achieve criteria of >80% correct challenge period responding (n=14). Finally, subjects were trained to perform the Go constituent (Go) of the Go/No-Go task (Figure 2). This component of the task required an average of 14 daily training sessions to achieve a criteria of >80% correct 2 s challenge period responding for 2 consecutive days (n=13). Raster plots of five consecutive representative No-Go trials (Figure 3A) and histograms of lever pressing and licking during the initiation tone, challenge period, and reward tone are shown (Figure 3B). The histograms represent cumulative responding across a 90 minute training session. Subjects lever pressed in response to an initiation tone and received an initial small sucrose reinforcement (8 µl), withheld from lever pressing (No-Go) during the challenge period, and emitted a final lever press during the final reward tone to receive a larger 40 µl sucrose reinforcement. One example of a failed No-Go trial is shown (4th trial on the raster plot). The subject emitted a lever press during the challenge period of the No-Go trial, resulting in a failed trial and no final 40µl sucrose delivery. Raster plots of five consecutive Go trials (Figure 4A) and histograms of lever pressing and licking during the initiation tone, challenge period, and reward tone are shown (Figure 4B). Subjects initiated a trial by lever pressing during the initiation tone, emitted a lever press during the challenge period (Go), and lever pressed during the final reward tone to receive sucrose reinforcement. One example of a failed Go trial is shown (1st trial on the raster plot). The subject failed to lever press during the challenge period of the Go trial and the final 40 µl sucrose reward was not delivered. The data set from all rats is presented.\n\nThe average and standard error of the mean training sessions required to achieve specified criteria for acquisition of each head-fixed training condition of the behavioral task is shown. Lever: n=24, Lever/Tone: n=20, No-Go: n=14, Go: n=13.\n\nRaster plot of five consecutive No-Go trials (A) and histograms of lever pressing and licking during initiation tone, challenge period, and reward tone (B). Green bar: initiation tone and reward tone, orange bar: challenge period, magenta bar: reward delivery, black: lick, blue: lever press. (Histogram bin size=100 ms)\n\nRaster plot of five consecutive Go trials (A.) and histograms of lever pressing and licking during the initiation tone, challenge period, and reward tone (B.). Green bar: initiation tone and reward tone, orange bar: challenge period, magenta bar: reward delivery, black: lick, blue: lever press. (Histogram bin size=100 ms). The second peak on the histogram of lever pressing during the challenge period represents lever presses during the final reward tone. Note the emergence of non cued lever presses and licking during the challenge period that represents the idiosyncratic variable initiation during the Go challenge tone and subsequent reward tone delivery under the Go condition.\n\n\n\n4 Data Files\n\nhttp://dx.doi.org/10.6084/m9.figshare.654019",
"appendix": "Author contributions\n\nSDD, SN, and DCC designed experiments and analyzed the data. SDD and SN performed the experiments. SDD wrote the paper. All authors agreed the final manuscript for publication.\n\n\nCompeting interests\n\nNo competing interests were declared.\n\n\nGrant information\n\nThis work was supported by R01 DA24040 (DCC).\n\n\nAcknowledgments\n\nThe authors would like to thank Dr. Mike Baratta and Matthew Pomrenze for their insightful commentary and intellectual faculty throughout the duration of the project.\n\n\nReferences\n\nSchwarz C, Hentschke H, Butovas S, et al:The head-fixed behaving rat - Procedures and pitfalls. Somatosens Mot Res. (2010); 27: 131–148.\n\nWinstanley CA: The utility of rat models of impulsivity in developing pharmacotherapies for impulsive control disorders. Br J Pharmacol. (2011); 164: 1301–21.\n\nChaniary KD, Baron MS, Robinson P, et al:A novel stereotaxic apparatus for neuronal recordings in awake head-restrained rats. J Neurosci Methods. (2011); 198: 29–35.\n\nZhang F, Wang LP, Brauner M, et al:Multimodal fast optical interrogation of neural circuitry. Nature. (2007); 446: 633–9.\n\nBoyden ES, Zhang F, Bamberg E, et al:Millisecond-timescale, genetically targeted optic control over neural activity. Nat Neurosci. (2005); 8: 1263–8."
}
|
[
{
"id": "1038",
"date": "03 Jul 2013",
"name": "Kenichiro Tsutsui",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis interesting paper describes the details of a go/no-go task designed for head-restrained rats. A detailed description about the training procedure is obviously beneficial for the biological community since the use of behaving rats is emerging in optogenetics and electrophysiology studies. I would like to ask the authors to consider the following points for the improvement of the paper. (All are “minor” comments.)1. Go/no-go tasks have long been commonly used in behavioral psychology, and its application to behavioral electrophysiology with head-restrained monkeys is as early as in 1980s. The authors could briefly mention the history of go/no-go task in the 'Introduction' section. 2. I am not entirely sure which part of the described go/no-go task is “novel”. Please point it out clearly in the manuscript which part of this protocol is different from the conventional go/no-go task procedure. 3. How stable is the performance of a typical subject that has reached the criteria of training? It would be helpful to show the weekly or monthly performance of a representative subject after the completion of training.",
"responses": []
},
{
"id": "1050",
"date": "09 Jul 2013",
"name": "Jeff Dalley",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting and well-conducted study on the development and validation of a novel, symmetrically-reinforced Go/No-Go task in rats. The novelty of this procedure presumably rests with head fixing the animals to limit the impact of extraneous behaviour, and the configuration of the task itself, which enables several component cognitive and behavioural processes to be independently assessed. One could imagine using such a task in a variety of settings to assess attention (e.g. based on Posner’s task), inhibitory response control, and working memory. However, this is not a radically new approach; for example, head restraint is often used to avoid the use of a general anaesthetic agent in MR and PET imaging and head restraint has been used for decades to facilitate neurophysiological experiments in non-human primates. Furthermore, restraint itself is a potent activator of the stress response which may have unintended consequences for brain-behaviour relationships. It would have been interesting to compare blood corticosterone in head-fixed and non-head-fixed animals. The reported behavioural findings suggest that sucrose was sufficient as a reinforcer but crucially, how stable was performance both within and between sessions? Overall, the article is clear and concise with methods that are fully transparent. Although the Title and Abstract reflect the content of the article it would be helpful to explain the novel aspects of the present protocol.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-125
|
https://f1000research.com/articles/3-75/v1
|
18 Mar 14
|
{
"type": "Commentary",
"title": "Advancing the activity cliff concept, part II",
"authors": [
"Dagmar Stumpfe",
"Antonio de la Vega de León",
"Dilyana Dimova",
"Jürgen Bajorath",
"Dagmar Stumpfe",
"Antonio de la Vega de León",
"Dilyana Dimova"
],
"abstract": "We present a follow up contribution to further complement a previous commentary on the activity cliff concept and recent advances in activity cliff research. Activity cliffs have originally been defined as pairs of structurally similar compounds that display a large difference in potency against a given target. For medicinal chemistry, activity cliffs are of high interest because structure-activity relationship (SAR) determinants can often be deduced from them. Herein, we present up-to-date results of systematic analyses of the ligand efficiency and lipophilic efficiency relationships between activity cliff-forming compounds, which further increase their attractiveness for the practice of medicinal chemistry. In addition, we summarize the results of a new analysis of coordinated activity cliffs and clusters they form. Taken together, these findings considerably add to our evaluation and current understanding of the activity cliff concept. The results should be viewed in light of the previous commentary article.",
"keywords": [
"Over the past decade",
"the activity cliff concept has been increasingly discussed in the chemoinformatics and medicinal chemistry literature1–3. In the practice of medicinal chemistry",
"activity cliffs",
"which are formed by structurally similar or analogous compounds with large potency differences for a given target1",
"2",
"have long been considered during chemical optimization efforts",
"typically for individual compound series2. However",
"the increasing popularity of the activity cliff concept can at least in part be attributed to computational exploration and large-scale analysis2",
"3. In fact",
"much of our current knowledge about activity cliffs has resulted from compound data mining and other chemoinformatics investigations2–4. Hence",
"in addition to supporting practical applications in compound development",
"activity cliff research is an area where chemoinformatics and medicinal chemistry meet."
],
"content": "Introduction\n\nOver the past decade, the activity cliff concept has been increasingly discussed in the chemoinformatics and medicinal chemistry literature1–3. In the practice of medicinal chemistry, activity cliffs, which are formed by structurally similar or analogous compounds with large potency differences for a given target1,2, have long been considered during chemical optimization efforts, typically for individual compound series2. However, the increasing popularity of the activity cliff concept can at least in part be attributed to computational exploration and large-scale analysis2,3. In fact, much of our current knowledge about activity cliffs has resulted from compound data mining and other chemoinformatics investigations2–4. Hence, in addition to supporting practical applications in compound development, activity cliff research is an area where chemoinformatics and medicinal chemistry meet.\n\nIn a previous commentary4, we have summarized key aspects of the activity cliff concept and discussed further extensions and refinements. Among others, discussed topics have included the current frequency of occurrence of activity cliffs, their dependence on chosen molecular representations, their target distributions, and associated structure-activity relationship (SAR) information4. Herein, we present a follow up to this commentary, which has been catalyzed by the availability of new results concerning the ligand efficiency and lipophilic efficiency of activity cliff partners as well as the topology of coordinated activity cliffs formed across currently available bioactive compounds. These findings should also be considered as further advancements of the activity cliff concept and viewed on the basis of the previous commentary article.\n\n\nActivity cliff definition\n\nThe definition of activity cliffs requires the specification of a similarity criterion (when are two compounds “similar”?) and a potency difference criterion (when is a potency difference “large” and “significant”?)1,2. Molecular similarity can be assessed in a variety of ways, which can roughly be divided into chemical descriptor-based approaches, which require the calculation of similarity values based on the comparison of chosen molecular representations2, and substructure-based approaches, which directly establish structural relationships (on the basis of molecular graphs)2. Among substructure-based approaches, the matched molecular pair (MMP) formalism5 has become very popular in recent years. An MMP is defined as a pair of compounds that are distinguished by the exchange of a substructure at a single site5 termed a chemical transformation6. The formation of an MMP can thus be considered as a possible similarity criterion for activity cliff formation. To define activity cliffs, transformation size-restricted MMPs have been introduced in which transformations are limited to small and chemically meaningful replacements7. Accordingly, transformation size-restricted MMPs mostly account for structural analogs.\n\nIn the following, we consistently adhere to our previously rationalized preferred activity cliff definition4:\n\n(a) Similarity criterion: Formation of a transformation size-restricted MMP.\n\n(b) Potency difference criterion: At least two orders of magnitude (≥ 100-fold).\n\n(c) Activity measurements: Equilibrium constants (Ki values).\n\nSo defined activity cliffs have also been termed MMP-cliffs7.\n\n\nLigand efficiency and lipophilic efficiency\n\nFor the assessment of activity cliffs, compound potency has thus far been a focal point, consistent with the original activity cliff definition. However, during compound optimization, other criteria are often applied to monitor progress that relate potency to changes in molecular weight (MW) or hydrophobicity8. These criteria are formalized as optimization indices and include, among others, ligand efficiency (LE)8,9 or ligand lipophilic efficiency (LLE)8,10, which is also termed lipophilic efficiency (LipE)8,11,12. For an active compound, LE yields the fraction of potency per non-hydrogen atom or MW unit9. In the presence of strong and specific ligand-target interactions, LE should increase during compound optimization; in other words, a gain in potency should not primarily be attributed to molecular size effects. Herein, LE was calculated using the binding efficiency index (BEI)13 defined as:\n\nBEI (LE) = pKi/MW [log unit/kDa]\n\nFurthermore, LipE was calculated as10:\n\nLipE (LLE) = pKi – cLogP [log unit].\n\nLipE is obtained by subtracting the logarithm of the calculated octanol/water partition coefficient, a measure of hydrophobicity, from the logarithm of the equilibrium constant. Hence, LipE indirectly accounts for the influence of hydrophobicity on potency. LipE should also increase during compound optimization because a gain in potency should not primarily be attributed to increasing hydrophobicity of a compound (which often gives rise to non-specific binding effects).\n\nBecause LE and LipE are important and widely applied measures for compound optimization in drug discovery, it makes sense to also consider them in the context of activity cliff formation, given their immediate relevance for SAR analysis. In a recent analysis14, 18,208 activity cliffs were extracted from more than 41,000 unique ChEMBL15 (release 15) compounds with activity against the current spectrum of human targets. Then, differences in LE between lowly and highly potent cliff partners were systematically assessed. For activity cliffs based upon our preferred definition, one would hope that favorable changes in LE might be observed in many instances. However, whether or not systematic trends might be detectable was an open question. The analysis revealed that the formation of 99.1% of all activity cliffs across different targets was accompanied by consistent increases in LE values between lowly and highly potent cliff partners, with, on average, ∆LE = 6.2514. For activity cliffs defined on the basis of calculated (molecular fingerprint-based) similarity values, comparable observations were made14.\n\nHere, we report results of LE and, in addition, LipE analysis for the most recent release of the ChEMBL database (version 17). From a total of ~45,000 unique ChEMBL compounds active against 661 different human targets (~77,000 Ki values), 20,080 activity cliffs were isolated. For the highly and lowly potent partners of each cliff, LE and LipE were calculated. The resulting value distributions are displayed in Figure 1. An increase in LE and LipE values for the highly potent cliff partner was detected for 99.1% and 96.7% of all activity cliffs, respectively, with, on average, ∆LE = 6.27 and ∆LipE = 2.42. Hence, similarly positive LE and LipE trends were observed for activity cliff formation (for LipE, this was difficult to predict). These findings further emphasize the relevance of activity cliff information for medicinal chemistry applications because chemical modifications encoded by activity cliffs consistently increase potency, LE, and LipE.\n\nLE (top) and LipE (bottom) value distributions are shown for lowly (red line) and highly potent (green) cliff partners.\n\n\nCoordinated activity cliffs\n\nThe assessment of activity cliffs has conventionally focused on compound pairs. Hence, cliffs are typically considered on an individual basis (including statistical analysis). However, activity cliffs are only formed in isolation if participating compounds have no structural neighbors with which they also form cliffs. This is unlikely for many compound series and data sets, especially those resulting from compound optimization efforts. In earlier studies, series of highly and lowly potent analogs have been identified in different data sets that formed multiple overlapping activity cliffs16. These cliff arrangements have been termed coordinated activity cliffs17. Indeed, based upon global statistical analysis, we have determined that ~97% of all activity cliffs are formed in a coordinated manner4. In principle, coordinated activity cliffs have higher SAR information content than and are thus of particular interest for medicinal chemistry. However, only very little information has thus far been available about how coordinated activity cliffs are formed and what the size of coordinated cliff arrangements might be.\n\nTherefore, in a recent study, all activity cliffs extracted from active compounds in ChEMBL (version 17) were subjected to network analysis18. Activity cliff forming compounds were represented as nodes that were connected by edges accounting for individual activity cliffs. The global network is depicted in Figure 2A. It consisted of activity cliffs formed by compounds with activity against a total of 293 targets, and more than 93% of all activity cliffs were found to be single-target cliffs18. Only 769 (3.8%) of a total of 20,080 activity cliffs were formed in isolation. Coordinated activity cliffs appeared as different-sized disjoint clusters of varying topologies. In total, 19,311 coordinated activity cliffs formed 1303 separate clusters. Among these were 26 clusters consisting of more than 50 compounds and 420 clusters containing six to 15 compounds, hence reflecting a high degree of activity cliff coordination.\n\nIn (A), the complete activity cliff network is shown. Nodes represent compounds and edges activity cliffs. Nodes of highly and lowly potent cliff partners are colored green and red, respectively, and nodes representing a compound that is a highly and lowly potent partner in different activity cliffs are colored yellow. Small sections of the network containing exemplary activity cliff cluster topologies are magnified. On the right, examples of the three most frequently occurring (main) topologies are displayed, which include so-called star, chain, and rectangle topology. In (B), main activity cliff cluster topologies and observed extensions as well as hybrid and irregular topologies are schematically illustrated (pink nodes: star; light blue: chain; purple: rectangle topology; gray: no topology assignment). Dual-color nodes indicate compounds belonging to cluster components with hybrid topologies. Squared nodes represent variable compound numbers (n) for a given topology. For each topology, the number (#) of instances in the network is reported.\n\nThe activity cliff clusters displayed 449 distinct topologies with different frequency of occurrence. Examples are provided in Figure 2A and 2B. The majority of activity cliff clusters, i.e., 861, were assigned to only three recurrent main topologies, termed the star, chain, and rectangle topology, and a limited number of extensions and combinations of these topologies, as illustrated in Figure 2B. The recurrent topologies covered many clusters of small to medium size. Topologies of increasingly large size often had hybrid character or became irregular, as also illustrated in Figure 2B.\n\nThe star topology reflects the presence of a highly or lowly potent compound and multiple analogs having opposite potency, a situation frequently observed in compound optimization. In total, the star topologies and its extensions were detected 351 times. Different from clusters with star topology, chains with more than three compounds and rectangles require the presence of alternating highly and lowly potent compounds forming sequences of activity cliffs or circular arrangements, which are less likely than stars. However, these topologies were also recurrent.\n\nActivity cliff network analysis has revealed how coordinated activity cliffs are formed across currently available compound activity classes. Taken together, the results clearly indicate that many coordinated activity cliffs occur as well-defined clusters with recurrent topologies, which can be easily isolated and subjected to SAR exploration. For a detailed characterization of the global activity cliff network and individual cluster topologies, the interested reader is referred to the original publication18.\n\n\nConclusions\n\nHerein, we have presented an update on the state-of-the-art in rationalizing activity cliffs. Focal points of our analysis have been the large-scale characterization of activity cliffs in terms of ligand efficiency and lipophilic efficiency as well as the visualization and systematic assessment of coordinated activity cliffs. The finding that activity cliff formation is generally accompanied by improvements in ligand and lipophilic efficiency further increases the attractiveness of activity cliff information for compound optimization. In addition, the observation that coordinated activity cliffs often form clusters of well-defined topology, irrespective of specific compound activities, is relevant for SAR analysis. Because activity clusters are rich in SAR information, an important topic for future research will be how such SAR information might be systematically extracted from clusters with different topology.",
"appendix": "Author contributions\n\n\n\nJB designed the study, DS, AVL, and DD collected and organized the data, JB wrote the manuscript, all authors examined the manuscript and agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were declared.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe authors thank Ye Hu for discussions and help with data sets.\n\n\nReferences\n\nMaggiora GM: On outliers and activity cliffs--why QSAR often disappoints. J Chem Inf Model. 2006; 46(4): 1535. PubMed Abstract | Publisher Full Text\n\nStumpfe D, Bajorath J: Exploring activity cliffs in medicinal chemistry. J Med Chem. 2012; 55(7): 2932–2942. PubMed Abstract | Publisher Full Text\n\nStumpfe D, Hu Y, Dimova D, et al.: Recent progress in understanding activity cliffs and their utility in medicinal chemistry. J Med Chem. 2014; 57(1): 18–28. PubMed Abstract | Publisher Full Text\n\nHu Y, Stumpfe D, Bajorath J: Advancing the activity cliff concept [v1; ref status: indexed, http://f1000r.es/1wf]. F1000Res. 2013; 2: 199. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKenny PW, Sadowski J: Structure modification in chemical databases. Chemoinformatics in Drug Discovery. Oprea, T. I., Ed.; Wiley-VCH: Weinheim, Germany, 2005; pp 271–285. Publisher Full Text\n\nHussain J, Rea C: Computationally efficient algorithm to identify matched molecular pairs (MMPs) in large data sets. J Chem Inf Model. 2010; 50(3): 339–348. PubMed Abstract | Publisher Full Text\n\nHu X, Hu Y, Vogt M: MMP-Cliffs: systematic identification of activity cliffs on the basis of matched molecular pairs. J Chem Inf Model. 2012; 52(5): 1138–1145. PubMed Abstract | Publisher Full Text\n\nHopkins AL, Keserü GM, Leeson PD, et al.: The role of ligand efficiency metrics in drug discovery. Nat Rev Drug Discov. 2014; 13(2): 105–121. PubMed Abstract | Publisher Full Text\n\nHopkins AL, Groom CR, Alex A: Ligand efficiency: a useful metric for lead selection. Drug Discov Today. 2004; 9(10): 430–431. PubMed Abstract | Publisher Full Text\n\nLeeson PD, Springthorpe B: The influence of drug-like concepts on decision-making in medicinal chemistry. Nat Rev Drug Discov. 2007; 6(11): 881–890. PubMed Abstract | Publisher Full Text\n\nFreeman-Cook K, Hoffman RL, Johnson TW: Lipophilic efficiency: the most important efficiency metric in medicinal chemistry. Future Med Chem. 2013; 5(2): 113–115. PubMed Abstract | Publisher Full Text\n\nShultz MD: The thermodynamic basis for the use of lipophilic efficiency (LipE) in enthalpic optimizations. Bioorg Med Chem Lett. 2013; 23(21): 5992–6000. PubMed Abstract | Publisher Full Text\n\nAbad-Zapatero C, Metz JT: Ligand efficiency indices as guideposts for drug discovery. Drug Discov Today. 2005; 10(7): 464–469. PubMed Abstract | Publisher Full Text\n\nde la Vega de León A, Bajorath J: Formation of activity cliffs is accompanied by systematic increases in ligand efficiency from lowly to highly potent compounds. AAPS J. 2014; 16(2): 335–341. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGaulton A, Bellis LJ, Bento AP, et al.: ChEMBL: a large-scale bioactivity database for drug discovery. Nucleic Acids Res. 2012; 40(Database issue): D1100–D1107. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVogt M, Huang Y, Bajorath J: From activity cliffs to activity ridges: informative data structures for SAR analysis. J Chem Inf Model. 2011; 51(8): 1848–1856. PubMed Abstract | Publisher Full Text\n\nNamasivayam V, Bajorath J: Searching for coordinated activity cliffs using particle swarm optimization. J Chem Inf Model. 2012; 52(4): 927–934. PubMed Abstract | Publisher Full Text\n\nStumpfe D, Dimova D, Bajorath J: Composition and topology of activity cliff clusters formed by bioactive compounds. J Chem Inf Model. 2014; 54(2): 451–461. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "4181",
"date": "21 Mar 2014",
"name": "Gerhard Müller",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDagmar Stumpfe, Antonio de la Vega de Leon, Dilyana Dimova, and Jürgen Bajorath refer in the submitted commentary entitled “Follow up: Advancing the activity cliff concept, part II” to a previously published commentary on the activity cliff concept, now reporting on (i) how this concept correlates with ligand efficiency measures of “cliff-forming” compound pairs, and (ii) how \"coordinated activity cliffs\" give new insight into a putative course of optimization by tracing the activity-critical compounds introducing activity cliff network and cluster topologies.The group around Jürgen Bajorath has made major contributions over the last few years to the development and establishment of the activity cliff concept within Medicinal Chemistry, documented by numerous seminal publications in the field, that provide a constantly maturing cheminformatics tool useful for the medicinal chemist to assess the steepness of an unfolding structure-activity relationship landscape. Activity cliff-forming compounds give rise to sharp curvatures in the structure-activity relationship surface and thus might suggest that affected groups establish significant pharmacophoric features in an underlying compound class. It is especially the introduction and consequent application of so-called “transformation size-restricted matched molecular pairs” that renders this concept useful for the end-user, since an abstract cheminformatics tool becomes more tangible for the practitioner in the field.In this context the authors set out to consider as to whether the occurrence of activity cliffs is associated with a general and systematic increase in molecular weight, as this is often found in consecutive medicinal chemistry optimization rounds (e.g. transforming a primary hit into a structurally more elaborate lead-like compound).This optimization process is often accompanied by a significant increase in molecular weight, as well as in lipophilicity. For that reason, normalization of detected binding affinities or inhibition data onto e.g. the number of heavy atoms in an underlying molecule, allows to better assess and cross-compare the true efficiency of a given compound acting at a specific target. Compounds of high ligand efficiency but low molecular weight and low lipophilicity are preferred in that they show fewer liabilities in e.g. unspecific binding to other targets, or in the number of putative metabolic soft spots.By applying stringent quality criteria for data mining, the authors have carried out a systematic analysis within the ChEMBL version 17 database identifying MMP cliffs. Approximately 20,000 activity cliffs were generated and systematically analysed for ligand efficiency changes. In more than 99% of all activity cliffs, the more potent cliff-forming compound exhibits consistently higher ligand efficiency, and in more than 96% of all identified MMP cliffs, the more active compound showed an increased lipophilic efficiency. And this finding is derived from more than 45,000 distinct compounds with biological activities reported for more than 661 targets, rendering the underlying dataset truly diverse in its chemical and biological nature.Activity cliff analyses by their algorithmic nature focus on compound pairs, in this contribution so-called matched molecular pairs with a size-restricted transformation accounting for a single structural difference between the two cliff-forming compounds. It seems obvious that the identified and isolated 20,000 MMP-cliffs might cluster into common underlying optimization programs, since the majority of medicinal chemistry optimization campaigns cover a number of subsequent iterative feedback cycles with many structurally related compounds belonging to consecutive design generations. To account for this interdependence of isolated cliff-forming compound pairs, the group around Jürgen Bajorath has embarked into the concept of “coordinated activity cliffs”.Again, this is a very helpful attempt to back-translate an abstract and simplified view on a compound-activity space into the operational world of medicinal chemistry in which those formerly isolated pairs appear in a broader context of more comprehensive chemistry campaigns. By generating and analysing activity cliff network and cluster topologies, a hypothetical optimization pathway can be unfolded that provides additional guidelines to the practitioner on the optimization philosophy.Admittedly, the naive end-user being confronted for the first time with the activity cliff concept will require some time to fully appreciate the intrinsic value of this cheminformatics-based approach towards the analysis of structure-activity relationships. However, the concept becomes more and more intuitive and as such is a true asset that should be applied in small-molecule drug discovery projects.As in the previous commentary of the Bajorath group, I see the attempt to reach high user-friendliness (e.g. by explaining the concept of coordinated activity cliffs) which renders this commentary helpful in alerting the medicinal chemistry community to this very useful, but still under-appreciated concept. The group around Jürgen Bajorath continue to qualify as advocates in that sense, and the community of practicing medicinal chemists should start to move in their direction accordingly.",
"responses": []
},
{
"id": "4182",
"date": "02 Apr 2014",
"name": "J. Richard Morphy",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe twin concepts of molecular matched pair (MMP) analysis and activity cliffs have been widely embraced by medicinal chemists in recent years as a means to explore and understand complex SAR landscapes. The contributions of the Bajorath group have been pivotal to the acceptance of these approaches. Activity cliffs are large changes in a biological activity arising from modest structural differences. In this study size restrictions are imposed on the activity cliff/MMP partners to ensure that structural changes are relatively small. Therefore it might be expected that the activity cliff trends based upon ligand efficiency would mirror those from activity alone. Still it is reassuring that this paper confirms that activity cliffs are indeed associated with large benefits in terms of ligand efficiency. The more interesting observation is that activity cliffs are also associated with significant improvements in lipophilic ligand efficiency (LLE or LipE) since this is less intuitive based on the selection criteria used for the MMP analysis. Therefore this latest study adds real weight to the argument that MMPs and activity cliffs can be very useful concepts within hit and lead optimisation projects.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-75
|
https://f1000research.com/articles/2-285/v1
|
27 Dec 13
|
{
"type": "Research Article",
"title": "Modeling enzootic raccoon rabies from land use patterns - Georgia (USA) 2006-2010",
"authors": [
"John E. Duke",
"Jesse D. Blanton",
"Melissa Ivey",
"Charles Rupprecht",
"Jesse D. Blanton",
"Melissa Ivey",
"Charles Rupprecht"
],
"abstract": "We analyzed how land-use patterns and changes in urbanization influence reported rabid raccoons in Georgia from 2006 - 2010. Using Geographical Information Systems and rabies surveillance data, multivariate analysis was conducted on 15 land-use variables that included natural topography, agricultural development, and urbanization to model positive raccoon rabies cases while controlling for potential raccoon submission bias associated with higher human population densities. Low intensity residential development was positively associated with reported rabid raccoons while a negative association was found with evergreen forest. Evergreen forests may offer a barrier effect where resources are low and raccoon populations are not supported. Areas with pure stands of upland evergreen forest might be utilized in baiting strategies for oral rabies vaccination programs where fewer or no baits may be needed. Their use as a barrier should be considered carefully in a cost-effective strategy for oral rabies vaccination (ORV) programs to contain the western spread of this important zoonotic disease.",
"keywords": [
"enzootic",
"environmental heterogeneity",
"GIS",
"land-use",
"raccoon",
"urbanization"
],
"content": "Introduction\n\nHere we use Geographical Information Systems (GIS) analysis to exhibit how both natural topography and urbanization potentially affect the dynamics of a zoonotic disease in its reservoir species. Today, the primary reservoir for rabies in Georgia, and, in fact, the entire eastern part of the USA, is the raccoon (Procyon lotor). Although dogs previously served as the main reservoir for rabies throughout the USA, dog rabies virus variants in the USA have been eliminated through persistent vaccination programs and policies1. Rabies virus has evolved to adapt to a wildlife meso-carnivore in a population that can be defined by geographical regions where transmission is sustained (Figure 1)2. Over the second half of the last century, the eastern raccoon rabies variant has spread northward and, in Georgia, the raccoon rabies enzootic has been established for over 50 years3.\n\nInfection from the reservoir species to another mammalian host can, and still does occur, but the non-reservoir animal most likely becomes a dead-end host4. Cases of rabies in Georgia in non-reservoir wildlife, such as foxes, coyotes and skunks, and in domesticated animals, such as dogs, cats, and farm animals, can almost always be traced either to the raccoon variant, or to one of the circulating variants among several bat species, through DNA molecular typing5. Using Georgia Department of Public Health laboratory data on raccoon specimens submitted for rabies testing, and the US Geological Survey Land Cover Database, we report a model to predict rabid raccoon cases from land use patterns.\n\nIt is well documented that higher densities of raccoon populations exist in urbanized areas than in more natural habitats, and, that the highest densities are found at the urban-rural interface6–9. An opportunistic omnivore, eating anything from crayfish in creeks to acorns, bird eggs, garden vegetables and even garbage, raccoons have been essentially subsidized by urbanization by the creation of an edge ecosystem. An edge ecosystem is created when habitat is broken up. This can occur naturally, such as disturbance by fire, or can be part of the geomorphology, such as the demarcation between uplands and wetlands. Or it can occur when development creates a new transition between forest and open space. Although some of the open space is replaced with impervious surfaces, i.e. roads and rooftops, higher raccoon populations can be sustained because they have increased access to food resources, and fewer predators, while still using the adjacent natural habitat for protective cover10.\n\nThis raccoon ecological theory, and the way it relates to land use patterns, has been supported in numerous studies where raccoon rabies epidemics occur during the initial introduction of rabies into naïve raccoon populations. These studies have described “sensational” epizootics so fulminating that natural barriers such as rivers can only slow them down11,12. Once the infection becomes enzootic in a raccoon population, large outbreaks rarely occur and the disease becomes incessant (Figure 2)13.\n\nStage a: Pre-raccoon variant – rare cases in raccoons; cases of raccoon rabies might spill over from other wildlife. b1: initial epizootic of raccoon variant rabies as it moves through the county. c1: sequential epizootic. d1: sequential inter-epizootics. (Adapted from Gordon et al., 200413).\n\nThe number of raccoons submitted for rabies testing listed by county and month was acquired from the Georgia Department of Public Health for a five-year period from 2006 to 2010. This database only represents results from raccoons involved in human or domestic animal exposure incidents that are submitted to the Georgia Public Health Laboratories (GPHL). Testing is done solely to inform medical professionals how to proceed with rabies prophylaxis for potential human exposure cases. The presence of rabies virus in the raccoon specimens is tested by direct fluorescent-antibody staining of brain tissue. [http://www.cdc.gov/rabies/diagnosis/direct_fluorescent_antibody.html].\n\nThe mosaic of urban and suburban development poses challenges to this type of surveillance of the raccoon rabies reservoir because higher human population densities bias reporting to passive public health surveillance systems (Figure 3); detection of positive cases in developed areas is likely increased due to the probability of more human-raccoon and domestic animal-raccoon interactions14. Due to potential confidentiality issues concerning the person exposed, this data is aggregated and reported at the county level. The number of raccoons submitted for testing closely parallels the human population densities.\n\nPrior to 2006, only positive cases were reported by GPHL. Because the 2006–2010 GPHL dataset reported both positive and negative submissions, there was opportunity to possibly mitigate the surveillance bias to some extent by incorporating a standardizing variable. The Getis-Ord Gi statistical tool in GIS (ESRI ArcMap 10) was used to exhibit spatial clustering of counties with higher or lower than expected number of positive cases, positive cases per person per square kilometer, and the number of submissions per person per square kilometer (Figure 4)15.\n\nThe clustering of positive cases is mostly away from major population centers when analyzed as a rate of population density. Clustering of submissions/density closely parallels positive cases/density.\n\nWhen the number of positive cases is put at a rate of population density, the clustering disperses away from the metropolitan areas. However, because our model counted the number of positive cases as the outcome variable, we wanted to use some form of the submissions data as the “weighted” standardizing variable on the right side of the regression model to normalize counties.\n\nTable 1 summarizes the human demographic and GPHL testing data. There were forty-six counties that were inclusive in the upper quartile for positive cases. Forty counties were inclusive in the upper quartile for the number of submissions for testing, as well as all other independent land use variables. The high odds ratio for both submissions, and population density, confirms that as there is more testing, there are more positive cases; it is a matter of more intense surveillance, and does not necessarily reflect the pattern of the rabies reservoir in the raccoon. However, when density is used as the denominator to analyze the effect of rate of submissions on disease predictability, the odds ration falls (3.1). The drop in the odds ratio, though still significant, could reflect two possibilities: that there truly are higher raccoon densities in more populated areas and/or that the database bias has been mitigated to some extent.\n\nSub/density is the weighted variable used in the regression model. High enzootic is the number of counties in the upper quartile of positive cases (≥ 8).\n\napeople per km2\n\nbnumber of submitted raccoons for testing per person per km2\n\nOR: odds ration\n\nCI: confidence interval\n\nUsing GIS, we extracted fifteen land use/land cover categories from the National Land Cover Database 2006 [http://www.mrlc.gov/nlcd06_leg.php]. Variable construction was based on the method of Jones et al. 200316, to define low intensity residential, high intensity residential and commercial land use. These three development variables are determined from measuring the impervious surface, and constitute less than 50% (combining “open space” and “low intensity”), 50 – 79% (“medium intensity”), and 80% or greater impervious surface area (“high intensity”), respectively. The agricultural variable was also constructed on the method of Jones et al. 2003, and is a combination of pasture/hay and cultivated crops. Both, demographic data, and land area of each county, were obtained from the 2010 US Census [http://quickfacts.census.gov/qfd/states/13000.html].\n\nThere were 1011 rabid raccoons from a total of 2064 raccoon specimens submitted from 150 counties over the five year period. A univariate analysis was conducted for all variables, including land-use data, human population density, and the number of rabid raccoons (outcome variable), by dichotomizing at the upper quartile to determine crude odds ratios (OR).\n\nUrbanization variables (“Low intensity res”, “High intensity res”, and “Com/Ind/Trans”) had the highest crude odds ratios among all the land use variables examined (Table 2). Shrub and the woody wetlands had similar protective odds ratios (0.2–0.3; CI 0.1–0.9) while evergreen forest had the lowest crude odds ratio at 0.04 (CI 0.006–0.3).\n\napercentage of land use\n\nOR: odds ratio\n\nCI: confidence interval\n\nThe independent variables with a p-value less than 0.1 were selected to build a regression model for predicting the number of rabid raccoons. Variables were eliminated through a manual backwards stepwise regression. Although the selection of independent variables (percentage land-use) to begin the model was determined with dichotomized data (odds ratios), the regression model predicted the number of rabid cases from continuous data. Potential autocorrelation between variables in the model were considered in the backwards stepwise regression by first removing co-variables with a correlation coefficient greater than 0.6; the co-variable with the highest p-value was eliminated. Because of high variance among the counties, including zeros, in the number of rabid raccoons, both Poisson and negative binomial distributions were evaluated for the regression analysis17.\n\nA negative binomial distribution was chosen for the final regression model after comparing the goodness of fit statistics (AIC=795, deviance/degrees of freedom=1.1, Pearson chi-square/degrees of freedom=0.96) to a model using the Poisson distribution (961, 3.2, and 3.2, respectively). The final negative binomial regression model included:\n\n+ 1.9(Sub/density)[p<0.001] – 1.8(Evergreen forest)[p=0.02]– 52.2(Com/Ind/Trans)[p<0.001] + 11.5(Low intensity res)[p<0.001] + 43.5(Barren)[p=0.019] as predictors of total positive raccoon rabies cases.\n\n\nConclusion\n\nWe have presented one example of how anthropogenic changes to the landscape impacts the natural ecology of the earth. This study supports the hypothesis that the majority of rabid raccoons are associated with low intensity urbanized areas. If raccoon populations were not “subsidized” and the raccoon densities were allowed to remain at normal carrying capacities, rabies as a reservoir in raccoons might possibly eradicate itself. Our finding that upland evergreen forests are inversely associated with the presence of rabid raccoons supports this possibility too, and, coincides with epidemiological theory that a population threshold is needed to maintain transmission of any infectious disease18. For example, pine forests that are managed for timber production generally have lower raccoon utilization as compared to other forested habitats19. Therefore, compelling evidence exists that the pure stands of managed pine forests in western and southern Alabama have been attributed as being the major barrier to the spread of the raccoon enzootic further west into Mississippi20.\n\nThe finding that commercial development was a positive predictor of raccoon rabies in the univariate analysis but had a negative coefficient in the final model suggests that, alone, commercial development is a co-associate of the low intensity urbanization phenomenon. Since the commercial development variable remained in the regression model with a negative coefficient however, it seems that as mixed land patterns give way to highly impacted landscapes, with high levels of impervious surface, there reaches a point at which protective cover falls below a minimal threshold.\n\nThe finding that barren land-cover is a predictor of positive raccoon rabies cases has no precedence in the literature, and contributed very little to the model, even with a high coefficient, due to the low proportion of this land type within our entire study area (0.34%). Barren land can mean rock that is exposed either naturally, or from strip mines and gravel pits. There seems to be no ecological reason for these land types to be associated with raccoons. However, “barren” also consists of land with less than 15% vegetative cover that has been temporarily cleared in preparation for development (future urbanization) and can include landfills (food resources) [http://www.hq.nasa.gov/iwgsdi/Barren_Land.html].\n\nThe submissions/population density variable was an attempt to normalize all counties in the multivariate model. We added this variable into the regression equation because the crude odds ratio for submissions/population (alone) was higher (4.1). The lower odds ratio using density as the denominator indicates that testing bias is better controlled. The odds of a county being in the upper quartile of percent evergreen forest coverage and having submitted fewer raccoon specimens for testing per person per square kilometer (the standardized variable) as compared to any other county is 1.1 (CI: 0.5–2.6 CI). This insignificant odds ratio means that although there are fewer submissions for testing in those counties with high coverage of evergreen forest as compared to other counties there is no difference in submission rate among the counties, and surveillance bias has been mitigated.\n\nFinally, our study model has implications for the enzootic areas along an oral rabies vaccination (ORV) zone. This vaccination zone lies along the entire western edge of the eastern raccoon rabies reservoir (see Figure 1). Oral vaccination baits are dropped along roadways and from planes along this front to prevent the westward spread of the raccoon rabies variant. Evidence here suggests support for possibly using few or no baits in a pure stand of upland evergreen forest, at least in the southern US. In a similar fashion to dropping baits along a river in a riparian area, baits could be dropped up to the edge of an upland evergreen forest, as those raccoons in a pure upland evergreen forest may primarily use adjacent habitats for their resource needs21. The baits saved through this procedure could be distributed elsewhere with no loss in control effort. Conversely, bait density could be increased in areas that have been fragmented due to low intensity development.",
"appendix": "Author contributions\n\n\n\nJohn Duke performed all the GIS work, compiled the Excel spreadsheet, wrote the program for the SAS analysis that gave us the model, and wrote the initial draft and some revisions. Jesse Blanton did the statistical analysis for autocorrelation and wrote many revisions. Melissa Ivey and Charles Rupprecht edited and revised the article for enzootic raccoon rabies specific content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe authors acknowledge the staff at the Georgia Department of Public Health for the raccoon rabies data and Richard Rothenberg (Epidemiology), Lisa Casanova (Environmental Health), and Dajun Dai (GIS) at Georgia State University, Institute of Public Health for their manuscript and statistical advice. Thanks to Andres Velasco-Villa and Sergio Recuenco at the Centers for Disease Control for their comments on the manuscript. Thanks to the Atlanta Regional Commission for GIS county shapefiles. The findings and conclusions in this article are those of the authors and do not necessarily reflect the position of the CDC.\n\n\nReferences\n\nVelasco-Villa A, Reeder SA, Orciari LA, et al.: Enzootic rabies elimination from dogs and reemergence in wild terrestrial carnivores, United States. Emerg Infect Dis. 2008; 14(12): 1849–1854. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBlanton JD, Palmer D, Rupprecht CE: Rabies surveillance in the United States during 2009. J Am Vet Med Assoc. 2010; 237(6): 646–657. PubMed Abstract | Publisher Full Text\n\nKappus KD, Bigler WJ, McLean RG, et al.: The raccoon an emerging rabies host. J Wildl Dis. 1970; 6(4): 507–509. PubMed Abstract | Publisher Full Text\n\nReal LA, Russell C, Waller L, et al.: Spatial dynamics and molecular ecology of North American rabies. J. Hered. 2005; 96(3): 253–260. PubMed Abstract | Publisher Full Text\n\nMcQuiston JH, Yager PA, Smith JS, et al.: Epidemiologic characteristics of rabies virus variants in dogs and cats in the United States, 1999. J Am Vet Med Assoc. 2001; 218(12): 1939–1942. PubMed Abstract | Publisher Full Text\n\nRiley SPD, Hadidian J, Manski DA: Population density, survival, and rabies in raccoons in an urban national park. Can J Zool. 1998; 76(6): 1153–1164. Publisher Full Text\n\nSmith HT, Engeman RM: An extraordinary raccoon, Procyon lotor, density at an urban park. USDA Nat Wildl Research Center - Staff Publications, Paper 487 2002. Reference Source\n\nRanda LA, Yunger JA: Carnivore occurrence along an urban-rural gradient: a landscape-level analysis. J Mammal 2006; 87(6): 1154–1164. Publisher Full Text\n\nRosatte R, Ryckman M, Ing K, et al.: Density, movements, and survival of raccoons in Ontario, Canada: implications for disease spread and management. J Mammal. 2010; 91(1): 122–135. Publisher Full Text\n\nSmith TM, Smith RL: Elements of Ecology, (7th ed.). Benjamin Cummings. 2008. Reference Source\n\nSmith DL, Waller LA, Russell CA, et al.: Assessing the role of long-distance translocation and spatial heterogeneity in the raccoon rabies epidemic in Connecticut. Prev Vet Med. 2005; 71(3–4): 225–240. PubMed Abstract | Publisher Full Text\n\nWheeler DC, Waller LA: Mountains, valleys, and rivers: The transmission of raccoon rabies over a heterogeneous landscape. J Agric Biol Environ Stat. 2008; 13(4): 388–406. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGordon ER, Curns AT, Krebs JW, et al.: Temporal dynamics of rabies in a wildlife host and the risk of cross-species transmission. Epidemiol Infect. 2004; 132(3): 515–524. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRecuenco S, Eidson M, Kulldorff M, et al.: Spatial and temporal patterns of enzootic raccoon rabies adjusted for multiple covariates. Int J Health Geogr. 2007; 6: 14–28. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGetis A, Ord JK: The analysis of spatial association by use of distance statistics. Geographical Analysis. 1992; 24(3): 189–206. Publisher Full Text\n\nJones ME, Curns AT, Krebs JW, et al.: Environmental and human demographic features associated with epizootic raccoon rabies in Maryland, Pennsylvania, and Virginia. J Wildl Dis. 2003; 39(4): 869–874. PubMed Abstract | Publisher Full Text\n\nCameron AC, Trivedi PK: Regression analysis of count data. Reference Source\n\nLloyd-Smith JO, Cross PC, Briggs CJ, et al.: Should we expect population thresholds for wildlife diseases? Trends Ecol Evol. 2005; 20(9): 511–519. PubMed Abstract | Publisher Full Text\n\nChamberlain MJ, Conner LM, Leopold BD: Seasonal habitat selection by raccoons (Procyon lotor) in intensively managed pine forests of central Mississippi. Am Midl Nat. 2002; 147(1): 102–108. Publisher Full Text\n\nArjo WM, Fisher CE, Armstrong J, et al.: Effects of natural barriers and habitat on the western spread of raccoon rabies in Alabama. J Wildl Manage. 2008; 72(8): 1725–1735. Publisher Full Text\n\nBeasley JC, Devault TL, Retamosa MI, et al.: A hierarchical analysis of habitat selection by raccoons in northern Indiana. J Wildl Manage. 2007; 71(4): 1125–1133. Publisher Full Text"
}
|
[
{
"id": "3369",
"date": "03 Mar 2014",
"name": "Colleen Jonsson",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI find the content and data of this article acceptable. I would add a few more sentences about the statistical approach to provide more background to those not familiar with a crude odds ratio",
"responses": [
{
"c_id": "730",
"date": "10 Mar 2014",
"name": "John Duke",
"role": "Author Response",
"response": "Thank you so much for your comments.We felt that a reader could quickly look up the term \"crude odds ratio\" to have enough understanding to proceed. I hope we made it clear that the crude odds ratio is a dichotomized, sort of \"quick and dirty\" way to decide which variables to use before proceeding with continuous data in the regression model."
}
]
},
{
"id": "3926",
"date": "06 Mar 2014",
"name": "Nicole Gottdenker",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral comments:This article provides an interesting analysis of the available data on raccoon rabies from a public health database in order to evaluate how land use impacts raccoon rabies transmission. In particular, results show that evergreen forests (likely managed pine stands) are negatively associated with raccoon rabies virus- a finding that has potential implications for habitat types used in the delivery of oral rabies vaccine. Can you justify/explain why you did backwards stepwise regression to eliminate variables versus other approaches for data/model analysis of raccoon rabies predictors?\n\nCan you find some more information from the literature for your discussion that evaluates the relative raccoon population densities/abundances in the types of habitats that you study, even if from other states, in addition to reference 19? Also, can you discuss potential differences in spatial aggregation in raccoon populations relative to the habitat types evaluated in your study?Minor comments:When describing table 1 in the fourth paragraph under “Mitigating the passive public health database”, change ‘odds ration’ to ‘odds ratio’. I suggest eliminating the first sentence in your conclusion.",
"responses": [
{
"c_id": "729",
"date": "10 Mar 2014",
"name": "John Duke",
"role": "Author Response",
"response": "Thank you so much for your comments.While the different statistical methodologies were discussed among the authors and others, the final decision was that, in our opinion, backwards stepwise regression was the most comprehensive way to include any independent variable that might influence reported cases.The National Land Cover Database provides land classification at such a high resolution that it offers more land classes than the literature currently uses to describe raccoon natural history. Current literature describes raccoon abundance/density in a little more broader/general terms. It seems that the Landsat technology used to classify land patterns can overpower the ability to provide on-the-ground, population data in wildlife. There are probably multiple reasons for this gap in information, including prioritization and practicality of research goals. The minor comments were accepted and the article revised."
}
]
}
] | 1
|
https://f1000research.com/articles/2-285
|
https://f1000research.com/articles/3-27/v1
|
27 Jan 14
|
{
"type": "Case Report",
"title": "Postoperative visual loss following cerebral arteriovenous malformation surgery: a case report",
"authors": [
"Nicolai Goettel",
"Jayati Ghosh",
"Pirjo H Manninen",
"Nicolai Goettel",
"Jayati Ghosh"
],
"abstract": "We report the case of a 46 year-old woman presenting with postoperative visual loss in the right eye after craniotomy for excision of an arteriovenous malformation.The intraoperative course was uneventful with hemodynamic stability and maintenance of blood pressure within 10% of the preoperative value. Blood loss was 300 ml; postoperative hemoglobin was 12.4 g/dl. In the recovery room, the ophthalmologic examination revealed decreased visual acuity, color vision, and visual field in the right eye. Assessment of the retina was normal, but the patient showed a relative afferent pupillary defect consistent with the clinical diagnosis of ischemic optic neuropathy.Anesthesiologists should be aware that this condition may follow uncomplicated intracranial surgeries in the supine position, and should obtain prompt ophthalmologic consultation when patients develop postoperative visual loss.",
"keywords": [
"Postoperative visual loss (POVL) is a known complication of surgery and anesthesia",
"its incidence varies from 0.03% after spine surgery to 0.086% after cardiac surgery1. Typically",
"this involves ischemic optic neuropathy (ION)",
"and is clinically characterized by the acute or subacute loss of visual acuity and/or visual field. Both anterior (diffuse optic disc swelling) and posterior (no optic disc swelling) ION have been reported after general anesthesia in spinal and other non-ocular surgeries1",
"2. It can present as both unilateral and bilateral. An association with systemic diseases",
"such as hypertension",
"diabetes",
"hypercholesterolemia",
"or atherosclerosis",
"is not well documented. The management of this entity has been described by an American Society of Anesthesiologists (ASA) task force3. The incidence of POVL in a general surgical population is low (0.0012%)1",
"to our knowledge",
"only one case has previously been reported in intracranial neurovascular surgery",
"and was included in the POVL registry of the ASA4."
],
"content": "Introduction\n\nPostoperative visual loss (POVL) is a known complication of surgery and anesthesia; its incidence varies from 0.03% after spine surgery to 0.086% after cardiac surgery1. Typically, this involves ischemic optic neuropathy (ION), and is clinically characterized by the acute or subacute loss of visual acuity and/or visual field. Both anterior (diffuse optic disc swelling) and posterior (no optic disc swelling) ION have been reported after general anesthesia in spinal and other non-ocular surgeries1,2. It can present as both unilateral and bilateral. An association with systemic diseases, such as hypertension, diabetes, hypercholesterolemia, or atherosclerosis, is not well documented. The management of this entity has been described by an American Society of Anesthesiologists (ASA) task force3. The incidence of POVL in a general surgical population is low (0.0012%)1; to our knowledge, only one case has previously been reported in intracranial neurovascular surgery, and was included in the POVL registry of the ASA4.\n\n\nCase report\n\nA 46 year-old woman initially presented with paresthesia in hands and legs, and was diagnosed with a right frontal, Spetzler-Martin grade 1, superficial, arteriovenous malformation (AVM). She was classified as ASA physical status 3, and underwent surgical excision of the AVM. The patient’s personal medical history revealed arterial hypertension, as well as a coronary artery disease with previous myocardial infarction (MI) and percutaneous coronary angioplasty (PTCA). Preoperative transthoracic echocardiography showed a grade II left ventricular function (ejection fraction 50–55%) and mild distal anteroseptal and distal anterior wall hypokinesia. The patient ceased to smoke one year ago with a 30 pack-year history. Medication consisted of metoprolol 12.5 mg twice daily, aspirin 81 mg and clopidogrel 75 mg. Aspirin and clopidogrel were stopped 7 days prior to the intervention. The patient’s preoperative blood pressure was 130/90 mmHg, and hemoglobin concentration was 14.2 g/dl. After induction of general anesthesia with midazolam 2 mg, fentanyl 150 µg, and propofol 150 mg, followed by neuromuscular blockade with rocuronium 50 mg, the patient’s trachea was orally intubated. Anesthesia was maintained with sevoflurane and a remifentanil infusion during the 4-hour surgical procedure. Surgery was performed in the supine position, with the head elevated to 30 degrees with fixation in a head frame. The patient’s intraoperative blood pressure was maintained stable around 100 mmHg systolic, and there was minimal blood loss (300 ml). Total crystalloid infusion was 2 l. Emergence from anesthesia was uneventful, and the patient was extubated while awake and obeying commands. There was no sign of external compression of the eyes during and after surgery.\n\nOn awakening in the recovery room, the patient complained of blindness in her right eye. Immediate computer tomography of the brain was inconclusive. Ophthalmological examination of the right eye showed a posterior ION in association with a relative afferent pupillary defect (RAPD), but fundoscopy was normal without optic disc edema or cherry-red spot. Upon diagnosis of ION, the patient immediately received digital massage of the right eye once for a few minutes. Anti-glaucoma treatment (timolol maleate ophthalmic solution 0.5% applied to the affected eye twice daily for 2 days) was given to decrease intraocular pressure (IOP). On the following day, her vision had somewhat improved; however, the right temporal visual field was still missing. Examination of the retina was again normal, and no further treatment was administered. She was discharged from the hospital 2 days after surgery. In a follow-up visit after 3 months, she was neurologically intact, and her vision was better. Her visual field had returned to normal with just some blurring of vision in the right eye.\n\n\nDiscussion\n\nThis patient experienced acute unilateral loss of visual acuity and visual field after AVM surgery in the supine position. Findings were consistent with the diagnosis of POVL due to posterior ION with RAPD. Fundo- and retinoscopy of the affected eye were normal, and there was no sign of external pressure on the ocular globe. In a review of the literature, we only found one case of unilateral visual loss after intracranial aneurysm surgery (ASA POVL Registry)4, suggesting that POVL is a very rare complication of intracranial neurovascular surgery in the supine position.\n\nSeveral potential causes of POVL have been described. Intraoperative corneal trauma may result in irritation, abrasion, or even laceration of the eye. Preventive measures include taping the eyes shut and careful patient positioning. Intraoperative stroke involving the visual tracts or the visual cortex may lead to hemianopsia and cortical blindness. Cerebral ischemia may be due to prolonged systemic hypotension or thromboembolism. Arteriosclerosis-related embolism also plays an important role in the etiology of central retinal artery occlusion (CRAO)5; however, CRAO may be caused by an acute and severe rise of IOP found in trauma or direct external pressure to the ocular globe as well. Clinical findings in CRAO are the unilateral painless loss of vision with signs of external periorbital swelling or ecchymosis, and a pathognomic cherry-red spot at the macula. Recently, posterior reversible encephalopathy syndrome (PRES) has also been proposed as potential cause of POVL6.\n\nRisk factors that have been implicated in the development of ION in spine surgery include male sex, obesity, prolonged intraoperative hypotension, long duration of surgery, substantial intraoperative blood loss, and the excessive use of intravenous replacement fluids, or anemia7,8.\n\nThe majority of POVL cases are reported after lumbar spine surgery1,2,5–9, suggesting a greater incidence of POVL associated with prone positioning. Increases in IOP during surgery in the supine position were thought to be an important factor for the development of visual loss; the current recommendation of the ASA task force3 is to keep the head elevated higher than the heart and in neutral position whenever possible. Intraoperative blood loss and prolonged arterial hypotension should be avoided. Intravenous administration of colloid solutions and avoidance of excessive crystalloid infusion has been recommended. Excess crystalloid may cause tissue edema, and compromise tissue oxygenation in the orbital cone. If prolonged duration of surgery in the prone position is necessary, the eyes should be examined for external compression or swelling at regular intervals.\n\n\nConclusion\n\nPOVL involving ION remains a rare, but devastating condition. Unfortunately, our limited knowledge of the pathophysiology restricts the treatment options. Type of surgery, patient-related and intraoperative risk factors have been identified, but absent in this case. Recognition of high-risk cases such as major spine surgery, intraoperative head elevation, use of colloids, avoidance of excessive crystalloid infusion, correction of anemia, and staging of surgery are preventive measures recommended by the ASA task force on POVL3. Cases of permanent loss of vision have occurred, therefore early diagnosis and treatment are paramount to increase chances of visual recovery in the event of POVL.\n\n\nPatient consent\n\nWritten informed consent for publication of their clinical details was obtained from the patient.",
"appendix": "Author contributions\n\n\n\nThe authors contributed equally to the writing of the manuscript.\n\n\nCompeting interests\n\n\n\nNo relevant competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nReferences\n\nShen Y, Drum M, Roth S: The prevalence of perioperative visual loss in the United States: a 10-year study from 1996 to 2005 of spinal, orthopedic, cardiac, and general surgery. Anesth Analg. 2009; 109(5): 1534–45. PubMed Abstract | Publisher Full Text\n\nRoth S: Perioperative visual loss: what do we know, what can we do? Br J Anaesth. 2009; 103(Suppl 1): i31–40. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAmerican Society of Anesthesiologists Task Force on Perioperative Visual Loss: Practice advisory for perioperative visual loss associated with spine surgery: an updated report by the American Society of Anesthesiologists Task Force on Perioperative Visual Loss. Anesthesiology. 2012; 116(2): 274–85. PubMed Abstract | Publisher Full Text\n\nLee LA: ASA Postoperative Visual Loss (POVL) Registry. Anesthesia Patient Safety Foundation Newsletter. 2001; 16(4). Reference Source\n\nBaig MN, Lubow M, Immesoete P, et al.: Vision loss after spine surgery: review of the literature and recommendations. Neurosurg Focus. 2007; 23(5): E15. PubMed Abstract | Publisher Full Text\n\nLee LA: Perioperative visual loss and anesthetic management. Curr Opin Anaesthesiol. 2013; 26(3): 375–81. PubMed Abstract | Publisher Full Text\n\nLee LA, Roth S, Posner KL, et al.: The American Society of Anesthesiologists Postoperative Visual Loss Registry: analysis of 93 spine surgery cases with postoperative visual loss. Anesthesiology. 2006; 105(4): 652–9; quiz 867–8. PubMed Abstract | Publisher Full Text\n\nPostoperative Visual Loss Study Group: Risk factors associated with ischemic optic neuropathy after spinal fusion surgery. Anesthesiology. 2012; 116(1): 15–24. PubMed Abstract | Publisher Full Text\n\nGoepfert CE, Ifune C, Tempelhoff R: Ischemic optic neuropathy: are we any further? Curr Opin Anaesthesiol. 2010; 23(5): 582–7. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "3378",
"date": "29 Jan 2014",
"name": "John Gaudet Van Driest",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors report a case of partially reversible right unilateral visual loss following elective resection of a right frontal arteriovenous malformation. Immediate ophthalmologic assessment in recovery is compatible with ischemic optic neuropathy (ION), a condition not commonly reported following neurosurgical surgery in supine position. The pathophysiology of ION remains unclear; it is most likely multifactorial. AbstractAlthough clearly specified in the case description, I would clarify the abstract by adding a couple of details about the procedure: right frontal craniotomy for resection of an AVM in supine position, under general anesthesia. This is just too properly set the table before digging in, so to speak… Also, I would mention that the patient partially recovered vision 3 months after surgery, for the sake of completeness. IntroductionI would leave the specifics of ION (presentation, assessment, causes & mechanisms) for the discussion altogether. POVL prevalence reports (0.0012% general surgery; 0.03% spine surgery; 0.086% cardiac surgery) and general classes of POVL (corneal, retinal including CRAO, optic, chiasma and tracts, cortical) are all that is needed for the introduction here. Case descriptionSince obesity is a known risk factor for ION, I would mention the patient’s height & weight. Were there any visual issues at baseline? I assume there weren’t any, but I would mention it anyway. Were any of the AVM feeders embolized prior to craniotomy? Were there any other abnormalities noticed on the cerebral angiogram? In particular, since the AVM was in proximity to the right optic area; were there any arterial branches coming off the feeders? If so, could arterial exclusion have caused optic ischemia? Was the circle of Willis normal on preoperative control? We typically perform an immediate postoperative angiogram following AVM resection to confirm complete resection of the AVM, as well as to assess perfusion of normal vessels. Did you perform such an exam before emergence and extubation? If so, I would mention it with a brief description of results. The discussion about potential mechanisms of ION is interesting. It appears to me that ION is a multifactorial process combining the focal effects of arterial hypotension, venous hypertension, and decreased oxygen capacity. Arterial blood pressure and oxygen capacity seem to have been managed properly in this case. The combination of elevated intra-ocular pressures and borderline low arterial pressures puts the optic nerve at risk of ischemia. You mention anti-glaucoma therapy having potentially helped to improve perfusion of the optic nerve; was this patient known for glaucoma? Venous hypertension remains a possibility; how was the head positioned during the case? Did mechanical ventilation require high peak pressures restricting cerebral venous drainage? In any case, I believe this case report deserves an ‘Approved’ status but needs minor additions imposed by the specific location of the AVM relative to the visual loss. Mainly:1. minor details about patient at baseline (height & weight, visual status)2. preoperative angiographic assessment (feeders, Willis) and management (embolization)3. postoperative angiographic assessment, if any.The general discussion about ION is interesting and most relevant to this case. I found it clear and well referenced.",
"responses": []
},
{
"id": "3377",
"date": "05 Feb 2014",
"name": "Lorri A Lee",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis case report of PION occurring ipsilaterally after a resection of a frontal AVM is very interesting. It is a reminder that PION can occur in many different types of procedures outside spine, cardiac, and head and neck surgery. During craniotomies, more direct insult to the blood supply of the optic nerve may be an additional etiologic factor via direct trauma to the nerve or blood supply, or compromise of the blood supply with edema or brain retractors. It is difficult to ascertain with the available information the proximity of the AVM to the optic nerve and its blood supply.For the readers of this case report, it might be helpful to discuss the blood supply of the optic nerve with a diagram, including both the superior hypophyseal artery and the opthalmic artery and branches. The patient's preoperative angiogram and MRI/CT of the head, showing the proximity of the AVM and venous drainage to the optic nerve and its blood supply would be helpful information for the clinician.Other useful information would include a statement re: any other postoperative complications; any signs of edema/ischemia on later CT/MRI of the head; later opthalmologic findings such as optic nerve pallor on fundoscopy (confirmation that it was PION); and if formal assessment of visual fields was performed after discharge.It will remain difficult to definitively ascertain the etiology of PION in this case, even if all the requested information is provided. Fortunately, perioperative PION is relatively rare; however, its low incidence, high severity of injury, and the multitude of perioperative and patient variables make it exceedingly unlikely that studies with a high level of evidence will be conducted in the future. Animal models of PION simulating perioperative conditions may provide important insights into etiology, prevention and treatment of this problem. Unlike most patients with perioperative PION, this patient seems to have had good recovery of vision.(N.B. The lack of reports of craniotomies and POVL in the ASA POVL Registry is more likely a result of exclusion of cases in that Registry where direct trauma to the optic nerve or its blood supply was possible.)",
"responses": []
},
{
"id": "3374",
"date": "05 Feb 2014",
"name": "Sergio Bergese",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAfter reading \"Postoperative visual loss following cerebral arteriovenous malformation surgery: a case report\" I have some minor additions to propose to the authors:Extend discussions based on patient medical history linked to POVL - neurological, ophthalmological, and concomitant medication. Explain the standardized grading scheme: Spetzler-Martin.The authors covers a topic of significant scientific interest, with original data and good quality of presentation.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-27
|
https://f1000research.com/articles/3-70/v1
|
13 Mar 14
|
{
"type": "Case Report",
"title": "Antifungal therapy and surgical drainage for the treatment of a cerebral abscess caused by Scedosporium apiospermum in a renal transplant patient - a case report",
"authors": [
"Maria Isabel Garzón",
"Claudio Abiega",
"Abel H. Zarate",
"Pablo E. Sanchez",
"Marcela F. Medeot",
"Carlos Chiurchiu",
"Jorge de La Fuente",
"Juan Pablo Caeiro",
"Maria Isabel Garzón",
"Claudio Abiega",
"Abel H. Zarate",
"Pablo E. Sanchez",
"Marcela F. Medeot",
"Carlos Chiurchiu",
"Jorge de La Fuente"
],
"abstract": "Scedosporium apiospermum, the asexual form of Pseudallescheria boydii, is a filamentous, opportunistic fungus which can be found in environmental sources all over the world. It is a human pathogen mostly associated with lung, bone and joint infections and less frequently with infections of the central nervous system (CNS). The latter is generally related to the patient’s immune state, and occurs most frequently in immunocompromised patients. We present the case of a 64-year-old male patient with a background of chronic kidney failure secondary to nephroangiosclerosis and a renal transplantation who presented with left-sided hemiplegia and dysarthria. A brain MRI revealed a hyperintense lesion with ring enhancement at the right paramedian posterior frontal subcortical area with an associated vasogenic edema. A stereotactic biopsy of the lesion revealed the presence of S. apiospermum. The patient received a combined therapy of voriconazole and terbinafine with surgical drainage, which led to temporarily clinical and radiological improvement.",
"keywords": [
"Scedosporium apiospermum",
"Pseudallescheria boydii",
"cerebral abscess",
"kidney transplant"
],
"content": "Introduction\n\nScedosporium apiospermum is a filamentous fungus causing a rare but serious opportunistic infection. It is the asexual form of Pseudallescheria boydii and is found in many environmental sources including soil and fresh water, but most commonly in stagnant or contaminated water1. The infection may be acquired by inhaling the microorganism or after traumatic inoculation through the skin2. The sites of infection include the lungs, sinuses, bones, skin, joints and notoriously, the central nervous system (CNS)3. S. apiospermum can infect the CNS of both healthy4 and immunocompromised hosts. Cerebral abscess is the most common clinical manifestation of S. apiospermum brain infections, although cases of meningitis and, less frequently, ventriculitis have also been reported5. Brain abscesses may be found as one or multiple lesions6. The overall mortality rate of patients infected with this pathogen is higher than 70%7. Solid organ transplant and its associated immunosuppression are important risk factors for infections with Scedosporium species8. Here we present a case of CNS infection caused by S. apiospermum in a patient who had received a kidney transplant and was treated with dual antifungal therapy and surgical drainage. The patient initially responded well to the therapy.\n\n\nClinical case\n\nA 64 year-old male patient underwent deceased-donor kidney transplantation following a chronic kidney failure secondary to nephroangiosclerosis. The past medical history was significant for hypertension, hyperlipidemia, peripheral vascular disease, chronic anemia and deep venous thrombosis of right lower extremity. Family history was significant for cardiomyopathy in one brother and diabetes in another. The immunosuppressive medication consisted of tacrolimus 3 mg every 12 hours, prednisone 20 mg daily and mycophenolate mofetil 500 mg every 12 hours. Seventeen days after transplantation, he presented left-sided hemiplegia and dysarthria. A brain MRI was performed, which revealed a hyperintense lesion with ring enhancement at the right paramedian posterior frontal subcortical area with an associated vasogenic edema (Figure 1).\n\nA hyperintense lesion with ring enhancement at the right paramedian posterior frontal subcortical area with an associated vasogenic edema is shown.\n\nA stereotactic biopsy was performed and tissue examination revealed the presence of a filamentous fungus that was identified as S. apiospermum (Figure 2). The sample was also cultured in Sabouraud’s dextrose agar medium at 25°C for a period of 14 days (Figure 3), the culture turned a dark brown color.\n\nMethylene blue staining showing the morphology of S. apiospermum: unicellular microconidia attached to filaments by conidiophores (original magnification 400×).\n\nThe culture grew in Sabouraud’s dextrose agar medium at 25°C and turned a dark brown color on the 14th day.\n\nThe patient started a treatment with voriconazole (6 mg/Kg po q12h for two days, then 4 mg/Kg q12h VO) and terbinafine (250 mg po daily) and subsequently was subjected to surgical drainage by craniotomy in order to remove the infected tissue.\n\nThe patient was on terbinafine for 4 months and continued to be on voriconazole for almost a year. At a follow up visit he showed significant recovery from the left-sided palsy and also an absence of dysarthria. The brain MRI follow-up images showed an improvement in the brain lesion.\n\nUnfortunately, 8 months later the patient clinical course was complicated and he eventually died of problems unrelated to fungal CNS disease.\n\n\nDiscussion\n\nSolid organ transplant recipients are highly susceptible to invasive fungal infections9.\n\nDuring the last few decades there has been a marked increase in the number of immunocompromised patients who have suffered Scedosporium infections, the most frequent cases being infections of the CNS10.\n\nSolid organ transplant patients are susceptible to invasive fungal infections as their immunity might be compromised due to the use of immunosuppressant drugs11. Therefore S. apiospermum should be considered in the differential diagnosis of immunocompromised patients presenting with a brain abscess12.\n\nWithin the nervous system, abscesses may be located in brain hemispheres, the cerebellum, the brain stem or the spinal cord, where they may cause alterations of consciousness levels, signs of meningeal irritation or focal neurological deficits13.\n\nIf not adequately treated, fungal brain abscesses in immunocompromised patients often result in poor prognosis. The diagnosis of an invasive fungal infection such as S. apiospermum is based on the combination of histopathological, microbiological and clinical findings14. As the clinical and histopathological presentations of S. apiospermum infections are similar to those of other fungi such as Aspergillus and Fusarium spp., a culture is necessary for accurate diagnosis. Furthermore, while most species of Aspergillus (except for Aspergillus terreus) are sensitive to amphotericin, S. apiospermum is usually resistant15. In addition, PCR techniques are important to diagnose as well as to distinguish between different species16.\n\nThere are many treatment options described in the literature, but there is an ongoing controversy over which treatment is most suitable for S. apiospermum infections. Voriconazole has emerged as a possible treatment option, since it shows high activity against several species of fungi, including S. apiospermum17. Several reports have shown the successful use of voriconazole synergistically combined with terbinafine against S. apiospermum18 and S. prolificans. Furthermore, many authors recommend the surgical drainage of brain abscesses caused by S. apiospermum19. Therefore, a combined antifungal therapy along with an aggressive surgical approach is recommended for therapeutic success20.\n\n\nConclusion\n\nS. apiospermum infection of the CNS is a rare but it is an extremely serious medical condition. Immediate diagnosis in the event of brain abscess in an immunocompromised patient is crucial and the choice of a suitable medical treatment is a priority. Despite the aggressive surgical treatment and the appropriate anti-fungal therapy used, mortality rates continue to be high.\n\n\nConsent\n\nWritten informed consent for publication of clinical details and clinical images was obtained from the patient’s family.",
"appendix": "Author contributions\n\n\n\nMIG, PES and JPC contributed to the design of the study. All the authors contributed to writing the manuscript and agreed to the final contents.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nHarun A, Gilgado F, Chen SC, et al.: Abundance of Pseudallescheria/Scedosporium species in the Australian urban environment suggests a possible source for scedosporiosis including the colonization of airways in cystic fibrosis. Med Mycol. 2010; 48(Suppl 1): S70–S76. PubMed Abstract | Publisher Full Text\n\nde Hoog GS, Marvin-Sikkema FD, Lahpoor GA, et al.: Ecology and physiology of the emerging opportunistic fungi Pseudallescheria boydii and Scedosporium prolificans. Mycoses. 1994; 37(3–4): 71–78. PubMed Abstract | Publisher Full Text\n\nTroke P, Aguirrebengoa K, Arteaga C, et al.: Treatment of scedosporiosis with voriconazole: clinical experience with 107 patients. Antimicrob Agents Chemother. 2008; 52(5): 1743–1750. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWilson HL, Kennedy KJ: Scedosporium apiospermum brain abscesses in an immunocompetent man with silicosis. Med Mycol Case Rep. 2013; 2: 75–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCortez KJ, Roilides E, Quiroz-Telles F, et al.: Infections caused by Scedosporium spp. Clin Microbiol Rev. 2008; 21(1): 157–97. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLeechawengwongs M, Milindankura S, Liengudom A, et al.: Multiple Scedosporium apiospermum brain abscesses after near-drowning successfully treated with surgery and long-term voriconazole: a case report. Mycoses. 2007; 50(6): 512–516. PubMed Abstract | Publisher Full Text\n\nKantarcioglu AS, Guarro J, De Hoog GS: Central nervous system infections by members of the Pseudallescheria boydii species complex in healthy and immunocompromised hosts: epidemiology, clinical characteristics and outcome. Mycoses. 2008; 51(4): 275–290. PubMed Abstract | Publisher Full Text\n\nShoham S: Emerging fungal infections in solid organ transplant recipients. Infect Dis Clin North Am. 2013; 27(2): 305–16. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPappas PG, Alexander BD, Andes DR, et al.: Invasive fungal infections among organ transplant recipients: results of the Transplant-Associated Infection Surveillance Network (TRANSNET). Clin Infect Dis. 2010; 50(8): 1101–1111. PubMed Abstract | Publisher Full Text\n\nKubak BM, Huprikar SS; the ASTIDCoP: Emerging & rare fungal infections in solid organ transplant recipients. Am J Transplant. 2009; 9(Suppl 4): S208–S226. PubMed Abstract | Publisher Full Text\n\nCastiglioni B, Sutton DA, Rinaldi MG, et al.: Pseudallescheria boydii (Anamorph Scedosporium apiospermum). Infection in solid organ transplant recipients in a tertiary medical center and review of the literature. Medicine (Baltimore). 2002; 81(5): 333–348. PubMed Abstract\n\nHusain S, Muñoz P, Forrest G, et al.: Infections due to Scedosporium apiospermum and Scedosporium prolificans in transplant recipients: clinical characteristics and impact of antifungal agent therapy on outcome. Clin Infect Dis. 2005; 40(1): 89–99. PubMed Abstract | Publisher Full Text\n\nBerenguer J, Diaz-Mediavilla J, Urra D, et al.: Central nervous system infection caused by Pseudallescheria boydii: case report and review. Rev Infect Dis. 1989; 11(6): 890–896. PubMed Abstract\n\nTarrand JJ, Lichterfeld M, Warraich I, et al.: Diagnosis of invasive septate mold infections. A correlation of microbiological culture and histologic or cytologic examination. Am J Clin Pathol. 2003; 119(6): 854–858. PubMed Abstract | Publisher Full Text\n\nCaggiano G, Cantisani P, Rolli M, et al.: The importance of a proper aetiological diagnosis in the management of patients with invasive mycoses: a case report of a brain abscess by Scedosporium apiospermum. Mycopathologia. 2011; 172(4): 317–22. PubMed Abstract | Publisher Full Text\n\nCastelli MV, Buitrago MJ, Bernal-Martinez L, et al.: Development and validation of a quantitative PCR assay for diagnosis of scedosporiosis. J Clin Microbiol. 2008; 46(10): 3412–3416. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchaenman JM, DiGiulio DB, Mirels LF, et al.: Scedosporium apiospermum soft tissue infection successfully treated with voriconazole: potential pitfalls in the transition from intravenous to oral therapy. J Clin Microbiol. 2005; 43(2): 973–977. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMorio F, Horeau-Langlard D, Gay-Andrieu F, et al.: Disseminated Scedosporium/Pseudallescheria infection after double-lung transplantation in patients with cystic fibrosis. J Clin Microbiol. 2010; 48(5): 1978–1982. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNesky MA, McDougal EC, Peacock JE Jr: Pseudallescheria boydii brain abscess successfully treated with voriconazole and surgical drainage: case report and literature review of central nervous system pseudallescheriasis. Clin Infect Dis. 2000; 31(3): 673–677. PubMed Abstract | Publisher Full Text\n\nGosbell IB, Toumasatos V, Yong J, et al.: Cure of orthopaedic infection with Scedosporium prolificans, using voriconazole plus terbinafine, without the need for radical surgery. Mycoses. 2003; 46(5–6): 233–236. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "4093",
"date": "03 Jun 2014",
"name": "Corrado Girmenia",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting case report of an unusual fungal infection in an immunocompromised patient. The metodology in the diagnostic approach and the description of the case are both adequate.",
"responses": []
},
{
"id": "4590",
"date": "02 Jul 2015",
"name": "Emmanuel Roilides",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting rare case of brain abscess due to Scedosporium apiospermum in a kidney transplant immunocompromised patient, indeed relatively early (17 days) post-transplantation. The report reads nicely and the Discussion is thoughtful. However, there are a few points that the authors should take into consideration:Have the authors performed an antifungal susceptibility testing? In addition, any molecular identification? It is apiospermum complex now. Please, add ESCMID Guidelines for rare fungi as a reference. This is a document that should be definitely cited and used in clinical practice. Tortorano AM et al. Clin Microbiol Infect. 2014 Apr;20 Suppl 3:27-46. Page 2. VO should be po",
"responses": []
}
] | 1
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https://f1000research.com/articles/3-70
|
https://f1000research.com/articles/3-69/v1
|
11 Mar 14
|
{
"type": "Data Article",
"title": "Compound data sets and software tools for chemoinformatics and medicinal chemistry applications: update and data transfer",
"authors": [
"Ye Hu",
"Jürgen Bajorath",
"Ye Hu"
],
"abstract": "In 2012, we reported 30 compound data sets and/or programs developed in our laboratory in a data article and made them freely available to the scientific community to support chemoinformatics and computational medicinal chemistry applications. These data sets and computational tools were provided for download from our website. Since publication of this data article, we have generated 13 new data sets with which we further extend our collection of publicly available data and tools. Due to changes in web servers and website architectures, data accessibility has recently been limited at times. Therefore, we have also transferred our data sets and tools to a public repository to ensure full and stable accessibility. To aid in data selection, we have classified the data sets according to scientific subject areas. Herein, we describe new data sets, introduce the data organization scheme, summarize the database content and provide detailed access information in ZENODO (doi: 10.5281/zenodo.8451 and doi:10.5281/zenodo.8455).",
"keywords": [
"The compound data sets reported in our original article1 and the new data sets presented herein have resulted from research in the chemoinformatics and medicinal chemistry area and have mostly been generated from public domain repositories of compound structures and activity data. In addition",
"software tools made publicly available have also been developed in our laboratory1. Data sets reported in the scientific literature in the context of computational method development and evaluation are often not publicly available",
"which limits the reproducibility of computational investigations and comparisons of different computational methods. We believe that it is important to provide such data to the scientific community to further improve the transparency and credibility of computational studies and support method development. In addition to the data sets designed for the development and evaluation of computational methods",
"we also make available data sets that were generated as a resource and knowledge base for medicinal chemistry applications. Our data sets and tools are provided via the ZENODO platform (https://zenodo.org/) to ensure easy and stable access."
],
"content": "Introduction\n\nThe compound data sets reported in our original article1 and the new data sets presented herein have resulted from research in the chemoinformatics and medicinal chemistry area and have mostly been generated from public domain repositories of compound structures and activity data. In addition, software tools made publicly available have also been developed in our laboratory1. Data sets reported in the scientific literature in the context of computational method development and evaluation are often not publicly available, which limits the reproducibility of computational investigations and comparisons of different computational methods. We believe that it is important to provide such data to the scientific community to further improve the transparency and credibility of computational studies and support method development. In addition to the data sets designed for the development and evaluation of computational methods, we also make available data sets that were generated as a resource and knowledge base for medicinal chemistry applications. Our data sets and tools are provided via the ZENODO platform (https://zenodo.org/) to ensure easy and stable access.\n\n\nMaterials and methods\n\nThe data sets reported herein were predominantly generated from ChEMBL2,3, BindingDB4 and PubChem5 (a few exceptions are specified in the original data article1). Compound structures are represented as SMILES6 strings or SD files7. Activity information and other (data set-dependent) annotations are provided in the individual data files. For software tools (written in different languages), the source code is also made available.\n\n\nData description\n\nTable 1 provides the updated list and classification of all freely available data sets and programs. Entries were organized according to the following scientific subject areas: data sets for structure-activity relationship (SAR) and structure-selectivity relationship (SSR) analysis, SAR visualization (SAR_VZ), and virtual screening via similarity searching or machine learning (VS_ML). In addition, the programs are provided separately (PROG). Data sets and programs are contained in separate ZENODO deposition sets with a unique reference. Three matched molecular pair (MMP)-based data sets also included in our update have recently been reported and described in detail8. Entries 1–30 in Table 1 represent the data sets and programs that we initially provided via our website1 and entries 31–43 represent new data sets. In the following, the new data sets are described:\n\nData entries are organized according to scientific subject areas: structure-activity relationship (SAR) and structure-selectivity relationship (SSR) analysis, SAR visualization (SAR_VZ), virtual screening via similarity searching or machine learning (VS_ML), and programs (PROG). References in the Entry column provide the original publication introducing the program and/or data set. Program entries are described in more detail in Table 2 of our original data article1. The new compound data sets 31–43 are discussed in the text. Programs and data sets reported herein have been separately deposited in ZENODO for access and download.\n\n50 compound activity classes (AC) are prioritized for the evaluation of scaffold hopping potential in ligand-based virtual screening38. These AC contain the largest proportion of scaffold pairs with largest chemical inter-scaffold distances38 that can be derived from current bioactive compounds and hence present challenging test cases for scaffold hopping analysis.\n\n596 SAR transfer series with regular potency progression (SAR-TS-RP) are extracted from 61 AC39. Each SAR-TS-RP represents two compound series with different core structures and pairwise corresponding substitutions that yield comparable potency progression against a given target. These series provide a knowledge base for the analysis and prediction of SAR transfer events.\n\nFour sets of molecular scaffolds (with each scaffold representing more than ten compounds) are provided that are active against a single target (ST), multiple targets from the same family (SF), or multiple targets from different families (MF)40. Data sets are separately assembled for different types of potency measurements (i.e., Ki and IC50 values) and provide a resource of scaffolds representing compounds with varying degrees of target promiscuity.\n\nTwo multi-target compound data sets consist of confirmed screening hits41. Each set contains compounds with single-, dual-, and triple-target activity, or no activity. These data provide test cases for machine learning or other approaches to differentiate between compounds with overlapping yet distinct activity profiles.\n\nFour multi-target compound data sets are provided42. Each set contains compounds tested in three different assays. Compounds are organized into eight different subsets according to their activity profiles, i.e., single-, dual-, and triple-target activity, or no activity. In addition, three multi-mechanism compound sets are designed42. In the latter case, compounds are organized into four subsets according to their mechanism-of-action. These data sets also represent test cases for machine learning to distinguish compounds with different activity profiles or mechanisms.\n\n2337 non-redundant compound series matrices (CSMs) are generated covering compounds active against a wide spectrum of targets43. Each matrix contains at least two analogous matching molecular series (MMS) with structurally related yet distinct cores. A matrix consists of known active compounds and structurally related virtual compounds and hence provides suggestions for compound design.\n\n128 target-based data sets are assembled that consist of at least 100 compounds with precisely specified equilibrium constants (Ki values) below 1 µM for human targets44. These high-confidence activity data sets provide a sound basis for SAR exploration.\n\n30,452 and 45,607 target-based MMS with Ki and IC50 values, respectively, are extracted from bioactive compounds45.\n\n221 scaffolds are identified that only occur in approved drugs but are not found in currently available bioactive compounds46. Accordingly, these scaffolds have been termed drug-unique scaffolds.\n\n92,734 MMPs are generated from 435 AC on a basis of retrosynthetic rules47. These MMPs consider chemical reaction information and should be useful for practical medicinal chemistry applications.\n\n20,073 and 25,297 MMP-based activity cliffs (i.e. pairs of structurally analogous compounds with an at least 100-fold difference in potency) are extracted from specifically active compounds based upon Ki and IC50 values, respectively8. The MMP-based activity cliffs provide a large knowledge base for SAR analysis.\n\n157 and 513 MMP-based SAR transfer series with approximate potency progression plus 60 and 322 SAR transfer series with regular potency progression based upon Ki and IC50 values, respectively, are isolated from bioactive compounds. These transfer series are active against individual targets8. Similar to MMP-based activity cliffs, SAR transfer series provide a resource for SAR analysis and compound design.\n\n169,889 and 240,322 transformation size-restricted MMPs based upon retrosynthetic rules with Ki and IC50 values, respectively, are systematically extracted from available AC8. Different from the retrosynthetic rule-based MMPs presented above, applied transformation size-restrictions ensure that chemical changes distinguishing compounds in pairs are small.\n\n\nSummary\n\nHerein we have provided an updated release of data sets and programs for chemoinformatics and medicinal chemistry that we make freely available. In total, 13 new data sets are introduced. Transferring all data entries in an organized form to the ZENODO platform makes them easily accessible. We hope that our current release might be of interest and helpful to many investigators in academia and the pharmaceutical industry.\n\n\nData availability\n\nZENODO: Programs for chemoinformatics and computational medicinal chemistry, doi: 10.5281/zenodo.845148.\n\nZENODO: Data sets for chemoinformatics and computational medicinal chemistry, doi: 10.5281/zenodo.845549.",
"appendix": "Author contributions\n\n\n\nJB designed the study, YH collected and organized the data, YH and JB wrote the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were declared.\n\n\nGrant information\n\n\n\n\nAcknowledgments\n\nWe are grateful to current and former members of our research group who have contributed to the development of the data sets and programs reported herein.\n\n\nReferences\n\nHu Y, Bajorath J: Freely available compound data sets and software tools for chemoinformatics and computational medicinal chemistry applications [v1; ref status: indexed, http://f1000r.es/Mu9krs]. F1000Res. 2012; 1: 11. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGaulton A, Bellis LJ, Bento AP, et al.: ChEMBL: a large-scale bioactivity database for drug discovery. Nucleic Acids Res. 2012; 40(Database issue): D1100–D1107. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBento AP, Gaulton A, Hersey A, et al.: The ChEMBL bioactivity database: an update. Nucleic Acids Res. 2014; 42(Database issue): D1083–D1090. PubMed Abstract | Publisher Full Text\n\nLiu T, Lin Y, Wen X, et al.: BindingDB: a web-accessible database of experimentally determined protein–ligand binding affinities. Nucleic Acids Res. 2007; 35(Database issue): D198–D201. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang Y, Xiao J, Suzek TO, et al.: PubChem: a public information system for analyzing bioactivities of small molecules. Nucleic Acids Res. 2009; 37(Web Server issue): W623–W633. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWeininger D: SMILES, a chemical language and information system. 1. Introduction to methodology and encoding rules. J Chem Inf Comput Sci. 1988; 28(1): 31–36. Publisher Full Text\n\nDalby A, Nourse JG, Hounshell WD, et al.: Description of several chemical structure file formats used by computer programs developed at Molecular Design Limited. J Chem Inf Comput Sci. 1992; 32(3): 244–255. Publisher Full Text\n\nHu Y, de la Vega de León A, Zhang B, et al.: Matched molecular pair-based data sets for computer-aided medicinal chemistry [v2; ref status: indexed, http://f1000r.es/309]. F1000Res. 2014; 3: 36. Publisher Full Text\n\nTovar A, Eckert H, Bajorath J: Comparison of 2D fingerprint methods for multiple-template similarity searching on compound activity classes of increasing structural diversity. ChemMedChem. 2007; 2(2): 208–217. PubMed Abstract | Publisher Full Text\n\nWang Y, Godden JW, Bajorath J: A novel descriptor histogram filtering method for database mining and the identification of active molecules. Lett Drug Design Discov. 2007; 4(4): 286–292. Publisher Full Text\n\nStumpfe D, Ahmed H, Vogt I, et al.: Methods for computer-aided chemical biology. Part 1: Design of a benchmark system for the evaluation of compound selectivity. Chem Biol Drug Des. 2007; 70(3): 182–194. PubMed Abstract | Publisher Full Text\n\nVogt I, Ahmed HE, Auer J, et al.: Exploring structure-selectivity relationships of biogenic amine GPCR antagonists using similarity searching and dynamic compound mapping. Mol Divers. 2008; 12(1): 25–40. PubMed Abstract | Publisher Full Text\n\nStumpfe D, Geppert H, Bajorath J: Methods for computer-aided chemical biology. Part 3: analysis of structure-selectivity relationships through single- or dual-step selectivity searching and Bayesian classification. Chem Biol Drug Des. 2008; 71(6): 518–528. PubMed Abstract | Publisher Full Text\n\nWang Y, Geppert H, Bajorath J: Random reduction in fingerprint bit density improves compound recall in search calculations using complex reference molecules. Chem Biol Drug Des. 2008; 71(6): 511–517. PubMed Abstract | Publisher Full Text\n\nNisius B, Göller AH, Bajorath J: Combining cluster analysis, feature selection and multiple support vector machine models for the identification of human ether-a-go-go related gene channel blocking compounds. Chem Biol Drug Des. 2009; 73(1): 17–25. PubMed Abstract | Publisher Full Text\n\nAhmed H, Geppert H, Stumpfe D, et al.: Methods for computer-aided chemical biology. Part 4: selectivity searching for ion channel ligands and mapping of molecular fragments as selectivity markers. Chem Biol Drug Des. 2009; 73(3): 273–282. PubMed Abstract | Publisher Full Text\n\nPeltason L, Weskamp N, Teckentrup A, et al.: Exploration of structure-activity relationship determinants in analogue series. J Med Chem. 2009; 52(10): 3212–3224. PubMed Abstract | Publisher Full Text\n\nNisius B, Bajorath J: Molecular fingerprint recombination: generating hybrid fingerprints for similarity searching from different fingerprint types. ChemMedChem. 2009; 4(11): 1859–1863. PubMed Abstract | Publisher Full Text\n\nBatista J, Tan L, Bajorath J: Atom-centered interacting fragments and similarity search applications. J Chem Inf Model. 2010; 50(1): 79–86. PubMed Abstract | Publisher Full Text\n\nHu Y, Bajorath J: Exploring target-selectivity patterns of molecular scaffolds. ACS Med Chem Lett. 2010; 1(2): 54–58. Publisher Full Text\n\nWassermann AM, Peltason L, Bajorath J: Computational analysis of multi-target structure-activity relationships to derive preference orders for chemical modifications toward target selectivity. ChemMedChem. 2010; 5(6): 847–858. PubMed Abstract | Publisher Full Text\n\nLounkine E, Wawer M, Wassermann AM, et al.: SARANEA: a freely available program to mine structure-activity and structure-selectivity relationship information in compound data sets. J Chem Inf Model. 2010; 50(1): 68–78. PubMed Abstract | Publisher Full Text\n\nPeltason L, Iyer P, Bajorath J: Rationalizing three-dimensional activity landscapes and the influence of molecular representations on landscape topology and the formation of activity cliffs. J Chem Inf Model. 2010; 50(6): 1021–1033. PubMed Abstract | Publisher Full Text\n\nWassermann AM, Bajorath J: Chemical substitutions that introduce activity cliffs across different compound classes and biological targets. J Chem Inf Model. 2010; 50(7): 1248–1256. PubMed Abstract | Publisher Full Text\n\nWawer M, Bajorath J: Similarity-potency trees: a method to search for SAR information in compound data sets and derive SAR rules. J Chem Inf Model. 2010; 50(8): 1395–1409. PubMed Abstract | Publisher Full Text\n\nVogt M, Stumpfe D, Geppert H, et al.: Scaffold hopping using two-dimensional fingerprints: true potential, black magic, or a hopeless endeavor? Guidelines for virtual screening. J Med Chem. 2010; 53(15): 5707–5715. PubMed Abstract | Publisher Full Text\n\nWawer M, Bajorath J: Extracting SAR information from a large collection of anti-malarial screening hits by NSG-SPT analysis. ACS Med Chem Lett. 2011; 2(3): 201–206. Publisher Full Text\n\nHu Y, Bajorath J: Combining horizontal and vertical substructure relationships in scaffold hierarchies for activity prediction. J Chem Inf Model. 2011; 51(2): 248–257. PubMed Abstract | Publisher Full Text\n\nDimova D, Wawer M, Wassermann AM, et al.: Design of multitarget activity landscapes that capture hierarchical activity cliff distributions. J Chem Inf Model. 2011; 51(2): 258–266. PubMed Abstract | Publisher Full Text\n\nWawer M, Bajorath J: Local structural changes, global data views: graphical substructure-activity relationship trailing. J Med Chem. 2011; 54(8): 2944–2951. PubMed Abstract | Publisher Full Text\n\nHeikamp K, Bajorath J: Large-scale similarity search profiling of ChEMBL compound data sets. J Chem Inf Model. 2011; 51(8): 1831–1839. PubMed Abstract | Publisher Full Text\n\nRipphausen P, Wassermann AM, Bajorath J: REPROVIS-DB: a benchmark system for ligand-based virtual screening derived from reproducible prospective applications. J Chem Inf Model. 2011; 51(10): 2467–2473. PubMed Abstract | Publisher Full Text\n\nHu Y, Bajorath J: Activity profile sequences: a concept to account for the progression of compound activity in target space and to extract SAR information from analogue series with multiple target annotations. ChemMedChem. 2011; 6(12): 2150–2154. PubMed Abstract | Publisher Full Text\n\nLi R, Stumpfe D, Vogt M, et al.: Development of a method to consistently quantify the structural distance between scaffolds and to assess scaffold hopping potential. J Chem Inf Model. 2011; 51(10): 2507–2514. PubMed Abstract | Publisher Full Text\n\nStumpfe D, Bajorath J: Assessing the confidence level of public domain compound activity data and the impact of alternative potency measurements on SAR analysis. J Chem Inf Model. 2011; 51(12): 3131–3137. PubMed Abstract | Publisher Full Text\n\nGupta-Ostermann D, Hu Y, Bajorath J: Introducing the LASSO graph for compound data set representation and structure-activity relationship analysis. J Med Chem. 2012; 55(11): 5546–5553. PubMed Abstract | Publisher Full Text\n\nHu Y, Bajorath J: Extending the activity cliff concept: structural categorization of activity cliffs and systematic identification of different types of cliffs in the ChEMBL database. J Chem Inf Model. 2012; 52(7): 1806–1811. PubMed Abstract | Publisher Full Text\n\nLi R, Bajorath J: Systematic assessment of scaffold distances in ChEMBL: prioritization of compound data sets for scaffold hopping analysis in virtual screening. J Comput Aided Mol Des. 2012; 26(10): 1101–1109. PubMed Abstract | Publisher Full Text\n\nZhang B, Wassermann AM, Vogt M, et al.: Systematic assessment of compound series with SAR transfer potential. J Chem Inf Model. 2012; 52(12): 3138–3143. PubMed Abstract | Publisher Full Text\n\nHu Y, Bajorath J: Systematic identification of scaffolds representing compounds active against individual targets and single or multiple target families. J Chem Inf Model. 2013; 53(2): 312–326. PubMed Abstract | Publisher Full Text\n\nHeikamp K, Bajorath J: Prediction of compounds with closely related activity profiles using weighted support vector machine linear combinations. J Chem Inf Model. 2013; 53(4): 791–801. PubMed Abstract | Publisher Full Text\n\nNamasivayam V, Hu Y, Balfer J, et al.: Classification of compounds with distinct or overlapping multi-target activities and diverse molecular mechanisms using emerging chemical patterns. J Chem Inf Model. 2013; 53(6): 1272–1281. PubMed Abstract | Publisher Full Text\n\nGupta-Ostermann D, Hu Y, Bajorath J: Systematic mining of analog series with related core structures in multi-target activity space. J Comput Aided Mol Des. 2013; 27(8): 665–674. PubMed Abstract | Publisher Full Text\n\nDimova D, Stumpfe D, Bajorath J: Quantifying the fingerprint descriptor dependence of structure-activity relationship information on a large scale. J Chem Inf Model. 2013; 53(9): 2275–2281. PubMed Abstract | Publisher Full Text\n\nde la Vega de León A, Hu Y, Bajorath J: Systematic identification of matching molecular series and mapping of screening hits. Mol Inf. 2014; In press.\n\nHu Y, Bajorath J: Many drugs contain unique scaffolds with varying structural relationships to scaffolds of currently available bioactive compounds. Eur J Med Chem. 2014; 76: 427–434. Publisher Full Text\n\nde la Vega de León A, Bajorath J: Matched molecular pairs derived by retrosynthetic fragmentation. Med Chem Commun. 2014; 5(1): 64–67. Publisher Full Text\n\nHu Y, Bajorath J: Programs for chemoinformatics and computational medicinal chemistry. 2014. Data Source\n\nHu Y, Bajorath J: Data sets for chemoinformatics and computational medicinal chemistry. 2014. Data Source"
}
|
[
{
"id": "4079",
"date": "13 Mar 2014",
"name": "Ajay Jain",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHu and Bajorath offer an update to their resource for computational chemistry. The curated data, and its engineered availability, will be of great interest, especially to methods developers. Even those researchers that are interested in exploring larger data sets that illuminate issues such as activity cliffs and small-molecule structural motifs will find the resource of interest.",
"responses": []
},
{
"id": "4409",
"date": "17 Apr 2014",
"name": "Chris J. Swain",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nBuilding and testing novel computer models requires access to suitable datasets. The authors have compiled a very useful set of interesting datasets and made them readily available in standard formats (SMILES and SDF). This allows others to both test existing algorithms and to develop new ones.",
"responses": []
},
{
"id": "4077",
"date": "22 Apr 2014",
"name": "Patrick Walters",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe ability to compare multiple computational methods across a series of consistent, high-quality datasets is critical to the progress of computational chemistry and cheminformatics. In the past, each paper published in the field seemed to present yet another new dataset. This dataset heterogeneity made it difficult, if not impossible, to objectively compare methods, and impeded the progress of the field. The availability of large repositories of carefully curated data is critical to the progress of the field. The datasets described in this paper will provide an invaluable resource for future studies. It is refreshing to see the emergence of platforms like ZENODO dedicated to hosting this data.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-69
|
https://f1000research.com/articles/3-68/v1
|
11 Mar 14
|
{
"type": "Research Article",
"title": "Nigrostriatal dynein changes in A53T alpha-synuclein transgenic mice",
"authors": [
"Yan Liu",
"Yu-He Yuan",
"Jian-Dong Sun",
"Jing Li",
"Zhi-Peng Li",
"Nai-Hong Chen",
"Yan Liu",
"Yu-He Yuan",
"Jian-Dong Sun",
"Jing Li",
"Zhi-Peng Li"
],
"abstract": "The accumulation of misfolded a-synuclein is mechanistically linked to neurodegeneration in Parkinson’s disease (PD) and other alpha-synucleinopathies. However, how alpha-synuclein causes neurodegeneration is unresolved. Several studies have supported the involvement of dynein, the major motor for retrograde axonal transport in alpha-synuclein-dependent neurodegeneration, especially in the nigrostriatal system. Therefore, we examined the nigrostriatal dyneins in transgenic mice that overexpress human A53T alpha-synuclein and recapitulate key features of a PD-like neuronal synucleinopathy. Age-matched nontransgenic littermates were used as controls. The results demonstrated that the protein level of dynein was decreased in the striatum, whereas it was elevated in the substantia nigra. Double immunostaining results revealed that the reduction in dynein level was associated with aggregation of A53T a-synuclein in the striatum. Furthermore, we performed a quantitative analysis of motor behaviors in A53T alpha-synuclein transgenic mice and controls using a modified open field test. We demonstrated that the protein level of dynein in the striatum was significantly correlated with the motor behaviors. Together, our data indicate that dynein changes in the nigrostriatal system of A53T alpha-synuclein transgenic mice may contribute to their severe movement disorder.",
"keywords": [
"Dynein",
"alpha-Synuclein",
"Open field test",
"Parkinson’s disease"
],
"content": "Introduction\n\nα-Synuclein abnormalities are mechanistically linked to the pathogenesis of Parkinson’s disease (PD) and other α-synucleinopathies. α-Synuclein is the major component of Lewy bodies, the neuropathological hallmarks of PD1,2. Duplication, triplication or mutations in the α-synuclein gene cause some forms of familial PD3,4. However, the mechanism whereby α-synuclein promotes neurodegeneration remains unclear.\n\nDyneins are minus end-directed microtubule motors that move cargoes such as mitochondria, organelles and proteins from the distal ends of axons toward neuronal cell bodies5,6. Dynein is the major motor of retrograde axonal transport, and it is also the molecular motor responsible for the transport of misfolded proteins to be degraded. Therefore, it is crucially involved in the appearance and clearance of protein aggregates7. Several studies have supported the involvement of dynein in the neurodegeneration associated with PD. First, recent studies suggest that axonal transport disruption may be causal to disease progression in PD8,9. Alterations in axonal transport motor proteins have been observed in postmortem patient brain samples9 and treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a PD-relevant injury, leads to defective axonal transport, including increased dynein-dependent transport10. Second, a number of dynein-dependent processes, including autophagy or clearance of aggregation-prone proteins, are found to be defective in PD11. In all cases, defects in axonal transport and autophagy occurring in PD indicate that dynein may be a central factor in PD pathology. Interestingly, experimental evidence suggests that α-synuclein mutations (A53T, A30P) might lead to axonal transport defects both in vivo and in vitro8,9,12. Therefore, we hypothesized that the neurodegeneration observed in the A53T mutant human α-synuclein transgenic (Tg) mouse model of α-synucleinopathy was associated with alterations of dynein.\n\nIn this study, we first evaluated the motor function of A53T human α-synuclein Tg mice and age-matched non-transgenic (nTg) littermates using a modified open field test. Unlike previous studies that only provided behavioral descriptions, in this study, we quantified behavior. We then examined the expression of dynein in the striatum and substantia nigra (SN), as dynein defects have been mostly studied in the nigrostriatal system in previous research9. To evaluate whether changes of dynein were related to α-synuclein aggregation, double immunostaining for α-synuclein and dynein was performed. In addition, we analyzed the correlation between the motor behaviors and the protein level of dynein in the striatum. Our findings reveal that dynein changes in the nigrostriatal system of A53T α-synuclein Tg mice may contribute to their dramatic motor phenotype.\n\n\nMaterials and methods\n\nAnimal experiments were conducted in accordance with the principles and procedures of the US National Institutes of Health Guide for the Care and Use of Laboratory Animals. All protocols were approved by the Institutional Animal Care and Use Committee of Peking Union Medical College and Chinese Academy of Medical Sciences.\n\nThe generation of Tg mice expressing high levels of mutant A53T α-synuclein under the control of the mouse prion protein (PrP) promoter has been described13. Mice expressing A53T α-synuclein (line M83), but not mice expressing wild type α-synuclein, develop adult-onset progressive motor deficits13. According to the first report of M83 mice13, about 50%–70% of mice at 10–14 months of age develop the motor phenotype. Original mice were obtained from The Jackson Laboratory. We purchased the mice from Model Animal research Center of Nanjing University. In all experiments 12 male M83 mice aged between 10–14 months were used. Eight control mice were age-matched, male, nTg littermates. No statistical method was used to pre-determine sample size; however, the sample size per experiment was based on our previous successful experiments and publications. Mice were raised on a 12-h light/dark cycle, with food and water available ad libitum and were housed in groups of four per cage.\n\nThe procedure was modified from the protocol previously described14. The apparatus consisted of a rectangular area of 32 × 32 cm which was divided into 64 squares of 4 × 4 cm. An uneven surface (32 cm in diameter) made of mesh wire (200 mesh) was raised by a ring in this area. A platform (4 cm in diameter) was placed in the middle of the uneven surface (Figure 1a). The day before the test, the mice were given two trials. During the test, the animals were placed on the platform and their activities were assessed during the subsequent 6 min period. The performance of mice was video recorded. Horizontal locomotion (number of grids crossed) and latency (time to get down from the platform) were analyzed thereafter.\n\n(a) 3-D model for modified open-field test apparatus. The data (mean ± SEM) show the number of grids crossed (b) and the time to get down from the platform (c) in a 6 min period in an open field (nTg, n = 8; A53T, n = 12). * p<0.05 compared to nTg.\n\nTotal protein extracts from striatum and ventral midbrain were prepared and western blot analyses were performed as described previously15,16. Primary antibodies were as follows: mouse anti-α-synuclein (Syn204) monoclonal antibody (Cell Signaling Technology, #2647), mouse anti-dynein monoclonal antibody (Millipore, #MAB1618), rabbit anti-dynein intermediate chain polyclonal antibody (Abcam, #ab81507). Horseradish peroxidase-conjugated secondary antibody (KPL, 1:5000) and enhanced chemiluminescence solution (Applygen Technologies Inc) were used for detection. Bands were quantified using Gel-Pro Analyzer software (Media Cybernetics).\n\nMice were anesthetized with 10% chloral hydrate and then perfused through the heart for 3 min with PBS followed by 4% paraformaldehyde (50–100 ml). The brains were then removed, postfixed with 4% paraformaldehyde for 4 hours, embedded in paraffin as described by OpenWetWare (http://openwetware.org/wiki/Paraffin_embedding_and_sectioning), and cut into 3μm serial sections using Leica tissue slicer (RM2235). Stainings were all performed according to our protocol for immunostaining16,17. Immunohistochemistry was performed with mouse anti-dynein antibody (Millipore, 1:100). Double immunofluorescence was performed by using rabbit anti-dynein antibody (Abcam, #ab121209, 1:50) and mouse anti-α-synuclein antibody (Cell Signaling Technology, #2647, 1:50). Immunostaining was visualized by 3,3-diaminobenzidine or by fluorescein isothiocyanate and Alexa 546 (Invitrogen, 1:100) and was examined by either regular light or laser-scanning confocal microscope (Zeiss, Germany).\n\nImage analyses were performed according to previously published procedures9,18,19. Briefly, a 1 × 0.5 mm2 contour was placed over the striatum at low magnification (×4 objective) and the optical density of dynein-immunoreactivity (dynein-ir) within the contour were measured under high magnification (×40 objective). Each subfield of the substantia nigra pars compacta (SNpc) was manually outlined at low magnification (×4 objective). 50% of the fields were randomly selected. Then, at high magnification (×40 objective), each selected field was retrieved automatically. Optical density measurements were performed on individual tyrosine hydroxylase-positive neurons which had been stained on adjacent sections. The optical density of dynein-ir in the striatum and SNpc was measured for six sections per animal (1 out of every 20 serial sections) using Image-Pro Plus software (Media Cybernetics).\n\nThe results are expressed as the mean ± SEM. Statistical significances were determined by two-tailed Student’s t-test. Pearson’s correlation test was used to analyze the correlation between motor behaviors and dynein protein level. The level of statistical significance was set at p<0.05. All analyses were conducted by the statistical software package SPSS 13.0 for Windows.\n\n\nResults\n\nConsistent with a previous report13, a few homozygous mice expressing A53T α-synuclein developed a progressively severe motor phenotype at 8 months of age. No behavioral tests were performed on the transgenic mice before 8 months of age. In the present study, we used a modified open field test14 to quantitatively evaluate the motor behaviors of the mice (Figure 1a). A53T human α-synuclein Tg mice showed a 60% decrease in the number of total grids crossed compared to nTg mice (p>0.05, Figure 1b), and A53T mice took much longer (3-fold) to get down from the platform than nTg mice (p<0.05, Figure 1c), indicating decreased motor function.\n\nWestern blotting was used to detect dynein protein expression in the striatum and ventral midbrain. The levels of dynein were significantly decreased in the striatum of A53T human α-synuclein Tg mice compared to nTg mice (p<0.001, Figure 2a, b). In contrast to the results in the striatum, the expression levels of dynein were upregulated in the ventral midbrain (p<0.001, Figure 2a, b).\n\n(a) Representative western blot images of dynein and β-actin in the striatum and midbrain are shown, and (b) the results were quantified (mean ± SEM) and normalized by the averaged value of nTg group. nTg, n = 5; A53T, n = 8; *** p<0.001 compared to nTg.\n\nNext, dynein immunohistochemistry was performed on the nigrostriatal sections of the two groups (Figure 3). An obvious suppression of the dynein immunoreactivity occurred in the striatum in A53T human α-synuclein Tg mice compared with that of nTg mice (Figure 3c, d). Consistent with the increased protein level of dynein in the ventral midbrain, dynein immunoreactivity in the SNpc was elevated in the transgenic mice (Figure 3e, f). Abundant dynein accumulation was seen in neuronal perikarya in the SNpc (Figure 3f). Quantitative analysis of dynein-ir optical density is shown in Figure 3g and h. The data showed that dynein-ir optical density was significantly decreased in the striatum of A53T α-synuclein Tg mice (p<0.001), whereas it was increased in the SNpc (p<0.001).\n\nOptical density of dynein-immunoreactivity was quantified in the striatum and SNpc as shown in (a) and (b)26. The photomicrographs show the distribution of immunoreactivity for dynein in the striatum (c–d) and SNpc (e–f). Sections were prepared from nTg mice (c, e) and A53T α-synuclein Tg mice (d, f). The immunoreactivity for dynein was decreased in the striatum of A53T α-synuclein Tg mice but increased in the SNpc. Insets in (e) and (f) are high-magnification images of the region indicated by arrows. Abundant dynein accumulation in neuronal perikarya in the SNpc is indicated by arrows in f. The optical density of dynein-ir in the striatum (g) and SN (h) was quantified (mean ± SEM) and normalized by the averaged value of nTg group (n = 3). ***p<0.001 compared to nTg. A section of the striatum from a 12-month-old homozygous M83 mouse was double labeled with anti-α-synuclein (i, red) and dynein (j, green) antibodies. Note that dynein immunofluorescence intensity was extensively reduced in cells with α-synuclein inclusions (arrows, i–k). Scale bar = 50 μm in (c–f); 20 μm in insets and (i–k).\n\nConsistent with a previous report13, we observed A53T α-synuclein inclusions in the striatum (Figure 3i). Co-localization studies revealed that a marked reduction in dynein immunoreactivity was observed in striatal cells featuring α-synuclein-immunoreactive inclusions compared to cells without α-synuclein inclusions (Figure 3i–k), suggesting that the reduction in dynein level was associated with accumulation of A53T α-synuclein.\n\nFigure 4 shows a significant positive correlation between the number of total grids crossed and the dynein protein level in the striatum (Figure 1a, r = 0.7688, p<0.01), as well as a negative correlation between the latency to get down from the platform and the dynein protein level in the striatum (Figure 1b, r = -0.6559, p<0.05). These data imply that the dynein protein level in the striatum was correlated with the motor behaviors.\n\nThe correlation analysis between number of grids crossed (a), time to get down from the platform (b), and the protein levels of dynein in the striatum was performed by the Pearson’s correlation test. Asterisks (*) show the significance of the correlation (two tailed). *p<0.05, **p<0.01.\n\n\nDiscussion\n\nThe major finding of this study is that dynein changes in expression occurred in the nigrostriatal system of A53T human α-synuclein Tg mice, with the level of dynein increasing in the SN, and decreasing in the striatum. At the behavioral level, the alterations were accompanied by significantly reduced horizontal locomotion and prolonged latency in modified open field test (Figure 1). A correlation analysis showed that the motor behaviors were significantly related to the protein level of dynein in the striatum (Figure 4). Furthermore, analysis of the co-localization of dynein and α-synuclein in the striatum indicated that the reduction in dynein level was associated with accumulation of A53T α-synuclein (Figure 3).\n\nThe severe motor phenotype was associated with the formation of α-synuclein inclusions in mice expressing A53T human α-synuclein; therefore M83 Tg mice represent an excellent model of α-synucleinopathies (especially familial PD)13. The 10–14 month old Tg mice were too weak to perform in the traditional behavior tests such as the rotarod test and the pole test20,21, and therefore we used a modified open field test that combines the traditional pole test and open field test. The number of grids crossed was equivalent to the square crossings in the traditional open-field test, a measure of general locomotor activity. The latency to get down from the platform was equivalent to the time to orient downwards in the traditional pole test, which has been used to assess basal ganglia-related movement disorders in mice20. The advantages of this method are the following: (i) the height of the platform is far lower than that of the pole, and, therefore, mice are protected from injuring themselves; (ii) the uneven surface increases the difficulty of movement, thus making the differences in motor function more obvious.\n\nOur data demonstrated that the critical retrograde axonal transport motor dynein was markedly reduced in the striatum of A53T α-synuclein Tg mice, whereas it was upregulated in the SN, indicating defects in retrograde axonal transport in the nigrostriatal pathway. Recent reports have demonstrated that α-synuclein can interact with dynein-containing complexes and its transport involves dynein motor protein22. The reduction in retrograde axonal transport might produce α-synuclein aggregation in neuronal processes (Lewy neurites in the striatum), which provides a reasonable explanation for the fact that aggregation of α-synuclein in neuronal processes was a major feature of M83 Tg mice13. On the other hand, mutant A53T α-synuclein is strongly dependent on autophagy for their clearance23, a dynein-dependent process7. In effect, the abnormalities of dynein would affect the clearance of A53T mutant α-synuclein protein by autophagy. Indeed, our co-localization results suggested that the reduction in dynein level was associated with accumulation of A53T α-synuclein in the striatum (Figure 3).\n\nCorrelation analysis between motor behaviors and dynein demonstrated that the motor behaviors were related to the alteration of dynein protein level in the striatum, which supports the involvement of dynein in neurodegeneration associated with PD and other α-synucleinopathies. In accordance with our findings, a large body of evidence has demonstrated that axonal transport machinery is impaired during neurodegeneration, and likely contributes to this condition24,25.\n\nDynein alterations have been detected in several α-synuclein-based models. A report indicated that viral over-expression of human mutant (A53T) α-synuclein resulted in an increase of dynein in striatum but no change in the SN 8 weeks after the injection8. Another report showed a decrease of dynein in the SN 6 weeks following viral A30P α-synuclein over-expression9. These divergent results may be related to the use of different mutant α-synuclein, different promoters, different animal species and different methods of protein evaluation.\n\nThis study also raises a few concerns that need to be mentioned. First, the complex changes exhibited by mice expressing A53T human α-synuclein suggest dysfunction in other neuronal systems. The presence of α-synuclein pathology in the motor neurons of the spinal cord may also contribute to motor deficits. Second, although dynein defects occur in the SN, TH-expressing neurons of the SN are spared from pathology. This population of neurons may be protected from the formation of inclusions due to the lack of neuromelanin formation in mice.\n\n\nConclusion\n\nOur results support the idea that dynein changes in the nigrostriatal system of A53T α-synuclein transgenic mice may contribute to their severe movement disorder, which provides new information for understanding the role of dynein in α-synuclein-linked neurodegeneration.\n\n\nData availability\n\nfigshare: Raw data from behavior tests and dynein protein levels, doi: 10.6084/m9.figshare.95493327",
"appendix": "Author contributions\n\n\n\nYan Liu performed most experiments. Yan Liu and Jian-Dong Sun analyzed the data and prepared the manuscript. Jing Li and Zhi-Peng Li assisted in experiments. Nai-Hong Chen and Yu-He Yuan designed the study.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by National Natural Science Foundation of China Grants (No. 30973887, No. 81073078, Key Program No. U832008), National Key Sci-Tech Major Special Item (No. 2012ZX09301002-004), and Studies on Structure and function of Bioactive Substances from Natural Medicines (IRT1007).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nSpillantini MG, Schmidt ML, Lee VM, et al.: Alpha-synuclein in Lewy bodies. Nature. 1997; 388(6645): 839–840. PubMed Abstract | Publisher Full Text\n\nSpillantini MG, Crowther RA, Jakes R, et al.: alpha-Synuclein in filamentous inclusions of Lewy bodies from Parkinson's disease and dementia with lewy bodies. Proc Natl Acad Sci U S A. 1998; 95(11): 6469–6473. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSingleton AB, Farrer M, Johnson J, et al.: alpha-Synuclein locus triplication causes Parkinson's disease. Science. 2003; 302(5646): 841. PubMed Abstract | Publisher Full Text\n\nPolymeropoulos MH, Lavedan C, Leroy E, et al.: Mutation in the alpha-synuclein gene identified in families with Parkinson's disease. Science. 1997; 276(5321): 2045–2047. PubMed Abstract | Publisher Full Text\n\nEschbach J, Dupuis L: Cytoplasmic dynein in neurodegeneration. Pharmacol Ther. 2011; 130(3): 348–363. PubMed Abstract | Publisher Full Text\n\nSchroer TA, Steuer ER, Sheetz MP: Cytoplasmic dynein is a minus end-directed motor for membranous organelles. Cell. 1989; 56(6): 937–946. PubMed Abstract | Publisher Full Text\n\nRavikumar B, Acevedo-Arozena A, Imarisio S, et al.: Dynein mutations impair autophagic clearance of aggregate-prone proteins. Nat Genet. 2005; 37(7): 771–776. PubMed Abstract | Publisher Full Text\n\nChung CY, Koprich JB, Siddiqi H, et al.: Dynamic changes in presynaptic and axonal transport proteins combined with striatal neuroinflammation precede dopaminergic neuronal loss in a rat model of AAV alpha-synucleinopathy. J Neurosci. 2009; 29(11): 3365–3373. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChu Y, Morfini GA, Langhamer LB, et al.: Alterations in axonal transport motor proteins in sporadic and experimental Parkinson's disease. Brain. 2012; 135(Pt 7): 2058–2073. PubMed Abstract | Publisher Full Text\n\nMorfini G, Pigino G, Opalach K, et al.: 1-Methyl-4-phenylpyridinium affects fast axonal transport by activation of caspase and protein kinase C. Proc Natl Acad Sci U S A. 2007; 104(7): 2442–2447. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSon JH, Shim JH, Kim KH, et al.: Neuronal autophagy and neurodegenerative diseases. Exp Mol Med. 2012; 44(2): 89–98. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSaha AR, Hill J, Utton MA, et al.: Parkinson's disease alpha-synuclein mutations exhibit defective axonal transport in cultured neurons. J Cell Sci. 2004; 117(Pt 7): 1017–1024. PubMed Abstract | Publisher Full Text\n\nGiasson BI, Duda JE, Quinn SM, et al.: Neuronal alpha-synucleinopathy with severe movement disorder in mice expressing A53T human alpha-synuclein. Neuron. 2002; 34(4): 521–533. PubMed Abstract | Publisher Full Text\n\nLuchtman DW, Shao D, Song C: Behavior, neurotransmitters and inflammation in three regimens of the MPTP mouse model of Parkinson's disease. Physiol. Behav. 2009; 98(1–2): 130–138. PubMed Abstract | Publisher Full Text\n\nVila M, Jackson-Lewis V, Vukosavic S, et al.: Bax ablation prevents dopaminergic neurodegeneration in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease. Proc Natl Acad Sci U S A. 2001; 98(5): 2837–2842. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSun JD, Liu Y, Yuan YH, et al.: Gap junction dysfunction in the prefrontal cortex induces depressive-like behaviors in rats. Neuropsychopharmacology. 2012; 37(5): 1305–1320. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKong LL, Hu JF, Zhang W, et al.: Expression of chemokine-like factor 1 after focal cerebral ischemia in the rat. Neurosci Lett. 2011; 505(1): 14–18. PubMed Abstract | Publisher Full Text\n\nBiju K, Zhou Q, Li G, et al.: Macrophage-mediated GDNF delivery protects against dopaminergic neurodegeneration: a therapeutic strategy for Parkinson's disease. Mol Ther. 2010; 18(8): 1536–1544. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWu LM, Han H, Wang QN, et al.: Mifepristone repairs region-dependent alteration of synapsin I in hippocampus in rat model of depression. Neuropsychopharmacology. 2007; 32(12): 2500–2510. PubMed Abstract | Publisher Full Text\n\nHwang DY, Fleming SM, Ardayfio P, et al.: 3,4-dihydroxyphenylalanine reverses the motor deficits in Pitx3-deficient aphakia mice: behavioral characterization of a novel genetic model of Parkinson's disease. J Neurosci. 2005; 25(8): 2132–2137. PubMed Abstract | Publisher Full Text\n\nGorton LM, Vuckovic MG, Vertelkina N, et al.: Exercise effects on motor and affective behavior and catecholamine neurochemistry in the MPTP-lesioned mouse. Behav Brain Res. 2010; 213(2): 253–262. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUtton MA, Noble WJ, Hill JE, et al.: Molecular motors implicated in the axonal transport of tau and alpha-synuclein. J Cell Sci. 2005; 118(Pt 20): 4645–4654. PubMed Abstract | Publisher Full Text\n\nWebb JL, Ravikumar B, Atkins J, et al.: Alpha-Synuclein is degraded by both autophagy and the proteasome. J Biol Chem. 2003; 278(27): 25009–25013. PubMed Abstract | Publisher Full Text\n\nDe Vos KJ, Grierson AJ, Ackerley S, et al.: Role of axonal transport in neurodegenerative diseases. Annu Rev Neurosci. 2008; 31: 151–173. PubMed Abstract | Publisher Full Text\n\nMorfini GA, Burns M, Binder LI, et al.: Axonal transport defects in neurodegenerative diseases. J Neurosci. 2009; 29(41): 12776–12786. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPaxinos G, Franklin KBJ: The Mouse Brain in Stereotaxic Coordinates. San Diego: Academic Press; 1997. Reference Source\n\nLiu Y, Yuan YH, Sun JD, et al.: Raw data from behavior tests and dynein protein levels. figshare. 2014. Data Source"
}
|
[
{
"id": "4454",
"date": "06 May 2014",
"name": "James B Koprich",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummaryIn this paper by Liu et al. a relationship between alpha-synuclein accumulation, motor deficits and expression of dynein in the nigrostriatal pathway is described. The study used transgenic mice overexpressing A53T aSyn similar to the M83 line and age matched Wt littermate controls. A behavioural deficit was confirmed in the Tg mice using a modified open-field test. The Tg mice showed reduced exploration and increased latency to get down from the starting platform. Mice were then sacrificed and tissue was prepared for western blot of dynein and immunohistochemistry of dynein and aSyn. The results showed decreased levels of dynein in the striatum and increased levels in the SN, both from western blots and histology. Furthermore, the authors showed that dynein levels were reduced in neurons with higher level of aSyn.CommentsThe number of animals used in each analysis needs to be provided along with the degrees of freedom for each statistical test performed. The staining in Fig 3, panel F has much more background compared to its counterpart in panel E. Please confirm that cases from different groups were all stained together to rule out any histology related confounds. The results from Fig 3 panels I, J and K are taken from a single region of a single case. Random sampling within the region of interest should be performed on each case (compared to Wt controls) to arrive at any conclusion. Optical density or fluorescent intensity measures should be obtained in the process to provide a quantitative assessment. The co-staining in Fig 3, panel K should include TH to know that the neurons being analyzed are SN dopamine neurons as alluded to in the conclusion.",
"responses": []
},
{
"id": "4861",
"date": "05 Jun 2014",
"name": "Gerardo Morfini",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nA large body of pathological evidence has established a “dying-back” pattern on degeneration for neurons affected in Parkinson’s disease (PD). Indeed, axonal processes and synapses of dopaminergic neurons in the substantia nigra (SN) progressively degenerate long before these cells undergo cell death. Mechanisms underlying early degeneration of axons in PD remain unknown, but several lines of evidence have linked this critical pathogenic event to abnormalities in the function of conventional kinesin and cytoplasmic dynein (CDyn), major microtubule-based molecular motors responsible for axonal transport in adult neurons.Consistent with these observations above, this work by Liu and collaborators reports a striking alteration in the distribution of CDyn in brains of transgenic mice expressing a PD-related mutant form of alpha synuclein (A53T-Tg mice). Immunohistochemical and immunoblotting experiments demonstrated a marked increase in levels of a specific CDyn subunit (DIC) in the SN of A53T Tg mice, compared to age-matched, non-transgenic control mice (nTg mice). Conversely, A53T-Tg mice showed decreased levels of DIC in the striatum when compared to nTg mice. Co-localization studies further suggested that this reduction in striatal DIC levels was associated to accumulation of A53T alpha-synuclein. Additionally, a highly sensitive open-field test paradigm confirmed and further extended prior reports of motor defects in A53T-Tg mice, which significantly correlated with alterations in striatal DIC levels.Taken together, data from this paper provides novel evidence suggesting that alterations in CDyn function might contribute to disease pathogenesis associated with PD-related mutant alpha synuclein. Comments The data presented is consistent with increased activation of CDyn-dependent retrograde axonal transport by mutant alpha synuclein. Supporting this view, prior studies showed activation of CDyn-based retrograde transport by the parkinsonian drug MPP+ (Kim-Han JS et al., J Neurosci. 2011). However, this possibility is not considered in the Discussion section. CDyn is composed of multiple protein subunits including heavy (DHC) intermediate (DIC), and light (DLC) subunits, among others. In Materials and Methods, the authors report using both a monoclonal (clone 74.1; Millipore#MAB1618), and a polyclonal (Abcam ab81507) antibody against DICs, but is unclear which one was used for immunoblots shown in Figure 2. Throughout the manuscript and in the Figure 2 legend, the text should clearly indicate that data gathered in Figure 2 corresponds to DIC subunits, rather than “dynein”. The authors report using an antibody from Millipore (presumably #MAB1618 against DIC), and a polyclonal (Abcam ab121209) antibody against TcTtex (one of several DLC subunits). Again, the main text and Figure 3 legend should indicate which specific CDyn subunits are analyzed for each panel in Figure 3. Some type of quantitation could be provided to illustrate the negative correlation between TcTtex levels and A53T alpha-synuclein accumulation. Minor comments The authors mention that the major finding in their study is that changes in CDyn expression occurred in the nigrostriatal system of A53T human α-synuclein Tg mice. However, the experiments performed do not directly address protein expression. Rather, those evaluate overall DIC steady state levels. To ensure reproducibility of results by other investigators, please provide the lot number for Abcam polyclonal antibodies #ab81507 and #ab121209.",
"responses": []
},
{
"id": "5464",
"date": "28 Jul 2014",
"name": "Abraham Acevedo-Arozena",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDefects in axonal transport are associated with a number of neurodegenerative disorders, including Parkinson’s disease (PD). Retrograde transport in neurons is powered by the cytoplasmic dynein complex, a macromolecular structure that contains different heavy, intermediate and light dynein polypeptides. Here, Liu et al report changes in “dynein” protein levels in a widely used mouse model of PD over-expressing mutant (A53T) a-synuclein. Interestingly, the authors found an increase in a-synuclein levels via immunoblot and immunohistochemistry in the substantia nigra (SN) of symptomatic a-synuclein mutant transgenic mice. Conversely, they found a decrease in “dynein” from the striatum of the same mice. The authors used an interesting modification of the open field paradigm to test the mice for motor abnormalities. By comparing the levels of “dynein” in the striatum with the degree of motor deficits in the tested mice, the authors found a correlation between striatum dynein levels and motor performance.Comments:Although the authors refer to “dynein” levels throughout the manuscript, the antibodies they are using for immunoblot are against the cytoplasmic intermediate chain (DIC1?). This needs to be clarified in the materials and also throughout the manuscript. The same applies to the immunohistochemistry. The authors report using two different anti-“dynein” antibodies, but one is against DIC (as above) and the other one against a cytoplasmic dynein light chain (DYNLT3). The authors should clarify which antibody they are using in Figure 3, as well as clarify throughout the text which cytoplasmic dynein subunits they are referring to.\n\nThe authors state at the end of the discussion that “although dynein defects occur in the SN, TH expressing neurons of the SN are spared from pathology”. However, they do not show TH co-staining together with a-synuclein in Figure 3. If this statement is referring to previously published data, it should be referenced. If it is referring to their data, it should also appear in Figure 3. In Figure 4, it appears that non-transgenic mice have also been used, together with symptomatic A53T transgenics, for the correlation between the “dynein” levels and the motor abnormalities. I would like to see the same correlation using transgenic mice only, which have obvious motor abnormalities.",
"responses": []
}
] | 1
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https://f1000research.com/articles/3-68
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https://f1000research.com/articles/2-201/v1
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03 Oct 13
|
{
"type": "Short Research Article",
"title": "Implementation of a worksite educational program focused on promoting healthy eating habits",
"authors": [
"Dimitra Tanagra",
"Dimitris Panidis",
"Yannis Tountas",
"Elina Remoudaki",
"Evangelos C. Alexopoulos",
"Dimitra Tanagra",
"Dimitris Panidis",
"Yannis Tountas",
"Elina Remoudaki"
],
"abstract": "Objective: To estimate the effectiveness of a short-term educational-counseling worksite program focused on lipid intake, by monitoring the possible change on nutrition knowledge and eating habits.Methods: an 8-week educational program based on the Health Belief Model was implemented in a honey packaging and sales company in Greece. 20 out of the 29 employees initially enrolled completed the program. Knowledge level and eating habits were evaluated prior and after the intervention by the “Nutrition Knowledge Questionnaire” and the “Food Habits Questionnaire”. ANOVA, Spearman rho test and paired Wilcoxon test were employed in statistical analysis.Results: Non smokers and those with higher educational level had healthier eating habits. Knowledge following the intervention was significantly improved concerning recommendations and basic food ingredients but as far as eating habits were concerned, scores were not improved significantly, while intake of fried food was increased.Conclusions and Implications: Short-term interventions may produce substantial improvement in knowledge but not necessarily modifications in unhealthy eating habits.",
"keywords": [
"Greece",
"worksite intervention",
"eating habits",
"health promotion programs."
],
"content": "Introduction\n\nOver the last decades, obesity has rapidly turned into a global epidemic in both developed and developing countries, affecting adults, children and adolescents as well. Currently, the number of people suffering from obesity is estimated at approximately 400 million people worldwide1. Moreover, increased body mass index (BMI) is associated with higher risk of cardiovascular diseases, some types of cancer and type II diabetes2–4. Recent data from Greece have shown obesity is an epidemic problem5,6. In recent years, Greeks have abandoned the traditional Mediterranean diet; one study reports that only 33% of Greek men and 43% of Greek women adhere to a traditional Mediterranean diet7.\n\nAmong various individual and lifestyle factors, many work-related factors are responsible for the modification of dietary patterns including working conditions, such as: working overtime, high job demands, occupational stress and others8. On the other hand, the workplace has been identified as a promising setting for health promotion although the findings of many worksite health promotion (WHP) programs indicate that these programs are associated with only moderate improvement in dietary intake9. Furthermore, it was shown that diet mediterranisation is feasible in a food-at-work intervention, affecting lunch consumption at the workers canteen10. However in a systematic review, participation levels in health promotion interventions at the workplace were typically below 50%11.\n\nThe purpose of the present study was to evaluate the effectiveness of an educational worksite intervention focused on lowering fat intake, by affecting nutrition knowledge and eating habits.\n\n\nMethods\n\nThe 48 employees working in the factory premises of a honey company, were asked to participate in this study. Most of the 48 employees were employed in jobs that required mild to moderate manual and intellectual activity (blue collar workers) and five were employed as food scientists and technologists, and supervisors. No inclusion or exclusion criteria were used and as there were no medical contraindications for participation in the program, as judged by the occupational health physician, all employees were eligible for participation. Twenty-nine employees responded positively (60%) to the invitation and gave their written informed consent. The Medical School Review Board judged that further approval was not required, since this program was under the occupational physician’s supervision and control. During the program, seven employees failed to attend day 2 and/or 3 and another two did not return the final questionnaires and all nine were excluded from the final analysis (see Table 1).\n\nInitially, all employees were informed about the program and were asked to participate by signing informed consent. Two questionnaires were used in order to estimate (Q1) the employees’ nutrition knowledge level and (Q2) their eating habits (see Supplementary File Q1 and Supplementary File Q2). The questionnaires were translated into Greek by two bilingual expert nutritionists and were piloted in 10 college students and blue collar employees for linguistic validation.\n\nNutrition knowledge was assessed using the “Nutrition Knowledge Questionnaire” (see Supplementary File Q1)12. The questionnaire covers four sections: (i) knowledge on experts' recommendations regarding the optimum intake of different food groups (maximum score: 11); (ii) nutrient knowledge, (maximum score: 69); (iii) food choice (which asks people to choose between different options, e.g. to pick the snack that is low in fat and high in fibers), (maximum score: 10); and (iv) the relationships between diet and disease (maximum score: 20). This last section looks at beliefs about the associations between food type, food quantity and diseases.\n\nThe eating habits of the participants were assessed by the “Food Habits Questionnaire” (see Supplementary File Q2)13,14, which has been widely used to estimate dietary changes15,16. Questions were rated on a 4-point scale, where 1 reflects the healthiest and 4 the unhealthiest eating habits, respectively. The questionnaire included five sections regarding the following habits: (i) replacing high fat foods with low fat substitutes (score range: 7–28); (ii) modifying high fat foods, e.g. fat removal from meat (score range: 3–12); (iii) avoiding high fat cooking methods (fried food) (score range: 4–16); (iv) consumption of fresh fruit and vegetables as a snack (score range: 3–12); and (v) choosing specially manufactured low fat foods products instead of high fat ones (score range: 5–20). The total score of eating habits is calculated from the sum of section scores divided by 5 (ranged from 4.4 to 17).\n\nData on age, family status, children, educational level, job position, smoking habit, BMI, arterial blood pressure and number of cigarettes/years of smoking were also collected (see Table 2).\n\nThe intervention took place in three distinct phases over a total of 7–8 weeks (Table 1) and it was based on the Health Belief Model which suggests that health behaviors are determined by health beliefs and readiness to take action. Behavioral theory has increasingly been used to guide nutrition research to improve intervention efficacy. The Health Belief Model was developed in the 1950s to explain health behavior associated with the failure of people to participate in programs that would reduce disease risk. Constructs central to the HBM consist of perceived susceptibility, perceived severity, perceived benefits, perceived barriers, and other mediating variables. The construct of self-efficacy is frequently included in applications of the HBM17,18.\n\nIn our program (Phase 1), all employees who initially responded to the invitation attended a 30 minute meeting in which a brief presentation of the self-administered questionnaires was done and instructions about the proper completion of both questionnaires were given. Further clarifications were answered the following days during the collection, where necessary. Data on individual characteristics (age, marital status, children, education, smoking status etc.), were also collected and blood pressure, weight and height of the subjects were measured (Seca® 764, Sigma Medical Co, Athens, Greece) at the end of the meeting. Completed questionnaires were collected, recorded in an electronic database and statistically analyzed. In Phase 2, 15 days after the questionnaires were initially distributed, a 45-minute lecture on healthy eating and mediterranean diet was held followed by discussion and distribution of printed material with practical proposals for adoption of healthier eating habits. Overall, the whole session lasted approximately two hours. A week later a second meeting took place (Phase 3) in order to discuss and clarify issues derived from the conclusions of the initial analysis. Specifically, knowledge gaps and restrictive factors for the adoption of healthier nutritional choices were further discussed. The intervention was completed 22 days later (Phase 4) when the participants were asked to fill in the questionnaires again. In the last phase (5th), a final meeting took place to present and discuss the results, and for individual counseling by the research team.\n\nAnalysis of variance (ANOVA) was used to reveal statistically significant differences among various subgroups. Due to the small sample, Spearman rho test was used to examine correlations of the quantitative variables while the paired signed Wilcoxon test was used to compare average values of continuous variables for each category of nominal variables in the intervention group (before and after). A p-value of <0.05 was considered statistically significant. Statistical processing and data analysis were performed using commercial software (SPSS version 16.0, SPSS Inc., 2007).\n\n\nResults\n\nFrom the 29 workers who initially responded positively, 20 workers (67%) attended all phases and completed the WHP program (Table 1). Losses were mainly due to absences on the days of intervention, or inability or failure to return the study questionnaires in time. Between the final and initial groups there were no significant differences in any of the variables.\n\nAnalysis of the knowledge questionnaire, returned by the 29 workers who initially responded, showed a lack of knowledge of food composition in saturated fat, fibers, and salt, of the origin of fatty acids (monosaturated, polysaturated and saturated) and of the sources of antioxidant vitamins. Education level correlated significantly with the partial score, i.e. the higher the education level the higher the scores.\n\nAnalysis of the habits questionnaire showed that dietary habits included medium to large consumption of fatty foods. However, the score of the workers corresponding to the avoidance of fried foods was pretty high, reaching almost the excellent level. No significant correlations were found between variables under study and the two first subscales (replacement of fatty foods and meat modification). Men, people living with others, and those without children had a tendency to avoid the more fatty substances. Meanwhile, women consumed less fried foods.\n\nPost-intervention analysis was done in the 20 workers who had participated in all phases. Table 2 shows the demographic and individual characteristics of these workers. Women and blue collar workers accounted for 90% and 95%, respectively, a fact that limits the possibility of revealing significant effects of these variables (sex and job title) on the results of the intervention.\n\nAs expected, there were significant correlations between the sections (scales) of the two questionnaires prior to, and following, the intervention. By contrast, between the different questionnaires, scales were less and weakly correlated with the exemption of the group of the avoidance of fatty substances. Very high Cronbach alpha score (above 0,80) shows satisfactory reliability of all subscales of both questionnaires. Table 3 presents the scores per category (section) of nutritional knowledge prior to, and following, the intervention in the 20 workers. Significant improvement was seen in the sections of “dietary recommendations”, in “basic food ingredients” and in the total score. Prior to intervention, non-smokers had higher (better) scores concerning the subscales of the “basic food ingredients” (41.0 vs 33.1, p=0.08) and the “selection of healthier foods” (6.2 vs 4.8, p=0.06) but these differences did not reach statistical significance. Following the intervention, non-smokers improved more in the “selection of healthier foods” (6.8 vs 5.2, p=0.04).\n\nTable 4 presents the dietary habit scores prior to, and following, the intervention. The mean score was improved in the categories of “replacement of fatty foods”, “meat modification”, “consumption of food and vegetables”, “avoidance of fatty substances” and in the total score but the difference was far from significant. On the contrary in the habit of “avoidance of fried foods”, the score was significantly worse, a paradox that might be explained by the very high initial score (tendency towards regression to the mean).\n\nWorkers with a normal BMI exhibited better habits compared with overweight and obese subjects in terms of “meat modification” (4.6 vs 12.7, p=0.002). Non-smokers had lower scores compared with smokers in “fat avoidance” (12.6 vs 15.0, p=0.06) and in “avoidance of fried food” (6.5 vs 7.7, p=0.05).\n\n\nDiscussion\n\nIn the present study, a short-term intervention regarding eating knowledge and habits was implemented in a worksite. Knowledge was significantly improved following this intervention, while no significant improvement was achieved concerning dietary habits. The paradox regarding the fact that average consumption of fried, browned or breaded food (“avoidance of fried food”) increased following the intervention could be partially attributed to the very high (excellent) initial score combined with the fact that some vacations also coincided with the program. The finding that knowledge gain did not lead to habit modification may be explained by the short duration of the program and the complexity that characterizes the conscious or unconscious choices of adults. Health promotion programs in workplaces have shown to be cost-effective, especially for long-term interventions19,20.\n\nIn our study, a number of factors, i.e. education level, job title, family situation and smoking status was shown to be related to the level of nutrition knowledge and dietary habits. However, the small number of participants prevented these correlations to be concurrently analyzed in multivariate analysis. Other factors known to influence dietary behavior including socio-economic factors, stress and organizational factors (increased work demands, low skills motivation, overtime employment) were not analyzed in our study21,22 but in our setting, the population was highly homogeneous and most of these factors are not anticipated to have a significant discriminatory impact. However, recent findings show that these interventions can be easily incorporated into the daily working routine programs, and if combined with stress management programs, may result in better results23,24.\n\nLimitations of this study arise from the small sample size and the short duration of the program. For organizational reasons, we did not attempt to allocate a control group by randomization. On the other hand, the homogeneity of the population concerning socio-economic aspects, the supportive environment, and the good relationships between colleagues are considered to minimize confounding of the different factors.\n\nShort-term interventions may produce substantial improvement in knowledge but not necessarily accompanied by changes in unhealthy eating habits. These types of programs are not far from those commonly encountered in every day practice but do not seem to be effective in changing unhealthy eating habits. Participation by the employees in defining their needs and priorities; planning long-term interventions, and incorporating self-empowerment and stress management techniques might be necessary for cost-effective worksite health promotion programs to succeed in reducing unhealthy eating habits.",
"appendix": "Author contributions\n\n\n\nConception and design of the study: DT, YT and ECA; Questionnaire validation and data collection: DT, DP and ER; Intervention team: DT, ER and ECA; Data analysis: DP and ECA; Draft writing: DP, ER and ECA; Co-ordination: YT and ECA; Guarantor of the study: ECA.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe are grateful to all the employees for their participation.\n\n\nReferences\n\nWorld Health Organization. Obesity and overweight.2013. Reference Source\n\nGrundy SM, Pasternak R, Greenland P, et al.: Assessment of cardiovascular risk by use of multiple-risk-factor assessment equations: a statement for healthcare professionals from the American Heart Association and the American College of Cardiology. Circulation. 1999; 100(13): 1481–1492. PubMed Abstract | Publisher Full Text\n\nDietz WH, Franks AL, Marks JS: The obesity problem. N Engl J Med. 1998; 338(16): 1156–1158. PubMed Abstract | Publisher Full Text\n\nDixon JB: The effect of obesity on health outcomes. Mol Cell Endocrinol. 2010; 16(2): 104–108. PubMed Abstract | Publisher Full Text\n\nRoditis M, Parlapani E, Tzotzas T, et al.: Epidemiology and predisponding factors of Obesity in Greece: from the second World war until today. J Pediat Endocrinol Metabol. 2009; 22(5): 389–406. PubMed Abstract | Publisher Full Text\n\nKatsarou A, Tyrovolas S, Psaltopoulou T, et al.: Socio-economic status, place of residence and dietary habits among the elderly: the Mediterranean islands study. Public Health Nutr. 2010; 13(10): 1614–1621. PubMed Abstract | Publisher Full Text\n\nPitsavos C, Panagiotakos DB, Chrysochοou C, et al.: Epidemiology of cardiovascular risk factors in Greece: aims, design and baseline characteristics of the ATTICA study. BMC Public Health. 2003; 3: 32. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNishitani N, Sakakibara H, Akiyama I: Eating behaviour related to obesity and job stress in male Japanese workers. Nutrition. 2009; 25(1): 45–50. PubMed Abstract | Publisher Full Text\n\nNi Mhurchu C, Aston LM, Jebb SA: Effects of worksite health promotion interventions on employee diets: a systematic review. BMC Public Health. 2010; 10: 62. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLeighton F, Polic G, Strobel P, et al.: Health impact of Mediterranean diets in food at work. Public Health Nutr. 2009; 12(9A): 1635–1643. PubMed Abstract | Publisher Full Text\n\nRobroek SJ, van Lenthe FG, van Empelen P, et al.: Determinants of participation in worksite health promotion programmes: a systematic review. Int J Beh Nutr Phys Act. 2009; 6: 26. PubMed Abstract | Publisher Full Text | Free Full Text\n\nParmenter K, Wardle J: Development of a general nutrition knowledge questionnaire for adults. Eur J Clin Nutr. 1999; 53(4): 298–308. PubMed Abstract | Publisher Full Text\n\nKristal AR, Shattuck AL, Henry HJ: Patterns of dietary behavior associated with selecting diets low in fat reliability and validity of a behavioral approach to dietary assessment. J Am Diet Assoc. 1990; 90(2): 214–220. PubMed Abstract\n\nKristal AR, Beresford SA, Lazovich D: Assessing change in diet-intervention research. Am J Clin Nutr. 1994; 59(1 Suppl): 185S–189S. PubMed Abstract\n\nGlasgow R, Perry JD, Toobert DJ, et al.: Brief assessments of dietary behavior in field settings. Addict Behav. 1996; 21(2): 239–247. PubMed Abstract | Publisher Full Text\n\nShannon J, Kristal AR, Curry SJ, et al.: Application of a behavioral approach to measuring dietary change: the fat- and fiber-related diet behavior questionnaire. Cancer Epidemiol Biomarkers Prev. 1997; 6(5): 355–361. PubMed Abstract\n\nRosenstock IM, Strecher VJ, Becker MH: Social learning theory and the health belief model. Health Educ Q. 1988; 15(2): 175–183. PubMed Abstract | Publisher Full Text\n\nGlanz K, Patterson RE, Kristal AR, et al.: Stages of change in adopting healthy diets: fat fiber, and correlates of nutrient intake. Health Educ Q. 1994; 21(4): 499–519. PubMed Abstract | Publisher Full Text\n\nAtlantis E, Chow CM, Kibry A, et al.: An effective exercise-based intervention for improving mental health and quality of life measurers: a randomized controlled trial. Prev Med. 2004; 39(2): 424–434. PubMed Abstract | Publisher Full Text\n\nGoetzel RZ, Ozminkowski RJ: What's holding you back: why should (or shouldn't) employers invest in health promotion programs for their workers? N C Med J. 2006; 67(6): 428–430. PubMed Abstract\n\nWamala SP, Wolk A, Schenck-Gustafsson K, et al.: Lipid profile and socioeconomic status in healthy middle aged women in Sweden. J Epidemiol Community Health. 1997; 51(4): 400–407. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKouvonen A, Kivimäki M, Cox SJ, et al.: Relationship between work stress and body mass index among 45,810 female and male employees. Psychosom Med. 2005; 67: 577–583. PubMed Abstract | Publisher Full Text\n\nChristaki E, Kokkinos A, Costarelli V, et al.: Stress management can facilitate weight loss in Greek overweight and obese women: a pilot study. J Hum Nutr Diet. 2013; 26(Suppl 1): 132–9. PubMed Abstract | Publisher Full Text\n\nAlexopoulos EC, Zisi M, Manola G, et al.: Short-term effects of a randomized controlled worksite relaxation intervention in Greece. Ann Agric Environ Med. (in press).\n\n\n\n\n"
}
|
[
{
"id": "1969",
"date": "23 Oct 2013",
"name": "John Mooney",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMany thanks for the opportunity to review this paper. Tanagra and colleagues present a concise, interesting and well discussed piece of research on their attempts to improve dietary practices in the workplace, using a ‘health belief model’ approach to improving awareness in the first instance of the main components of a healthy diet and food items which it is advisable to consume more sparingly (e.g. fried food). I particularly liked the focus for the most part on blue collar occupational groups and the relative homogeneity of the target population. This holds promise for being able to target higher risk occupational groups that are known to be less receptive to health promotion type interventions.It is relatively well established among advocates of public health interventions, however, that changing actual behaviour is a much greater challenge than improving awareness of risk factors, particularly around diet and eating practices. While the contrast between the improved knowledge and no real benefit in behaviour is certainly of interest in this well controlled setting, it is not a terribly surprising or novel finding in its own right. As the authors themselves also point out, this is a study population which already has a relatively healthy diet, so their scope for encouraging and achieving improvements is necessarily limited. The narrow sample characteristics of the sub-group participating in the post-intervention follow-up (90% women) also restricts any meaningful inferences around the influence of gender and occupational group (a fact also acknowledged by the authors).My most significant misgiving about the article in its present form however surrounds the established limitations of the ‘health belief model’ as applied to preventative public health interventions:“The HBM is more descriptive than explanatory, and does not suggest a strategy for changing health-related actions. In preventive health behaviors, early studies showed that perceived susceptibility, benefits, and barriers were consistently associated with the desired health behavior; perceived severity was less often associated with the desired health behavior. The individual constructs are useful, depending on the health outcome of interest, but for the most effective use of the model it should be integrated with other models that account for the environmental context and suggest strategies for change.”[Extract from Boston University MPH content around ‘Limitations of the Health Belief Model’; http://sph.bu.edu/otlt/MPH-Modules/SB/SB721-Models/SB721-Models2.html].The call for looking at the environmental context in particular suggests a potential missed opportunity within the current study to examine the characteristics and quality of food catering within the workplace itself.Overall, as stated at the outset, I think this is an elegant and well-presented piece of research which certainly deserves to be published, but ideally needs some additional discussion around some of its methodological drawbacks. While the authors have acknowledged the short duration and sample size issues, there could be more discussion of desirability bias in self-reporting and ‘healthy-worker’ effects, the latter of which almost certainly will have diminished their scope for demonstrating improvements. Those relatively minor issues aside, there does need to be a fuller consideration of the limitations of the health belief model and the slightly confusing absence of any details on the types of catering and foods available within the workplace itself. In terms of specific essential revisions, I would suggest:Demographic characteristics (Table 2): the addition of a section within 'individual characteristics' on how this population compares with the general Greek population on BMI and smoking etc. Acknowledgement of the drawbacks of HBM and some description of the catering opportunities within the workplace itself, and if there are aspects of availability which could be improved to compliment the educational measures. Some additional discussion around the drawbacks of self reporting and to what extent other back-up measures, such as 24 hour recall diaries, had been considered to enhance and substantiate the quality of the data gathered.",
"responses": []
},
{
"id": "3399",
"date": "18 Feb 2014",
"name": "Marjorie Freedman",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review this paper. I have recently traveled to Greece and love traditional Greek food. It was distressing to read about low numbers of Greeks following a traditional Mediterranean diet, which has many health benefits. The authors are to be commended for undertaking this study in a worksite setting, aimed to lower fat intake. Overall, this research project represents a simple, non-invasive, low-cost method of attempting to improve health behaviors among a work force. It is unfortunate that the lack of evidence of significant change in actual health behavior is also typical of many such small-scale, short-term projects. The authors attempted to link change in knowledge with change in behavior. However, the literature is clear that change in knowledge alone is not sufficient to change behavior. The use of the Health Belief Model was presumably chosen to enhance motivation to change behaviors (even though over 60% of the population was at a normal weight, despite a “medium to large consumption of fatty foods.”). However, there was limited information presented in this paper as to how the constructs of the HBM were integrated into the classes presented to the employees. Did participants perceive any risks? Did they perceive susceptibility to a particular disease or to weight gain? This needs further explanation. Additional issues that need to be addressed are as follows:The Supplemental files (Q1 and Q2) are presented inversely from their description in text. The text indicates that the Knowledge questionnaire is File Q1, when it is in fact Q2, and the reverse is true for the Diet Habits questionnaire (actually Q1, listed as Q2 in text). [minor technicality] Q1, Diet Habits, indicates that some portions of this questionnaire were administered by an interviewer, while other portions were self-administered by participants. However, in text no mention is made of interviewer participation in the questionnaire, indicating only that participants received explanation in the Phase I session how to complete the form; greater clarification of procedure is required here. Furthermore, social pressures and positive presentation bias during personal interviews with a study administrator could have resulted in participants misrepresenting (toward a \"healthy\" direction) the quantities and frequencies of the foods they consumed. Particularly if the pre-intervention diet habit assessment was done via personal interview while the post-intervention was done via self-completed questionnaire, changes in dietary habits post-intervention may not have been accurately captured. This could also possibly explain the apparent increase in consumption of fried foods post-intervention. The diet habits questionnaire also asks participants about their food intake over the past month (~30 days) or 3 months; however, barely 30 days elapsed between the first day of intervention (Phase 2 - day 16) and the post-intervention questionnaire (Phase 4, day 45). If dietary changes are to be captured, either a shorter-term assessment (i.e. asking about intake over the past 2 weeks) or a greater delay between intervention and post-assessment should be considered. The authors note that some participants took vacations during the intervention/assessment period. Given the typical deviations from \"normal\" dietary habits that many people enjoy on vacation, and the recency (within the past month) of the diet habits questionnaire, this factor may function as a greater confounding variable in the observed lack of change in diet habits than the authors acknowledge. The 45-minute primary intervention lecture (Phase 2) was based on the Health Belief model, and it is the authors' claim that the HBM (which links beliefs and perceptions about health behaviors/risks to an individual's personal level of risk and his/her ability to control that risk through healthier behaviors) is an effective way to approach behavior change. However, Section 4 of the Knowledge questionnaire, which addresses the Diet-Health relationship, showed the least significant change following the intervention. Therefore, either the intervention lectures did not adequately target this subsection of knowledge, or else the participants were resistant to learning in this area for other reasons. The Discussion section, addressing the lack of observed behavior change, would be strengthened by acknowledging that the supposedly most critical piece of effecting behavior change was apparently not adequately targeted by the intervention, as evidenced by the lack of change in knowledge in this area. In addition, 61% of participants were normal weight. Thus, it is unclear why the authors chose the Health Belief Model, and how the intervention was tailored to address perceived risk, perceived susceptibility, perceived barriers or perceived benefits. [another technicality/clarification] Table 4 (comparison of dietary habits) would be improved with a caption or footnote clarifying that a lower score on this assessment indicates improved/more healthy eating habits.",
"responses": []
}
] | 1
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https://f1000research.com/articles/2-201
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https://f1000research.com/articles/2-252/v1
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21 Nov 13
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{
"type": "Research Article",
"title": "Monitoring ferumoxide-labelled neural progenitor cells and lesion evolution by magnetic resonance imaging in a model of cell transplantation in cerebral ischaemia",
"authors": [
"Rachael A Panizzo",
"David G Gadian",
"Jane C Sowden",
"Jack A Wells",
"Mark F Lythgoe",
"Patrizia Ferretti",
"Rachael A Panizzo",
"David G Gadian",
"Jane C Sowden",
"Jack A Wells"
],
"abstract": "Efficacy of neural stem/progenitor cell (NPC) therapies after cerebral ischaemia could be better evaluated by monitoring in vivo migration and distribution of cells post-engraftment in parallel with analysis of lesion volume and functional recovery. Magnetic resonance imaging (MRI) is ideally placed to achieve this, but still poses several challenges. We show that combining the ferumoxide MRI contrast agent Endorem with protamine sulphate (FePro) improves iron oxide uptake in cells compared to Endorem alone and is non-toxic. Hence FePro complex is a better contrast agent than Endorem for monitoring NPCs. FePro complex-labelled NPCs proliferated and differentiated normally in vitro, and upon grafting into the brain 48 hours post-ischaemia they were detected in vivo by MRI. Imaging over four weeks showed the development of a confounding endogenous hypointense contrast evolution at later timepoints within the lesioned tissue. This was at least partly due to accumulation within the lesion of macrophages and endogenous iron. Neither significant NPC migration, assessed by MRI and histologically, nor a reduction in the ischaemic lesion volume was observed in NPC-grafted brains. Crucially, while MRI provides reliable information on engrafted cell location early after an ischaemic insult, pathophysiological changes to ischaemic lesions can interfere with cellular imaging at later timepoints.",
"keywords": [
"contrast agent",
"ferumoxide Endorem-protamine sulphate",
"imaging",
"neural stem cell",
"MRI",
"stroke"
],
"content": "Introduction\n\nStroke remains a common cause of mortality and disability worldwide, with few effective treatments available. Cerebral ischaemia, caused by the reduction of blood flow in a cerebral artery, is the most common type of stroke, accounting for 80% of strokes1. Cellular therapy is an emerging and promising avenue for treatment of cerebral ischaemia. Several experimental studies have demonstrated that treatment with neural stem cells after an ischaemic event improves neurological score and behavioural recovery, and can reduce infarct volume2–5. These findings have led to a small number of clinical trials of cellular therapy in stroke patients, with modest effects on neurological outcome and motor skill recovery6–9. It is clear that further optimisation of cell type and cell delivery, and a greater understanding of the behaviour of engrafted cells in vivo is required for improved therapeutic outcome.\n\nPrevious research has demonstrated that fetal telencephalic neural progenitor cells engrafted into the ischaemic brain are capable of migration towards the infarct, and of improving behavioural recovery10. A small percentage of engrafted neural stem/progenitor cells (NPCs) have been shown to differentiate into neurons in the peri-infarct area and integrate into the neural network11–18. Developmentally, NPCs give rise to the cortical neuron layers in the normal developing embryo, and may have the appropriate migratory capacity and neurogenic potential to give rise to neurons after engraftment into host injury tissue.\n\nIn vivo monitoring of engrafted cells is an important tool that could be developed to correlate cell behaviour and distribution with clinical outcome. Cellular imaging with MRI contrast agents can be used to monitor the distribution of transplanted cells in models of cerebral ischaemia. Hoehn et al. demonstrated that mouse embryonic stem (ES) cells could be labelled with an iron oxide-based agent19 and subsequently migrate into the infarcted area from the contralateral hemisphere. Cell migration and functional improvement has been observed in rat models of stroke after intracisternal injection of superparamagnetic iron oxide (SPIO)-labelled neurospheres20. Engrafted cells labelled with SPIO have been shown to survive and differentiate into neural lineages in the ischaemic striatum21.\n\nFurther development of contrast agents with low toxicity, high labelling efficiency and retention is required for improved detectability and effective long-term cell tracking in vivo.\n\nThe ferumoxide Endorem is a dextran-coated superparamagnetic iron oxide agent that can form a complex with the polycationic protamine sulphate – the FePro complex22–24. Previous studies have suggested that the FePro complex has little effect on cell viability and behaviour23–25.\n\nTherefore we wished to assess the effect of FePro on NPC behaviour, and establish whether labelling and in vivo imaging of NPCs could be enhanced by using this complex. We studied the effect of FePro labelling on NPC metabolism, proliferation and differentiation capacity. FePro-labelled NPCs were engrafted into the ipsilateral parenchyma at 48 hours post-ischaemia and monitored over 4 weeks using MRI. Assessment of NPC proliferation and differentiation capacity demonstrated no difference in the behaviour of FePro-labelled NPC compared to unlabelled NPC. In vivo, we observed the evolution of hypointense T2 contrast in the ischaemic striatum over several weeks in both control and FePro-NPC engrafted animals.\n\n\nMaterials and methods\n\nFor initial cell labelling assays and quantification of iron, the ST14A rat neural progenitor cell (NPC) line was used, and cultured as previously described (generous gift from E. Cattaneo)26. For subsequent labelling, viability, proliferation and differentiation assays, and for the in vivo study, primary embryonic forebrain-derived NPCs were used.\n\nForebrain tissue was dissected from E13.5 CD1 mouse embryos (Charles Rivers UK) and digested in Trypsin with DNase. Cells were washed in trypsin inhibitor and Earl’s Balanced Salt Solution (EBSS), and plated in 6 well culture dishes. Proliferation medium contained DMEM:F12 with glutamax (Invitrogen), N2 supplement (GIBCO), 10 ng/ml EGF (Peprotech), 20 ng/ml FGF-2 (Peprotech), 0.05% heparin and 1% Penicillin-Streptomycin. Cells were incubated at 37°C with 5% CO2. Under these conditions neural stem/progenitor cultures (NPC) form aggregates called neurospheres that grow in suspension. Neurospheres were passaged every 7 days. The ST14A rat neural progenitor cell line was cultured as previously described26. All reagents were from Sigma, unless otherwise stated.\n\nFor neurosphere labelling, neurospheres were dissociated at 6 days after passage, by incubation in 1X trypsin with DNase for 7 minutes at 37°C. Cells were washed in trypsin inhibitor and EBSS, and then incubated with contrast agent (Endorem or the FePro complex) in proliferation medium for 24 hours to allow contrast agent internalisation. Cells were washed in EBSS, and dissociated for replating or use in intracerebral microinjections.\n\nFor adherent ST14A cell labelling, cells were incubated with contrast agent in culture medium for 24 hours to allow contrast agent internalisation, washed three times with EBSS, then passaged.\n\nEndorem: Cells were incubated with 500 µg/ml Endorem (Guerbet Laboratories Ltd, UK).\n\nFePro complex: 2 mg/ml protamine sulphate was incubated with Endorem in a ratio of 9:5 for 10 minutes at room temperature to form the complex (FePro), making a final concentration of 100 µg/ml iron and 10 µg/ml protamine sulphate in the culture medium.\n\nCells were incubated with Endorem, FePro, or no contrast agent (control) for 24 hours. Cells were washed and replated into 96 well plates at a density of 104 cells per well, 8 wells per treatment group, for cell metabolic activity assays as a measure of cell viability. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; Sigma) and Alamar Blue (Invitrogen) assays were performed as previously described27,28. For the Trypan Blue assay, cells were resuspended 10:1 in 0.5% Trypan Blue (Sigma), and transferred to a haemocytometer for counting. Under a light microscope (Zeiss), non-viable blue-stained cells and total viable cells were counted. For each assay, two samples were counted for each treatment group, and the assay was repeated three times.\n\nThe neurosphere-forming assays measured the number of neurospheres generated per 1000 cells and the average neurosphere diameter per sample, which are indicators of the number of stem and progenitor cells in the sample and their rate of proliferation, respectively. Cells were plated at a density of 104 cells per ml in neurosphere proliferation medium into 96 well plates, where each well contained 100 µl of proliferation medium and 103 cells. After 5 days in culture, the number of neurospheres generated per 103 cells was counted, and neurosphere diameter was measured using the imaging analysis software, ImageJ developed by U. S. National Institutes of Health, Bethesda, Maryland, USA (http://imagej.nih.gov/ij/, 1997–2012). For each assay, a minimum of 8 wells per treatment group was counted, and each assay was repeated twice.\n\nNPCs cultured at a density of 104 cells per ml for 7 days were transferred to polyornithine-coated coverslips in 24 well plates for one hour to allow neurosphere adherence to coverslips. NPC were then fixed in 4% paraformaldehyde (PFA) in PBS for 10 minutes, incubated in 6% hydrochloric acid and 4% potassium ferrocyanide for 20 minutes, then washed in PBS and mounted.\n\nNeurospheres at 6 days after passage were differentiated as previously described29. Differentiated neurospheres were fixed in 4% PFA at 4°C. For immunocytochemistry, coverslips were incubated in blocking solution (10% goat serum with 0.1% Triton-X) for 30 minutes, then incubated in primary antibody in blocking solution for one hour at 37°C: IgG1 monoclonal mouse anti-β3-tubulin (1:1000, Promega G7121: G-purified monoclonal antibody from clone 5G8 raised in mice against a peptide (EAQGPK) corresponding to the C-terminus of β3-tubulin; the antibody has been tested to perform in frozen and paraffin-embedded sections of rat brain, cerebellum and spinal cord, human and rat fetal CNS progenitor cell cultures and adult human paraffin-embedded brain); polyclonal rabbit anti-GFAP (1:1000, Chemicon AB5804; raised in rabbit against purified bovine GFAP; routinely evaluated by immunohistochemistry on brain tissue, astrocytes, and neurons by Chemicon); monoclonal O4 IgM (1:2, generous gift Prof Rhona Mirsky)30,31. Coverslips were washed three times in PBS, then incubated with secondary antibody for 30 minutes at 37°C. Secondary antibodies were: Cy3-conjugated goat anti-mouse (1:100, Invitrogen A10521); Alexa 488-conjugated goat anti-rabbit 1gG (1:1000, Invitrogen A11008); and Alexa 680-conjugated goat anti-mouse IgM (1:1000, Invitrogen A21048). Coverslips were washed in PBS and mounted onto slides for fluorescence imaging.\n\nST14A cells were labelled with Endorem or FePro using the labelling methods described above. Labelled cells were then washed and fixed in 4% PFA. Cells were transferred to gelatin capsules (size 0, Capsugel) containing cotton wool, at a density of 1.25 × 106 per gel cap. Iron content of samples was measured using a superconducting quantum interference device (SQUID) and the amount of iron per cell was calculated. Sample magnetization was measured over a range of magnetic field strengths at 300K (Kelvin; room temperature) and 10K, and hysteresis loops were plotted. To calculate the amount of iron in each sample, remnant magnetization and saturation magnetization of the samples were compared to the magnetization of a sample with Endorem alone, which had a known iron content against which cell magnetisation was calibrated.\n\nAll procedures were carried out under the Animals Scientific Procedures Act 1986 (project license: PPL 70/05617). Eleven wild type male Sprague-Dawley rats (250 g, 8 weeks old, Charles Rivers, UK; n=11) were anaesthetised with 2% isofluorane. Temperature was monitored with a rectal probe. The middle cerebral artery occlusion was performed as previously described32, to occlude the MCA for 30 minutes. Briefly, the right common carotid artery was exposed. A sterile 4-0 suture with an epoxy resin tip (Araldite®, Huntsman Advanced Materials) was inserted into the carotid artery and advanced 17 mm into the brain to occlude the MCA for 30 minutes. Following occlusion, the MCA was reperfused by slowly removing the suture, and animals were then recovered on a heated mat. Animal weight was monitored. Following MCAO surgery, animals were assigned a number and alternately allocated to control or treatment group. After surgery, animals were housed individually, provided with soft tissue bedding and had unrestricted access to water and softened food pellets.\n\nIn six animals, FePro-labelled NPCs (FePro-NPCs) were injected into the ipsilateral corpus callosum 48 hours following cerebral ischaemia. Animals were anaesthetised with 2% isofluorane, and secured in a stereotactic frame (Kopf, Germany). Animals were injected with 1.25 × 105 FePro-labelled cells in a maximum of 3 µl, as described above, at 0.2 l/min into the ipsilateral corpus callosum. The coordinates used were AP +1.0, ML -0.3; DV -0.2 to Bregma. Five animals received no intracerebral injection (control uninjected group).\n\nAnimals were imaged to determine whether injected cells could be identified and monitored over time with MRI, relative to the control uninjected group.\n\nFor in vitro MRI, contrast agent-labelled cells were washed and fixed in 4% PFA. Cells were transferred to 250 µl Eppendorf tubes and centrifuged to produce a cell pellet. Eppendorfs were placed in a custom-made probe and imaged using a 2.35T horizontal bore magnet (Oxford Instruments, UK) interfaced to an SMIS console (Guildford, UK). A 2D spin echo sequence was used with the following parameters: TR=1500 ms; TE=80 ms; FOV 25 mm; slice thickness 1 mm.\n\nFor in vivo MRI, animals were anaesthetised with 2% isofluorane, and secured on a stereotactic probe. Animals were imaged using the above 2.35T system. A 2D gradient echo (T2*-weighted) sequence was used with the following parameters: TR=500 ms; TE=30 ms; FOV 30 mm; 128 × 128 voxels; 11 slices; 1 mm slice thickness; and 16 averages. A 2D spin echo (T2-weighted) sequence was also used: TR=1500 ms; TE=120 ms; FOV 30 mm; 128 × 128 voxels; 7 slices; slice thickness 1 mm; 16 averages. Animals were imaged at 3, 7, 14, 21, and 28 days post MCA occlusion surgery. For lesion volume measurements, hyperintense lesion areas from T2-weighted images were segmented manually using ImageJ software available at http://rsb.info.nih.gov/ij (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD). Lesion areas per slice were used to determine the total brain lesion volume, taking into account the thickness of the 2D slices (total brain lesions covered a mean of 7.33 (1.44 SD) slices in FePro-NPC animals and 7.50 (1.27 SD) slices in control animals). The lesion volume of the central slice covering the central MCA territory was measured and compared between groups. Homologous regions of interest were defined in the contralateral hemisphere.\n\nAfter the final MRI time point, animals were euthanized by transcardial perfusion with 0.9% saline then perfusion fixed with 4% PFA. Brains were removed from the skull and transferred to 30% sucrose in PBS for 48 hours, then frozen and stored at -80°C for tissue processing.\n\nCoronal brain sections of 30 μm thickness were taken on a cryostat (Bright Instruments, UK). For Prussian blue staining, sections were incubated in 6% hydrochloric acid and 4% potassium ferrocyanide for 20 minutes, then washed in PBS. Brain sections were counterstained with nuclear fast red or hematoxylin-eosin, and mounted with DPX (distyrene, plasticizer, xylene) mountant.\n\nThe MCA occlusion surgery was performed and animals were imaged at 24 hours post-ischaemia to visualize the infarcted area. Four animals underwent MCA occlusion surgery and were euthanised using Schedule 1 method at 24 hours (n=2) or 48 hours (n=2) post-ischaemia. The area of infarct, as determined by MRI at 24 hours post-ischaemia, was isolated. The contralateral hemisphere was used as non-ischaemic control tissue. Tissue was homogenized and filtered through a 0.4 μm sterile filter. Protein content of the homogenate was calculated against an albumin standard using the Pierce BCA Protein Assay Kit.\n\nSingle neurospheres were transferred into polyornithine-coated wells of 96 well plates, in 100 μl of culture medium per well. Individual neurospheres were photographed on a Zeiss AxioVert inverted microscope with a Hamamatsu digital camera and using OpenLab imaging software, then tissue homogenate or growth factors were added to the control medium. Treatment groups were as follows: 1400, 700, 300 μg/ml MCAO homogenate from 24 hours time point; 1400, 700, 300 μg/ml MCAO homogenate from 48 hours time point. Control groups were as follows: DMEM:F12 with N2 medium; DMEM:F12 with control tissue homogenate. Neurospheres were photographed 72 hours following treatment and the area covered by the cells measured to quantify the extent of migration. Assays were repeated three times, with 8 wells per treatment group.\n\nData are expressed as mean +/- standard error. Data analysis was performed using ImageJ for MRI image analysis and neurosphere diameter measurements; Adobe Photoshop for microscopy image analysis, and SPSS software for statistical analysis. A one-way ANOVA was used for statistical comparisons, with P < 0.05 defined as significant. A two-way mixed ANOVA was used for statistical analysis of the change in lesion volume over time in control and FePro-NPC-treated animals.\n\n\nResults\n\nWe assessed whether the NPCs expanded as neurospheres from embryonic mouse forebrain were able to respond to cues from cerebral ischaemia. We investigated their migratory response to protein extracted from cerebral ischaemia infarct regions at two different timepoints – 24 and 48 hours post-ischaemia and from the corresponding contralateral control (Figure 1). The protein extracts were added directly to the wells containing the individual neurospheres; hence migratory cues were evenly distributed.\n\nA–B, Migration of individual neurosphere in response to 1400 μg/ml protein from 24 hour MCAO homogenate at t=0 hr (A) and t=72 hr (B). C–D, Migration of individual neurosphere in response to contralateral hemisphere MCAO proteins at t=0 hr (C) and t=72 hr (D). E, Cell migration from E14 neurospheres at 72 hours with no proteins or treatment with proteins from either normal brain homogenates or from 24 (light blue bars) and 48 (dark blue bars) hours MCAO homogenates at different concentrations; *: p<0.001. Scale bar, 400 µm.\n\nAs shown in Figure 1A–E, extensive migration in all directions had occurred by 72 hours from neurospheres exposed to ischaemia protein extracts (Figure 1A–B) but not in the presence of control proteins (Figure 1C–D). Figure 1E demonstrates the migratory response of NPC to 24 hr and 48 hours ischaemic tissue protein extracts at different protein concentrations. Migration in both 24 hours and 48 hours post-ischaemia groups was significantly different from control migration (F=6.680; p<0.001). Post hoc analysis showed that at 24 hr, the 1400 µg/ml concentration, and at 48 hours the 300 and 700 μg/ml MCAO concentrations, were significantly different from control non-ischaemic tissue protein extract. Hence NPCs are able to respond to cerebral ischaemia cues in vitro.\n\n\n\nThe effects of iron oxide-based MRI contrast agents on cell viability were first assessed in a fetal neural stem cell line and on primary NPCs. Incubation of ST14A cells with Endorem or FePro for 24 hours did not affect cell viability as assessed using metabolic activity assays (Supplementary Figure 1). Iron content was measured using a SQUID. Mean iron uptake was 3.93 pg Fe/cell for Endorem-labelled cells, and 14.5 pg Fe/cell for FePro-labelled cells. Therefore the iron content was 3.7-fold greater in FePro-labelled cells than in Endorem-labelled cells.\n\nWe then investigated the effect of cell labelling with FePro and Endorem on NPC viability and proliferative capacity (Figure 2A–D). Neither contrast agent affected NPC viability as shown by the Trypan Blue exclusion assay and Alamar blue assay (Figure 2A–B). Furthermore, neither neurosphere-forming ability nor neurosphere growth were negatively affected by FePro or Endorem (Figure 2C–D).\n\nA, Alamar Blue assay. No significant difference in NPC metabolism (F=0.736, p=0.49) is observed. B, Trypan Blue assay. No significant difference in cell survival (F=0.106, p=0.901) is observed. C, Neurosphere diameter. Diameter (µm) of Endorem-labelled neurospheres is significantly different from control unlabelled neurospheres (F=7.497; p<0.05). D, Neurosphere forming ability. No significant difference in neurosphere formation per 1000 cells (F=0.184; p=0.833) is observed. E, F. Prussian Blue stain for iron in Endorem (G) and FePro (H) labelled neurospheres after 7 days in culture.\n\nThe iron within NPC neurospheres was visualized after 7 days in culture by Prussian blue. As shown in Figure 2E–F, the contrast agent is retained within cells across cell divisions.\n\nTo further characterise the effects of Endorem and FePro, the differentiation capacity of contrast agent-labelled NPCs was assessed. Figure 3 shows that FePro and Endorem-labelled NPCs can differentiate into neurons, oligodendrocytes, and astrocytes (Figure 3A–F). The cells retain the contrast agent label after differentiation (Figure 3G–H) and FePro-labelled cells generate negative contrast on MRI (Figure 3I–J).\n\nA–F. Immunocytochemistry of FePro (A–C) and Endorem (D–F) labelled neurospheres. A,D, anti-β3-tubulin (neuron, red). B,E, anti-GFAP (astrocyte, green). C,F, anti-O4 (oligodendrocyte, red). Blue, Hoescht nuclear stain. FePro-labelled neurospheres can differentiate into neurons (A,D), astrocytes (B,E), and oligodendrocytes (C,F). G, immunocytochemistry of FePro-labelled astrocyte (GFAP, green). H, corresponding light micrograph showing Prussian blue stain. I,J MRI T2-weighted image of FePro-labelled neurosphere pellet (I) and control unlabelled cell pellet (J). FePro-labelled cell pellets produce hypointensity on MRI images. Arrows denote a GFAP-labelled cell co-labelled with Prussian blue, indicating that the FePro-labelled cell was capable of astrocyte differentiation. Scale bars, 20 µm.\n\n\n\nIn vivo MRI of injected FePro-labelled NPC was carried out 3, 7, 14, 21 and 28 days after cerebral ischaemia, to determine whether cells could be identified and monitored over time, relative to a control MCAO group without NPCs (Figure 4). The T2-weighted images revealed extensive heterogeneous ischaemic lesions in both groups, with hypointense regions developing in the striatum and middle cortical layers, from day 7 and persisting until day 28 (Figure 4A–B). This lesion heterogeneity was present in all control animals as well as in the FePro-NPC group. The FePro-labelled cells at the injection site were clearly detected in T2*-weighted images (Figure 4C–D).\n\nA,B, Serial T2-weighted imaging up to 28 days post-ischaemia in FePro-NPC and control animals. Regions of T2 hypointensity develop in the lesion at later timepoints (white arrows). C,D, Serial T2*-weighted imaging up to 28 days post-ischaemia in FePro-NPC and control animals. The injection site of FePro-labelled cells can be identified in the FePro-NPC group at all timepoints (black arrows).\n\nWe compared T2- and T2*-weighted images at 28 days in the FePro-NPC and control group. Figure 5A–D shows the profile of a FePro-NPC-treated animal and control animal at the 28 day time period. Lesion heterogeneity was observed in all animals in both T2 and T2*-weighted images. Signal intensity in hypointense ischaemic striatum regions was significantly different from the intensity in corresponding contralateral regions (Figure 5E; F=3.154, p=0.05).\n\nT2- (B,D) and T2*-weighted (A,C) images are shown. A,B, MCA region in one control. C,D. MCA region in one FePro-NPC animal at 28 days post-ischaemia. Hypointense regions within the ischaemic lesion (arrows) were observed in both T2- and T2*-weighted images. E, Signal intensity in regions of T2-weighted hypointense ipsilateral striatum at 28 days post-ischaemia, and in corresponding contralateral regions in control (green) and FePro-NPC (blue) groups. Signal intensity in the ischaemic striatum was lower than in the contralateral striatum. Signal intensity was significantly different between contralateral and ipsilateral hemispheres (p<0.001).\n\nWe investigated the origin of the hypointense signal on T2-weighted MRI by examining histological sections. Iron can be a source of T2-weighted hypointensity, and we assessed its distribution in both groups. In both groups, iron was detected in the ipsilateral (ischaemic) striatum at the ischaemic border, and in the ischaemic cortex in some animals (2 of 5 control, and 3 of 6 FePro-NPC animals)(Figure 6C–F). Iron accumulation was detected in the mid-striatum at the lesion border, which approximates the area of hypointensity on MRI. Additionally in the FePro-NSC group, iron was detected at the injection site (Figure 6G). No iron was detected in the contralateral cortex or striatum in the FePro-NSC group (Figure 6H).\n\nA,B, Prussian blue stain for iron and hematoxylin-eosin staining at the ipsilateral striatum. Iron detected in the ischaemic lesion. C,D, Prussian blue stain with nuclear fast red counterstain in the ipsilateral striatum. E,F, OX-42 immunohistochemistry stain for macrophage/microglia, and Prussian blue stain for iron. Prussian blue-positive and – negative macrophage/microglia, detected by OX-42, are present at the ischaemic lesion. G, Prussian blue stain at the injection site in FePro-NPC ipsilateral hemisphere. H, OX-42 immunohistochemistry for macrophage/microglia and Prussian blue stain in the contralateral hemisphere. No macrophage/microglia or iron were observed in the striatum or cortex of the contralateral hemisphere. Scale bars, 30 µm.\n\nWe investigated whether the MRI signal hypointensity was associated with the distribution of macrophage/microglia in the brain. We observed accumulated macrophage/microglia in the infarcted cortex and striatum in both FePro-NSC and control groups, and some of them co-labelled with iron (Figure 6E–F). Iron-positive cells were a mixture of macrophage and non-macrophage cell types. No macrophage/microglia were observed in the contralateral hemispheres (Figure 6G–H).\n\nFinally to investigate the effect of cell injections on the outcome of cerebral ischaemia, MRI analysis was carried out. Changes in volume of the lesion at the level of the central MCA region and the volume of the whole lesion over time were compared between control ischaemic and FePro-NPC groups. No statistically significant difference in lesion volume over time was observed either for the whole lesion or at the central MCA territory (Figure 7A–B; p=0.275 and p=0.244, respectively). The change in hemisphere volume at the MCA territory was also assessed. We observed that the ipsilateral hemisphere volume was decreased by 28 days post-ischaemia relative to the contralateral hemisphere in both groups (F=17.18; p<0.005; Figure 7C), but we did not observe a difference between FePro-NPC and control groups. This reduction in ipsilateral hemisphere volume over time may represent secondary, delayed neuronal death in the ischaemic hemisphere, lesion compaction, brain reorganisation or a combination of these factors.\n\nA, Lesion volume (cm3/mL) of the central MCA region slice. There is no significant difference between controls (solid line, blue) and FePro-NPC (dashed line, green) lesion volumes at the MCA region (p=0.577). B, Total lesion volume. There is no significant difference between control and FePro-NPC lesion volumes (p=0.921). C, Hemisphere volume change. Change in ipsilateral:contralateral hemisphere volume ratio between 7 and 28 days (*:ANOVA, p<0.0001), but there is no significant difference between control and FePro-NPC in hemisphere volume.\n\n\nDiscussion\n\nThe main findings of this study were that i) FePro displays a high labelling efficiency and does not affect NPC behaviour in vitro; ii) following brain ischemia, the evolution of endogenous MRI T2-weighted contrast and heterogeneity can interfere with NPC detection and iii) soluble cues from injured tissue promote NPC migration in vitro, but no discernible NPC migration is observed in vivo following an extensive stroke.\n\nWe observed that NPC can be labelled with FePro with no significant effect on cell viability, metabolic activity or proliferation relative to control, unlabelled cells, and that labelled NPC can differentiate into neurons, oligodendrocytes and astrocytes. The FePro label is retained throughout multiple cell divisions during the formation of new neurospheres. In addition, data from the ST14A cell line (Supplementary Figure 1) also confirm that FePro does not affect cell viability, and has greater labelling efficiency than Endorem. Imaging NPC in vitro demonstrated that FePro uptake under the described labelling conditions was sufficient to generate MRI contrast. SQUID measurement of iron oxide uptake also confirmed and quantified cellular uptake of FePro and suggest that FePro may be a superior MRI contrast agent for NPC labelling compared with Endorem24. Our results indicate that there are no cytotoxic effects associated with FePro labelling, which is consistent with other studies that have similarly shown limited cytotoxicity of FePro in other cell types, and that FePro uptake is sufficient to generate contrast on T2*-weighted images20,21,33,34. Taken together with our previous work demonstrating that endogenous neural stem cells could be labelled with FePro and migrate from the subventricular zone (SVZ) to the olfactory bulb22, NPCs are ideally suited for labelling with FePro for in vivo detection of cell migration.\n\nThis study demonstrated that cellular imaging was in part complicated by the evolution of T2 and T2* heterogeneity in the ischaemic lesion in all animals, obscuring cell detection on MRI at later timepoints. The evolution of T2-weighted hypointensity could be related to the accumulation of macrophages and granulocytes to the lesion over time, leading to localized hypercellularity. Accumulation of MHC Class II-expressing cells (such as macrophages, dendritic and B cells) was shown to be spatially coincident with regions of T2-weighted hypointensity and reduced ADC (apparent diffusion coefficient) in a model of multiple sclerosis35. Hypercellularity was shown to increase from one week, peak at 3 weeks and persist in later timepoints, and regions of T2-weighted hypointensity develop by 3 weeks, which is consistent with our results35. In another study, T2 hypointensity was observed from 4 days post-ischaemia at the lesion border, which interfered with detection of SPIO-labelled injected cells, and accumulation of ferric iron was detected histologically from 6 days post-ischaemia around the lesion36. Although we did not observe haemorrhage in any of the animals, the T2 heterogeneity may also arise as a consequence of the accumulation of iron in the lesion, through the development of microhaemorrhages, the degradation of blood products, or neuronal degeneration.\n\nIn the FePro-NSC group, an additional factor is clearly the presence of iron oxide particles in the FePro contrast agent. Palweczyk et al. have shown that iron exchange can occur between labelled cells and macrophages in vitro, although the transfer of iron to macrophages is low37. Weber et al. observed the evolution of hypointensity in ischaemic striatum on T2*-weighted MRI at 2 and 10 weeks post-ischaemia in a MCA occlusion model, and found that T2* hypointense regions colocalised with iron-containing macrophages38. The authors attributed the iron in macrophages to the phagocytosis of red blood cells from damaged blood vessels. Danielisova et al. have observed iron deposition in the striatum and pyramidal cells of cortical layers III and V following 20 minute MCA occlusion39. Justicia et al. have reported iron accumulation, T2-weighted hypointensity and delayed neuronal death in the thalamus between 3 and 7 weeks following cerebral ischaemia40 and suggested that iron accumulation was mediated by heme oxygenase-1-positive (HO-1) microglial activity. These studies suggest that iron accumulation at lesion sites may be linked to microglial activity and neurodegeneration.\n\nWhereas our previous study has shown in normal brains a good correlation between MRI and FePro-labelled endogenous neural progenitor cells24, tracking FePro-labelled cells in the ischaemic brain is clearly more challenging. This study highlights the need to characterise the long-term profile of cerebral ischaemia, and points to confounding image contrast at later timepoints that must be overcome in order for iron oxide-based cellular imaging to be a viable method of cell tracking.\n\nThe NPC demonstrated a migratory response to cerebral ischaemia cues in vitro. These results indicate that fetal NPC are responsive to cerebral ischaemia factors, and the migratory cue is likely to be a soluble extracellular factor(s). In contrast, we did not observe any major cell migration from the injection site to the ischaemic striatum or cortex. It is possible that the soluble cues promoted cell migration in vitro because of the absence of inhibitory or toxic molecules present in the in vivo environment. It is also conceivable that the FePro-labelled NPCs were phagocytosed by macrophages in vivo41. The apparent lack of accumulation of NPCs at the infarcted site correlates with the lack of a significant reduction in infarct size. These findings are consistent with a recent report showing that reduction in infarct size is \"dose-dependent\" and that neural stem cell injection is effective in reducing neural damage only following a moderate infarct42. On the other hand, behavioural improvement following injection of NPC has been reported in the absence of a significant reduction in infarct size, and might be attributed to increased plasticity in the grafted brains, possibly via paracrine mechanisms as reported for both neural and non-neural stem cells16,43–45.\n\nIn our study, NPCs were engrafted at 48 hours post-ischaemia. Data from the in vitro NPC migration assay demonstrated that NPCs responded to migratory cues from this time point, except at the highest concentration. In a recent study, Darsalia et al. demonstrated that this time point was optimal for cell survival and migration in a similar 30 minute occlusion model and intrastriatal engraftment of human fetal NPCs3. Similarly, cell migration has been observed in the corpus callosum in other studies engrafting murine embryonic stem cells or human NPC at later timepoints in models of cerebral ischaemia. Injection at the corpus callosum allows for cell injection at a distance from the lesion, reducing exposure to potentially toxic inflammatory environment, while providing a potential pathway for migration towards the lesion site and subsequent migration into the lesion. Clearly, cell type and origin, engraftment timepoint, engraftment site and infarct size are important factors for survival and migration of engrafted cells in ischaemic environments, as well as the parameters used to assess recovery.\n\n\nConclusion\n\nWe have shown that the MRI cell tracking agent FePro does not affect NPC viability, proliferation or differentiation capacity in vitro. However, in the in vivo studies we have identified possible sources of T2-weighted image contrast in the ischaemic lesion that develops over time, creating a challenge for longitudinal cellular imaging studies. In conclusion, this study raises important issues surrounding the use of MRI and MRI contrast agents for longitudinal cell tracking studies in models of injury, where the evolution of endogenous contrast over time within lesioned tissue can be a source of uncertainty in image interpretation.\n\n\nList of abbreviations\n\nES cell – embryonic stem cell\n\nGFAP – glial fibrillary acidic protein\n\nIgG – immunoglobulin G\n\nIgM – immunoglobulin M\n\nMCA – middle cerebral artery\n\nSQUID – superconducting quantum interference device\n\nMTT – 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide\n\nPFA – paraformaldehyde\n\nSPIO – superparamagnetic iron oxide",
"appendix": "Author contributions\n\n\n\nRAP designed and performed experiments, analyzed data and wrote the manuscript; DGG provided advice on experimental design, data interpretation and critical reading of the manuscript; JCS provided advice on experimental design and critical reading of the manuscript; JAW optimized MRI sequences and contributed to data analysis; MFL planned research, analyzed data, obtained funding and provided critical feed-back on the manuscript; PF planned research, analyzed data, obtained funding, and wrote the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was funded by the Child Health Research Appeal Trust and the British Heart Foundation.\n\n\nAcknowledgements\n\nThe authors would like to thank E. Cattaneo for providing the ST14 cell line, R. Mirsky for providing the O4 antibody, and all the members of our laboratories for the helpful discussions.\n\n\nSupplementary material\n\nA, MTT assay. No decrease in the percentage of viable ST14A cells is observed between control, Endorem-, and FePro-labelled cells after 24 hour treatment with MRI contrast agents; a small increase is observed in FePro-labelled cells (p<0.05). B, Alamar Blue assay. No significant difference is observed between FePro-labelled and control unlabelled cells, but there is a decrease in metabolic activity of Endorem-labelled cells relative to control unlabelled cells (p<0.01).\n\n\nReferences\n\nGarcia JH, Ye ZR: Epidemiology and pathology of occlusive cerebrovascular disease. In: Crockard A, Hayward R,Hoff JT (eds). Neurosurgery. The scientific basis of clinical practice, vol. 2. Blackwell Scientific: Oxford, 2000; pp 704–726.\n\nArvidsson A, Collin T, Kirik D, et al.: Neuronal replacement from endogenous precursors in the adult brain after stroke. Nat Med. 2002; 8(9): 963–70. PubMed Abstract | Publisher Full Text\n\nDarsalia V, Allison SJ, Cusulin C, et al.: Cell number and timing of transplantation determine survival of human neural stem cell grafts in stroke-damaged rat brain. J Cereb Blood Flow Metab. 2011; 31(1): 235–42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKokaia Z, Lindvall O: Neurogenesis after ischaemic brain insults. Curr Opin Neurobiol. 2003; 13(1): 127–32. PubMed Abstract | Publisher Full Text\n\nLindvall O, Kokaia Z: Stem cells for the treatment of neurological disorders. Nature. 2006; 441(7097): 1094–6. PubMed Abstract | Publisher Full Text\n\nAarum J, Sandberg K, Haeberlein SL, et al.: Migration and differentiation of neural precursor cells can be directed by microglia. Proc Natl Acad Sci U S A. 2003; 100(26): 15983–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBang OY, Lee JS, Lee PH, et al.: Autologous mesenchymal stem cell transplantation in stroke patients. Ann Neurol. 2005; 57(6): 874–82. PubMed Abstract | Publisher Full Text\n\nKondziolka D, Wechsler L: Stroke repair with cell transplantation: neuronal cells, neuroprogenitor cells, and stem cells. Neurosurg Focus. 2008; 24(3–4): E13. PubMed Abstract | Publisher Full Text\n\nSavitz SI, Dinsmore J, Wu J, et al.: Neurotransplantation of fetal porcine cells in patients with basal ganglia infarcts: a preliminary safety and feasibility study. Cerebrovasc Dis. 2005; 20(2): 101–7. PubMed Abstract | Publisher Full Text\n\nDarsalia V, Allison SJ, Cusulin C, et al.: Cell number and timing of transplantation determine survival of human neural stem cell grafts in stroke-damaged rat brain. J Cereb Blood Flow Metab. 2011; 31(1): 235–42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAbrous DN, Koehl M, Le Moal M: Adult neurogenesis: from precursors to network and physiology. Physiol Rev. 2005; 85(2): 523–69. PubMed Abstract | Publisher Full Text\n\nDarsalia V, Kallur T, Kokaia Z: Survival, migration and neuronal differentiation of human fetal striatal and cortical neural stem cells grafted in stroke-damaged rat striatum. Eur J Neurosci. 2007; 26(3): 605–14. PubMed Abstract | Publisher Full Text\n\nIshibashi S, Sakaguchi M, Kuroiwa T, et al.: Human neural stem/progenitor cells, expanded in long-term neurosphere culture, promote functional recovery after focal ischemia in Mongolian gerbils. J Neurosci Res. 2004; 78(2): 215–23. PubMed Abstract | Publisher Full Text\n\nKelly S, Bliss TM, Shah AK, et al.: Transplanted human fetal neural stem cells survive, migrate, and differentiate in ischemic rat cerebral cortex. Proc Natl Acad Sci U S A. 2004; 101(32): 11839–44. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLee HJ, Kim KS, Park IH, et al.: Human neural stem cells over-expressing VEGF provide neuroprotection, angiogenesis and functional recovery in mouse stroke model. PloS One. 2007; 2(1): e156. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSmith EJ, Stroemer RP, Gorenkova N, et al.: Implantation site and lesion topology determine efficacy of a human neural stem cell line in a rat model of chronic stroke. Stem Cells. 2012; 30(4): 785–96. PubMed Abstract | Publisher Full Text\n\nTakahashi K, Yasuhara T, Shingo T, et al.: Embryonic neural stem cells transplanted in middle cerebral artery occlusion model of rats demonstrated potent therapeutic effects, compared to adult neural stem cells. Brain Res. 2008; 1234: 172–82. PubMed Abstract | Publisher Full Text\n\nZhu W, Mao Y, Zhou LF: Reduction of neural and vascular damage by transplantation of VEGF-secreting neural stem cells after cerebral ischemia. Acta Neurochir Suppl. 2005; 95: 393–7. PubMed Abstract | Publisher Full Text\n\nHoehn M, Kustermann E, Blunk J, et al.: Monitoring of implanted stem cell migration in vivo: a highly resolved in vivo magnetic resonance imaging investigation of experimental stroke in rat. Proc Natl Acad Sci U S A. 2002; 99(25): 16267–72. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang ZG, Jiang Q, Zhang R, et al.: Magnetic resonance imaging and neurosphere therapy of stroke in rat. Ann Neurol. 2003; 53(2): 259–63. PubMed Abstract | Publisher Full Text\n\nDaadi MM, Li Z, Arac A, et al.: Molecular and magnetic resonance imaging of human embryonic stem cell-derived neural stem cell grafts in ischemic rat brain. Mol Ther. 2009; 17(7): 1282–91. PubMed Abstract | Publisher Full Text | Free Full Text\n\nArbab AS, Yocum GT, Kalish H, et al.: Efficient magnetic cell labeling with protamine sulfate complexed to ferumoxides for cellular MRI. Blood. 2004; 104(4): 1217–23. PubMed Abstract | Publisher Full Text\n\nArbab AS, Yocum GT, Rad AM, et al.: Labeling of cells with ferumoxides-protamine sulfate complexes does not inhibit function or differentiation capacity of hematopoietic or mesenchymal stem cells. NMR Biomed. 2005; 18(8): 553–9. PubMed Abstract | Publisher Full Text\n\nPanizzo RA, Kyrtatos PG, Price AN, et al.: In vivo magnetic resonance imaging of endogenous neuroblasts labelled with a ferumoxide-polycation complex. Neuroimage. 2009; 44(4): 1239–46. PubMed Abstract | Publisher Full Text\n\nGuzman R, Uchida N, Bliss TM, et al.: Long-term monitoring of transplanted human neural stem cells in developmental and pathological contexts with MRI. Proc Natl Acad Sci U S A. 2007; 104(24): 10211–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCattaneo E, Conti L: Generation and characterization of embryonic striatal conditionally immortalized ST14A cells. J Neurosci Res. 1998; 53(2): 223–34. PubMed Abstract | Publisher Full Text\n\nKyrtatos PG, Lehtolainen P, Junemann-Ramirez M, et al.: Magnetic tagging increases delivery of circulating progenitors in vascular injury. JACC Cardiovasc Interv. 2009; 2(8): 794–802. PubMed Abstract | Publisher Full Text\n\nLee KM, Santos-Ruiz L, Ferretti P: A single-point mutation in FGFR2 affects cell cycle and Tgfbeta signalling in osteoblasts. Biochim Biophys Acta. 2010; 1802(3): 347–55. PubMed Abstract | Publisher Full Text\n\nYoung KM, Fogarty M, Kessaris N, et al.: Subventricular zone stem cells are heterogeneous with respect to their embryonic origins and neurogenic fates in the adult olfactory bulb. J Neurosci. 2007; 27(31): 8286–96. PubMed Abstract | Publisher Full Text\n\nSommer I, Schachner M: Monoclonal antibodies (O1 to O4) to oligodendrocyte cell surfaces: an immunocytological study in the central nervous system. Dev Biol. 1981; 83(2): 311–27. PubMed Abstract | Publisher Full Text\n\nBansal R, Warrington AE, Gard AL, et al.: Multiple and novel specificities of monoclonal antibodies O1, O4, and R-mAb used in the analysis of oligodendrocyte development. J Neurosci Res. 1989; 24(4): 548–57. PubMed Abstract | Publisher Full Text\n\nBadin RA, Modo M, Cheetham M, et al.: Protective effect of post-ischaemic viral delivery of heat shock proteins in vivo. J Cereb Blood Flow Metab. 2009; 29(2): 254–63. PubMed Abstract | Publisher Full Text | Free Full Text\n\nArbab AS, Bashaw LA, Miller BR, et al.: Intracytoplasmic tagging of cells with ferumoxides and transfection agent for cellular magnetic resonance imaging after cell transplantation: methods and techniques. Transplantation. 2003; 76(7): 1123–30. PubMed Abstract | Publisher Full Text\n\nArbab AS, Liu W, Frank JA: Cellular magnetic resonance imaging: current status and future prospects. Expert Rev Med Devices. 2006; 3(4): 427–39. PubMed Abstract | Publisher Full Text\n\nBroom KA, Anthony DC, Blamire AM, et al.: MRI reveals that early changes in cerebral blood volume precede blood-brain barrier breakdown and overt pathology in MS-like lesions in rat brain. J Cereb Blood Flow Metab. 2005; 25(2): 204–16. PubMed Abstract | Publisher Full Text\n\nVandeputte C, Thomas D, Dresselaers T, et al.: Characterization of the inflammatory response in a photothrombotic stroke model by MR: implications for stem cell transplantation. Mol Imaging Biol. 2011; 13(4): 663–71. PubMed Abstract | Publisher Full Text\n\nPawelczyk E, Arbab AS, Chaudhry A, et al.: In vitro model of bromodeoxyuridine or iron oxide nanoparticle uptake by activated macrophages from labeled stem cells: implications for cellular therapy. Stem Cells. 2008; 26(5): 1366–75. PubMed Abstract | Publisher Full Text\n\nWeber R, Wegener S, Ramos-Cabrer P, et al.: MRI detection of macrophage activity after experimental stroke in rats: new indicators for late appearance of vascular degradation?. Magn Reson Med. 2005; 54(1):59–66. PubMed Abstract | Publisher Full Text\n\nDanielisova V, Gottlieb M, Nemethova M, et al.: The effect of preconditioning on the iron deposition after transient forebrain ischemia in rat brain. Arch Ital Biol. 2004; 142(2): 87–94. PubMed Abstract\n\nJusticia C, Ramos-Cabrer P, Hoehn M: MRI detection of secondary damage after stroke: chronic iron accumulation in the thalamus of the rat brain. Stroke. 2008; 39(5): 1541–7. PubMed Abstract | Publisher Full Text\n\nDetante O, Valable S, de Fraipont F, et al.: Magnetic resonance imaging and fluorescence labeling of clinical-grade mesenchymal stem cells without impacting their phenotype: study in a rat model of stroke. Stem Cells Transl Med. 2012; 1(4): 333–41. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDaadi MM, Hu S, Klausner J, et al.: Imaging Neural Stem Cell Graft-Induced Structural Repair in Stroke. Cell Transplant. 2013; 22(5): 881–92. PubMed Abstract | Publisher Full Text\n\nAndres RH, Horie N, Slikker W, et al.: Human neural stem cells enhance structural plasticity and axonal transport in the ischaemic brain. Brain. 2011; 134(Pt 6): 1777–89. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPatkar S, Tate R, Modo M, et al.: Conditionally immortalised neural stem cells promote functional recovery and brain plasticity after transient focal cerebral ischaemia in mice. Stem Cell Res. 2012; 8(1): 14–25. PubMed Abstract | Publisher Full Text\n\nPrasongchean W, Bagni M, Calzarossa C, et al.: Amniotic fluid stem cells increase embryo survival following injury. Stem Cells Dev. 2012; 21(5): 675–88. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "2517",
"date": "21 Nov 2013",
"name": "Roger Barker",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis project is investigating the migration and effects of rat neural precursor cells on ischaemic brain lesions using an improved method for labelling these cells in vivo using an iron based marker. The authors show that the label does not interfere with the normal behaviour of the cells, and that it allows for cells to be seen in the grafted brain. However the study also highlights that the cells have no benefit on the lesion (when grafted 48 hours after the lesion) and in addition as the vascular lesion re-organises, the iron signal can be hard to see over endogenous iron deposition.This is a straightforward study with clear data, my only comments are:What is the efficiency of uptake on the iron label into cells, in terms of how many of the cells take up the label?How stable is the iron label within the cells in vitro? Is it lost over time independent of any cell death- namely are the number of cells with a label the same over time in the stem cell cultures?What factors present in the ischemic tissue facilitate NPC migration?It would be of interest to know if transplanting the cells into the non stroked hemisphere would have allowed for better visualisation of cell migration to the lesion.",
"responses": []
},
{
"id": "2840",
"date": "21 Jan 2014",
"name": "Keith Muir",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study highlights difficulties with the use of MRI contrast agents for the imaging of cells in vivo in stroke models.The evolution of ischaemic damage after stroke typically involves a degree of petechial haemorrhage with deposition of haemosiderin commonly evident histologically or on sensitive MRI sequences after an iscaemic event. Confounding of the signal from iron-labelled cell therapies is therefore likely, as described here.In vitro functional assays offer some evidence that the MRI contrast agent may not affect important properties of the NPC cell line, but the in vivo assessment is incomplete and deserves comment. Effects on infarct volume are highly unlikely with therapies delivered 48h after ischaemia, so this is unsurprising and neither supports nor argues against a therapeutic effect. Functional assays are more difficult and time consuming, but a rodent focal ischaemia model with delayed cell delivery would offer the only means of establishing whether any therapeutic effect is compromised by the cell label. This would be important for further study since it has previously been reported that intracellular MRI labels (a gadolinium conjugate) negated functional benefits from a neural stem cell line (Modo et al. Neuroimage. 2009;47 Suppl 2:T133-42).",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-252
|
https://f1000research.com/articles/3-15/v1
|
17 Jan 14
|
{
"type": "Case Report",
"title": "Pericardial synovial sarcoma: challenges in diagnosis and management",
"authors": [
"Prajakta Phatak",
"Jagadish Khanagavi",
"Wilbert S. Aronow",
"Sonam Puri",
"Yasmin Yusuf",
"Carmelo Puccio",
"Prajakta Phatak",
"Jagadish Khanagavi",
"Sonam Puri",
"Yasmin Yusuf",
"Carmelo Puccio"
],
"abstract": "Introduction: Pericardial synovial sarcoma is an extremely rare tumor with poor prognosis. Timely diagnosis and aggressive multimodal management improves patient outcome. We present our experience of diagnosis and management of a young patient with monophasic synovial sarcoma arising from pericardium.Case: A 27-year-old man presented with dyspnea and cough of three weeks duration. Examination revealed sinus tachycardia, distant heart sounds and elevated jugular venous pressure. Chest X-ray showed widened mediastinum. Transthoracic echocardiogram (TTE) noted large pericardial effusion with tamponade physiology. Therapeutic pericardiocentesis yielded hemorrhagic fluid. Computed tomography (CT) of the chest showed persistent pericardial effusion and a left anterior mediastinal mass. Left anterior thoracotomy, pericardial window and left anterior mediastinotomy were done, revealing a well-encapsulated gelatinous tumor originating from the pericardium. Histology and immunohistochemical profile showed the tumor to be a monophasic synovial sarcoma. Fluorescent in-situ hybridization (FISH) was positive for SS18 (SYT) gene rearrangement on chromosome 18q11, substantiating the diagnosis. Work-up for metastases was negative. Neo-adjuvant chemotherapy with high dose ifosfamide led to substantial reduction in the size of the tumor. The patient underwent surgical resection and external beam radiation therapy (EBRT) post surgery. He had symptom-free survival for 8 months prior to local recurrence. This was managed with left lung upper lobectomy and follow-up chemotherapy with docetaxel. The patient is currently stable with an acceptable functional status.Conclusion: In patients with pericardial effusions of unknown etiology, multiple modalities of cardiac imaging must be employed if there is suspicion of a pericardial mass. CT and magnetic resonance imaging (MRI) are useful to evaluate for pericardial thickening or masses in addition to TTE. Treatment of synovial sarcoma is not well established. Surgery is the cornerstone of treatment. In non-resectable tumors, aggressive neo-adjuvant chemotherapy with ifosfamide followed by surgical resection and EBRT may lead to improved outcome.",
"keywords": [
"pericardium",
"synovial sarcoma",
"tamponade",
"ifosfamide"
],
"content": "Introduction\n\nSynovial sarcomas are malignant soft tissue tumors in extremities with a predilection for juxta-articular location and are predominantly diagnosed in young adults. Synovial sarcoma arising from the pericardium is an extremely rare tumor. Although termed as synovial sarcoma, these tumors have now been recognized to arise from mesenchymal tissue and show epithelial differentiation1. To the authors’ knowledge, only 20 cases of synovial sarcomas originating from the pericardium have been described in the English literature to date2. This type of tumor is known to have a poor prognosis with no clarity about the best approach in managing this disease.\n\nThis case report describes a young man who presented to us with a cough and dyspnea and was diagnosed with cardiac tamponade due to this rare tumor. Pericardial effusions and cardiac tamponade secondary to malignancy are mostly secondary to metastases3. Primary synovial sarcoma leading to tamponade is very rare, and to the best of our knowledge, has been reported only twice prior to our report4,5. We delineate the systematic approach to diagnosis using imaging modalities, histopathology and cytogenetic analysis. Due to its site of origin and histology, it was essential to distinguish this tumor from other soft tissue tumors, and cytogenetic analysis was crucial with regard to this. Although surgery is the cornerstone of treatment of this tumor, we used multiple modalities of treatment such as neo-adjuvant chemotherapy using high-dose ifosfamide and external beam radiation therapy (EBRT). Our patient had a good initial response to therapy and had improved survival compared to previous patients4,5. We believe that this case report and the discussion about diagnosis, management and interval monitoring for recurrence will add to the current literature and help in managing such cases in the future.\n\n\nCase report\n\nA 27-year-old Hispanic man with no significant prior medical history presented to the emergency room with shortness of breath and a cough for three weeks. He also complained of mild precordial chest pain and generalized fatigue. His family history was contained type 2 diabetes mellitus in both parents but reported no history of cancer or genetic diseases. His social history revealed that he worked as a gardener, was married with two kids, was a non-smoker, occasionally consumed alcohol and had no history of drug abuse. He gave no history of any psychological illnesses. A physical exam revealed sinus tachycardia, distant heart sounds and elevated jugular venous pressure and a normal blood pressure. Chest X-ray showed an impressive mediastinal enlargement. Transthoracic echocardiogram (TTE) showed a large pericardial effusion (Figure 1a) and collapse of the right atrium during diastole along with respirophasic changes consistent with cardiac tamponade physiology. The patient underwent an emergent therapeutic pericardiocentesis that yielded hemorrhagic fluid. Cytological analysis of the fluid revealed no malignant cells. Computed tomography (CT) of the chest showed persistent pericardial effusion and a left anterior mediastinal mass (Figures 1b and 1c). Work-up for possible metastases performed using Gallium scintigraphy showed no evidence of involvement of any other organs. Left anterior thoracotomy, pericardial window and left anterior mediastinotomy were done, revealing a well-encapsulated gelatinous tumor originating from the pericardium. The tumor grew from the inner surface of the pericardium near the origin of the superior vena cava and ascending aorta (Figure 2). A portion of the pericardium where the tumor was attached was resected and the tumor was biopsied. Complete resection was not attempted due to the proximity and attachment to the great vessels.\n\nThe echocardiogram image (1a) shows large pericardial effusion with collapse of the free wall of right atrium. The coronal (1b) and axial (1c) CT images show the effusion and an enormous heterogeneous mass in the pericardial cavity.\n\nLeft anterior thoracotomy, pericardial window and left anterior mediastinotomy were done, revealing a well-encapsulated gelatinous tumor originating from the pericardium (5.2 × 1.3 × 0.7 cm).\n\nReview of pathology on routine hematoxylin-eosin stain showed that the tumor was monophasic and was composed of numerous spindle to oval cells in a fascicular growth pattern. The cells showed scant cytoplasm, oval to spindle nuclei with granular chromatin and with inconspicuous nucleoli (Figure 3a). The tumor was mitotically active and showed 3–4 mitosis per high power field (Figure 3b). Some areas showed myxoid background (Figure 3b) and others showed large areas of necrosis (Figure 3c). Immunohistochemistry showed that the tumor cells were strongly positive for BCL-2 (Figure 4a), CD 99 (Figure 4b), Vimentin (Figure 4c), FLI1 (Figure 4d) and neuron-specific enolase (NSE) (Figure 4e). Stain for epithelial membrane antigen (EMA) showed focal scattered positive cells (Figure 4f). Muscle markers (smooth muscle actin, desmin and myogenin), neural and melanoma marker (S-100), epithelial markers (AE1/AE3), CD57, CK5/6 and p63 were negative. Based on morphology and immunostaining results, a diagnosis of monophasic synovial sarcoma was made. Fluorescent in-situ hybridization (FISH) was done to confirm the diagnosis and to exclude Ewing sarcoma. It showed the presence of SS18 (SYT) gene rearrangement on chromosome 18q11, substantiating the diagnosis. Also the FISH assay for rearrangement or dosage abnormalities of the EWSR1 oncogene at Ewing sarcoma locus 22q12 was negative.\n\n(3a) (× 100 magnification) shows cellular monophasic tumor with spindle cells in fascicular growth pattern. (3b) (× 400 magnification) shows cells in a myxoid background with many mitosis (arrows). (3c) (× 100 magnification) shows large areas of necrosis (arrow).\n\nTumor cells were strongly positive for: BCL-2 (× 100 magnification) (4a), CD 99 (× 100 magnification) (4b), Vimentin (× 100 magnification) (4c), FLI1(× 100 magnification) (4d) and NSE (× 100 magnification) (4e). They also showed focal scattered positivity for EMA (× 400 magnification) (4f).\n\nThe patient was treated with 6 cycles of high dose ifosfamide (2000mg/m2) and mesna (1200mg/m2) between July and December 2012. This led to substantial reduction (>50%) in the size of the tumor. A repeat thoracotomy was done with an aim for curative resection and pathology revealed that the borders were positive for malignant cells. The patient recovered well after the repeat thoracotomy and EBRT was initiated 1-month post surgery. He had no recurrence of tumor on CT imaging at the first and second three-month intervals post surgery. Chest CT repeated at 9 months (third three-month interval) showed local recurrence. The patient was noted to have an anterior mediastinal mass extending to the left upper lobe of the lung and a left pleural effusion. There was no evidence for any distant metastases. The patient underwent left upper lobe resection and decortication of the left pleura. Chemotherapy was reinitiated with docetaxel (100mg/m2) every 21 days along with steroid premedication with dexamethasone (4mg) for 5 days prior to treatment. This resulted in minor improvement on repeat CT imaging, but more importantly there was no further symptomatic worsening. The patient is clinically stable 18 months after diagnosis.\n\n\nDiscussion\n\nPrimary cardiac tumors are rare, and large autopsy series have shown the prevalence of primary cardiac tumors to be approximately 0.02%6. Soft tissue sarcomas account for 37% of these primary cardiac tumors7. In descending order of frequency, these are angiosarcoma, fibrosarcoma, rhabdomyosarcoma, and malignant fibrous histiocytoma7,8. Synovial sarcomas of cardiac origin are rare, and those originating from the pericardium are exceedingly rare. The real incidence of pericardial synovial sarcoma may be underestimated though, as the precise site of origin of such tumors can be difficult to determine. To date, to the authors’ knowledge, only 20 cases have been reported in the English literature2.\n\nSynovial sarcomas are malignant soft tissue tumors with an aggressive growth pattern. Although they are predominantly diagnosed in extremities with a predilection for juxta-articular location, in young adults, they have been reported to occur in almost any organ9–12. They are classified as biphasic, monophasic and poorly differentiated based on their histology. Among the pericardial synovial sarcomas, the biphasic type (composed of an epithelial component and a spindle-cell component) appears to be more common than the monophasic type (only spindle-cells) and the poorly differentiated type (small round cells)2,9–12. Our patient had a monophasic variant with predominant spindle to oval cells in a fascicular pattern. Some areas showed myxoid background as well as large areas of necrosis and the tumor was mitotically active.\n\nVarious other mediastinal and pericardial tumors (lymphoma, mesothelioma, thymoma, metastatic carcinomas, other soft tissue sarcomas) can have a similar appearance to synovial sarcoma on histology. Further analysis using immunohistochemistry is commonly done to differentiate between them. The specimens of our patient were uniformly positive for Vimentin, BCL-2 and CD99 (markers which are typical for synovial sarcoma) and showed focal positivity for EMA. We also noted absence of immunostain positivity for muscle markers (smooth muscle actin, desmin and myogenin), neural and melanoma marker (S-100), pankeratin (AE1/AE3), cytokeratin (CK5/6), CD57 and p63. This helped in arriving at a diagnosis of synovial sarcoma and to exclude other possible soft tissue tumors. In our patient, the tumor cells also stained positive for NSE and FLI1. FLI1 is a fusion gene that is usually positive in Ewing sarcoma, but some cases of synovial sarcoma also show FLI1 positivity13.\n\nDespite arriving at a diagnosis based on histology and immunohistochemistry, in order to further confirm diagnosis, cytogenetic analysis was done in our patient. Cytogenetic analysis is increasingly being used, especially in diagnosing those tumors arising in unusual sites. Confirming the histological diagnosis is crucial in determining therapy. More than 90% of synovial sarcomas demonstrate the presence of a reciprocal translocation t (X; 18) (p11.2; q11.2), and detection of this SSX-SYT fusion gene confirms the diagnosis1,14–17. Reverse transcription-polymerase chain reaction (RT-PCR) or FISH can both be used to detect this gene. FISH has a higher sensitivity for detection and a better success rate than RT-PCR17. In our patient, FISH assay was performed, and it showed the presence of SS18 (SYT) gene rearrangement on chromosome 18q11. Also, in our patient in view of the immunostain positivity for markers of Ewing sarcoma, the FISH assay was conducted for rearrangement or dosage abnormalities of EWSR1 oncogene at Ewing sarcoma locus 22q12, and the result was negative. This helped us to further confirm the diagnosis of synovial sarcoma and exclude Ewing sarcoma13.\n\nManagement of synovial sarcoma of pericardial origin is challenging and needs a multimodal approach. It is an aggressive tumor with a poor prognosis. Most patients have a short survival time1,2. One exception is the case report of a patient whose survival exceeded 14 years and involved recurrences and multiple surgical resections4. Surgical resection can lead to curative therapy. Most often, due to the site of origin, tumors are adherent to adjacent vital organs and are non-resectable at the time of diagnosis. Therefore, EBRT and less commonly neo-adjuvant chemotherapy are used to make the tumor resectable. In our patient, we could not attempt complete surgical resection. Therefore, after the initial biopsy and pericardial window, neo-adjuvant chemotherapy was done with six cycles of high dose ifosfamide (2000mg/m2) and mesna. Due to the rarity of these tumors, it is very difficult to establish an optimal regimen for chemotherapy. In general synovial sarcomas are chemo-sensitive to ifosfamide and show a good response. A case series of 13 patients with synovial sarcoma treated with high-dose ifosfamide showed a response in all patients, with 4 clinical remissions achieved18. Combining doxorubicin with ifosfamide may achieve a higher response rate (58%)19 but patient tolerability and side effects need to be considered. The initial favorable response to neoadjuvant high dose ifosfamide in our patient made the tumor resectable and may be a viable alternative to EBRT alone in non-resectable tumors. Gu et al. have reported using the collagen gel droplet embedded culture-drug sensitivity test (CD-DST) prior to determining the therapeutic value of chemotherapy in their patient with synovial sarcoma20. It may be useful to have CD-DST as a screening method for the selection of effective anticancer drug regimen for each individual patient2.\n\nEBRT is a very useful modality of treatment post surgery when the margins of resection are positive. In our patient, EBRT was initiated one month after surgery. Previous reports have shown similar favorable results21. Close monitoring for recurrence is very crucial in management of this tumor. CT or magnetic resonance imaging should be performed every 6 months to detect subclinical recurrences4. We performed CT imaging every three months along with regular clinical follow-up. Repeat imaging is essential to be able to diagnose subclinical recurrence and initiate repeat broad surgical resection and follow-up chemotherapy if needed. We used the anti-mitotic agent docetaxel as well as left lung upper lobe resection for treating the recurrence.\n\n\nConclusions\n\nThis case report describes a case of monophasic synovial sarcoma of pericardial origin presenting as pericardial effusion with cardiac tamponade physiology in a young man. It is an extremely rare tumor, and systematic approach to diagnosis and management are crucial. We emphasize the use of cytogenetic analysis in confirming the diagnosis and excluding tumors that might mimic synovial sarcoma on immunohistochemistry. This case also highlights the use of neo-adjuvant chemotherapy using high dose ifosfamide as part of multimodal approach to treatment.\n\n\nConsent\n\nWritten informed consent for publication of clinical details and clinical images was obtained from the patient.",
"appendix": "Author contributions\n\n\n\nPrajakta Phatak, Carmelo Puccio, and Jagadish Khanagavi were involved in the diagnosis and treatment of the patient and in writing the manuscript. Wilbert S. Aronow, Yasmin Yusuf and Sonam Puri were involved in writing the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nHing SN, Marshall L, Al-Saadi R, et al.: Primary pericardial synovial sarcoma confirmed by molecular genetic studies: a case report. J Pediatr Hematol Oncol. 2007; 29(7): 492–495. PubMed Abstract | Publisher Full Text\n\nYoshino M, Sekine Y, Koh E, et al.: Pericardial synovial sarcoma: a case report and review of the literature. Surg Today. 2013; 595(5): 720–724. PubMed Abstract | Publisher Full Text\n\nBurke AP, Virmani R: Primary cardiac sarcomas. In: Burke AP, Virmani R, eds. Tumors of the Cardiovascular System. Atlas of Tumor Pathology, Third Series. Washington, DC: Armed Forces Institute of Pathology, 1996; (3): 9–11.\n\nVan der Mieren G, Willems S, Sciot R, et al.: Pericardial synovial sarcoma: 14-year survival with multimodality therapy. Ann Thorac Surg. 2004; 78(3): e41–e42. PubMed Abstract | Publisher Full Text\n\nConstantinou LL, Charitos CE, Lariou CM, et al.: Primary synovial cardiac sarcoma: a rare cause of tamponade. Eur Heart J. 1996; 17(11): 1766–8. PubMed Abstract\n\nReynen K: Frequency of primary tumors of the heart. Am J Cardiol. 1996; 77(1): 107. PubMed Abstract\n\nBurke AP, Cowan D, Virmani R: Primary sarcomas of the heart. Cancer. 1992; 69(2): 387–395. PubMed Abstract\n\nDemmy TL: Tumors of the heart and pericardium. In: Aisner J, Arriagada R, Green M, et al. (eds): “Comprehensive Textbook of Thoracic Oncology.” Philadelphia: Williams & Wilkins, 1996; 681–710. Reference Source\n\nAl-Rajhi N, Husain S, Coupland R, et al.: Primary pericardial synovial sarcoma: a case report and literature review. J Surg Oncol. 1999; 70(3): 194–198. PubMed Abstract | Publisher Full Text\n\nMoorjani N, Peebles C, Gallagher P, et al.: Pericardial synovial sarcoma. J Card Surg. 2009; 24(3): 349–51. PubMed Abstract | Publisher Full Text\n\nYokouchi Y, Hiruta N, Oharaseki T, et al.: Primary cardiac synovial sarcoma: a case report and literature review. Pathol Int. 2011; 61(3): 150–5. PubMed Abstract | Publisher Full Text\n\nCheng Y, Sheng W, Zhou X, et al.: Pericardial synovial sarcoma, a potential for misdiagnosis: clinicopathologic and molecular cytogenetic analysis of three cases with literature review. Am J Clin Pathol. 2012; 137(1): 142–9. PubMed Abstract | Publisher Full Text\n\nWarren M, Weindel M, Ringrose J, et al.: Integrated multimodal genetic testing of Ewing sarcoma--a single-institution experience. Hum Pathol. 2013; 44(10): 2010–9. PubMed Abstract | Publisher Full Text\n\nYano M, Toyooka S, Tsukuda K, et al.: SYT-SSX fusion genes in synovial sarcoma of the thorax. Lung Cancer. 2004; 44(3): 391–397. PubMed Abstract | Publisher Full Text\n\nFletcher CDM, Bruijn DRH, Kessel AG: Synovial sarcoma. In: Fletcher CDM, Unni KK, Mertens F, eds. World Health Organization Classification of Tumours. Pathology and Genetics, Tumours of Soft Tissue and Bone. Lyon: International Agency for Research on Cancer Press, 2002; 200–204. Reference Source\n\nOizumi S, Igarashi K, Takenaka T, et al.: Primary pericardial synovial sarcoma with detection of the chimeric transcript SYT-SSX. Jpn Circ J. 1999; 63(4): 330–332. PubMed Abstract | Publisher Full Text\n\nSun B, Sun Y, Wang J, et al.: The diagnostic value of SYT-SSX detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH) for synovial sarcoma: a review and prospective study of 255 cases. Cancer Sci. 2008; 99(7): 1355–1361. PubMed Abstract | Publisher Full Text\n\nRosen G, Forscher C, Lowenbraun S, et al.: Synovial sarcoma. Uniform response of metastases to high dose ifosfamide. Cancer. 1994; 73(10): 2506–2511. PubMed Abstract\n\nSpurrell EL, Fisher C, Thomas JM, et al.: Prognostic factors in advanced synovial sarcoma: an analysis of 104 patients treated at the Royal Marsden Hospital. Ann Oncol. 2005; 16(3): 437–444. PubMed Abstract | Publisher Full Text\n\nGu F, Ma Y, Fan Y, et al.: Synovial sarcoma individual chemotherapy directed by collagen gel droplet embedded culture drug sensitivity test: a case report. Oncol Lett. 2010; 1(5): 885–888. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAnand AK, Khanna A, Sinha SK, et al.: Pericardial synovial sarcoma. Clin Oncol (R Coll Radiol). 2003; 15(4): 186–188. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "3183",
"date": "27 Jan 2014",
"name": "Nikhil Mukhi",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a very well written article, and I think it makes a positive contribution to our current knowledge of cardiac sarcomas. The authors appropriately mention that these tumors are rare and no standard of care exists. However, this patient in the relapse setting was treated with single agent docetaxel. In phase II trials of soft tissue sarcomas, docetaxel has been used in combination with gemcitabine due to their synergistic effect (Maki RG et al., 2007). It is unclear why patient was only given docetaxel. It is however very interesting to see that patient responded well to therapy and is stable 18 months post treatment. This article may help guide therapy in these tumors.",
"responses": [
{
"c_id": "683",
"date": "29 Jan 2014",
"name": "Wilbert Aronow",
"role": "Author Response",
"response": "We thank Dr. Mukhi very much for his review and approval of our case report. Our patient was treated with docetaxel for his local recurrence based on evidence showing that docetaxel is an efficacious and tolerable treatment as rescue medication in ifosfamide-resistant locally advanced or metastatic soft tissue sarcoma (Kostler et al. 2001). Maki et al. (2007) showed that gemcitabine-docetaxel yielded a better response compared to gemcitabine alone when used for initial treatment rather than as a rescue treatment option. We are of the opinion that although our patient had a favourable response, it may be useful to have CD-DST as a method for the selection of an effective anticancer drug regimen for each individual patient for both primary treatment and to select the rescue chemotherapy."
}
]
},
{
"id": "3181",
"date": "12 Feb 2014",
"name": "Edwin Choy",
"expertise": [],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this case report, the authors offer a clinicopathologic description of a case of pericardial synovial sarcoma. There are several major points that need to be addressed:The patient was treated neoadjuvantly with \"high dose\" ifosfamide, but the authors describe using 2000 mg/m2 per cycle, which is actually considered quite a low dose for treatment of sarcomas. This is either an error (perhaps 2000 mg/m2 was given each day for 3, 4, or 5 days per cycle?) or a very low dose was used intentionally. Regardless, the patient apparently benefitted with some tumor shrinkage, which allowed for surgical resection. A description of the surgery is lacking. We do not know how much pericardium was removed or if lymph nodes were sampled, as sarcomas are known to metastasize lymphatically as well as hematogenously. Although postoperative EBRT was administered, there is no discussion about whether intraoperative XRT or proton therapy was considered. Upon disease recurrence with a mediastinal mass and pleural effusion, a lobectomy and decortication was performed. We are not told if malignant cells were seen in the pleural fluid, in which case the entire pleura would be considered contaminated. Post-op, the patient was treated \"adjuvantly\" with docetaxel. Here, the authors need to be careful to point out that single agent docetaxel is not considered standard therapy for synovial sarcoma, and adjuvant therapy after a metastasectomy is also quite a controversial strategy. It would be wrong if readers interpreted this report as an endorsement of post-op single agent docetaxel after a metastasectomy. In summary, it is not clear what we learn from this case report except that the patient apparently sustained a partial tumor response to ifosfamide chemotherapy, tolerated an R1 or R2 surgery, and despite post-op radiation, sustained a regional/metastatic recurrence. A more thorough discussion about the complexities and controversies of clinical decision making in patients such as this would make such case reports more informative to the audience.",
"responses": []
},
{
"id": "3725",
"date": "24 Feb 2014",
"name": "Gurpreet S Lamba",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting paper and is well written. It would definitely help future clinicians who might encounter this rare diagnosis. However I think that for the benefit of these clinicians the authors should include more details on the chemotherapy regimen, including the number of days ifosfamide was administered for and the frequency of the cycle. More details about the surgery and radiation would also be helpful.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-15
|
https://f1000research.com/articles/3-66/v1
|
03 Mar 14
|
{
"type": "Research Article",
"title": "First regional evaluation of nuclear genetic diversity and population structure in northeastern coyotes (Canis latrans)",
"authors": [
"Javier Monzón"
],
"abstract": "Previous genetic studies of eastern coyotes (Canis latrans) are based on one of two strategies: sampling many individuals using one or very few molecular markers, or sampling very few individuals using many genomic markers. Thus, a regional analysis of genetic diversity and population structure in eastern coyotes using many samples and several molecular markers is lacking. I evaluated genetic diversity and population structure in 385 northeastern coyotes using 16 common single nucleotide polymorphisms (SNPs). A region-wide analysis of population structure revealed three primary genetic populations, but these do not correspond to the same three subdivisions inferred in a previous analysis of mitochondrial DNA sequences. More focused geographic analyses of population structure indicated that ample genetic structure occurs in coyotes from an intermediate contact zone where two range expansion fronts meet. These results demonstrate that genotyping several highly heterozygous SNPs in a large, geographically dense sample is an effective way to detect cryptic population genetic structure. The importance of SNPs in studies of population and wildlife genomics is rapidly increasing; this study adds to the growing body of recent literature that demonstrates the utility of SNPs ascertained from a model organism for evolutionary inference in closely related species.",
"keywords": [
"Canis latrans",
"northeastern coyote",
"SNP",
"cryptic genetic structure",
"ascertainment bias"
],
"content": "Introduction\n\nHistorically restricted to the open deserts and plains of central and western North America, the coyote (Canis latrans) has colonized almost the whole continent in the last 100 years, with few exceptions (e.g. Long Island)1,2. The eastward range expansion was likely facilitated by widespread deforestation associated with agricultural development in the early 20th century and by the near extirpation of eastern wolves (Canis lupus lycaon or Canis lycaon) and red wolves (Canis rufus)3–5. The range expansion into northeastern North America advanced as two primary colonization fronts: the northern route through Ontario, where coyotes hybridized with resident eastern wolves, and the southern route through Ohio, where wolves were eradicated prior to coyote expansion2,5,6. Along with its geographic range expansion, the coyote also experienced a niche expansion by rapidly colonizing whole new biomes, including eastern temperate and boreal forests1. This complex scenario of colonization provides an interesting opportunity to explore the swift formation of population genetic structure following a rapid expansion in geographic distribution and ecological niche.\n\nRecent analyses of population structure in northeastern coyotes have described a general lack of genetic differentiation among sampling localities, except at the coarsest geographic scales. Way et al.7 examined genetic variation and structure in a sample of coyotes from eastern Massachusetts using mitochondrial DNA (mtDNA) and eight microsatellite loci. They found no genetic structure in coyotes within Massachusetts or even within the broader region of northeastern North America. Instead, northeastern coyotes seemed to constitute one uniform population slightly differentiated from western coyotes. In another analysis of genetic variation in northeastern coyotes, Kays et al.5 identified three coarse phylogeographic areas: Ohio, the northeast zone, and a contact zone in western Pennsylvania and New York where the colonization front from Ohio has spread into the northeastern population. Although Kays et al. surveyed genetic variation in a dense geographic sample of 687 coyotes, they only used one genetic marker, the hypervariable mtDNA control region. vonHoldt et al.8 conducted a genome-wide analysis of North American Canis species and detected population structure in C. latrans, but only at the broadest continental scale. Although vonHoldt et al. genotyped tens of thousands of loci, they only sampled 14 northeastern coyotes, making detection of finer levels of population structure in the region very improbable. Thus, all the previous studies of population structure in northeastern coyotes have adopted one of two strategies: sampling many individuals using one (mtDNA) or very few molecular markers, or sampling very few individuals using thousands of genomic markers. A regional analysis of genetic diversity and population structure in northeastern coyotes using many samples and many nuclear molecular markers is currently lacking.\n\nGenetic structure is a ubiquitous property of natural, domesticated, and human populations. Population genetic structure plays considerable roles in evolution, as both the basis and the consequence of local adaptation, the splitting of one species into two if the environments are markedly different, and the adaptability of a species as a whole across its range (i.e., transformation rather than speciation)9. The detection of genetic structure largely depends on the type and number of molecular markers examined, their variability in the target population, the number of individuals sampled, and the spatial sampling scheme10,11. Single nucleotide polymorphisms (SNPs) have become a popular and inexpensive tool in the field of molecular population genetics. SNPs have properties that make them a superior alternative to other widely used genetic markers, such as microsatellites and mtDNA sequences, in evaluating genetic diversity and population structure12–14. Furthermore, the sampling scheme can greatly influence the location and composition of genetic clusters, especially in species that are continuously distributed across a landscape11, as are northeastern coyotes. Thus the conclusion of no population structure from previous analyses based on limited individual or genomic sampling may be imprecise.\n\nThe objective of this study was to test the hypothesis that fine-scale population structure in northeastern coyotes exists, but remains undetected due to the small number of individuals or the low resolution of the genetic markers previously analyzed. I hypothesized that population structure would be detectable, at finer levels than in previous analyses, by using an array of 16 high-heterozygosity nuclear SNPs ascertained from the dog genome and a spatially dense sample of 385 coyotes. This is the first comprehensive regional survey of genetic diversity and population structure in northeastern coyotes that uses a dense geographic sampling scheme and several SNPs. This regional analysis reveals a cryptic population structure and a geographic pattern of nuclear genetic diversity that is discrepant with previous mtDNA- and microsatellite-based surveys. More generally, this study adds to the growing body of recent literature that demonstrates the utility of SNPs discovered in a model organism for evolutionary inference in wild relatives, as long as ascertainment bias is explicitly evaluated.\n\n\nMaterials and methods\n\nThe study area was located in northeastern North America. Coyotes were sampled from New York (N = 174), Pennsylvania (N = 103), Vermont (N = 34), Ohio (N = 30), New Jersey (N = 14), New Hampshire (N = 11), Connecticut (N = 8), Massachusetts (N = 5), southern Quebec (N = 4), and Rhode Island (N = 2) (Figure 1). All samples (Total N = 385) used in this study are archived and vouchered in the New York State Museum, Albany, NY, where they were kept at -80°C. Specimens (tissues or combination of skin, skull, skeleton) were obtained primarily through donations from licensed local hunters and trappers since 1999. Six samples came from previous scat surveys in New York15,16 (Data File). No Institutional Animal Care and Use Committee (IACUC) review was required for this study because the DNA samples came from scat or animals killed for reasons other than research.\n\nSymbol shapes represent phylogeographic zones as in Kays et al.5: circle, Ohio; square, contact zone; triangle, northeast zone.\n\nI selected molecular markers based on a genomics study that used the Affymetrix Canine Mapping Array to genotype 61,435 SNP loci in hundreds of wild and domestic canids, including 14 northeastern and 3 Ohio coyotes8,17. I used the program PLINK18 to compute observed and expected heterozygosity per locus in the subset of 17 northeastern and Ohio coyotes. I selected 16 unlinked SNPs, each on a different autosomal chromosome, with the highest observed heterozygosity, a measure of genetic variability in a population (Table 1). Ascertainment schemes that select highly variable SNP loci increase power to detect population structure13; accordingly, these 16 informative SNPs with high heterozygosity and high minor allele frequencies allowed me to assess genetic variation and population structure in the larger target sample. I designed primers using the Primer3 software19 and tested them in silico against the dog CanFam2 genome assembly (GenBank Assembly ID: GCA_000002285.1)20 using the University of California, Santa Cruz In-Silico PCR and BLAT web tools (http://genome.ucsc.edu).\n\nSNP ID chromosomal coordinates and dbSNP rs numbers correspond to the CanFam2 dog genome assembly. Measures of genetic variability correspond to the initial seventeen northeastern and Ohio coyotes (Canis latrans) that formed the ascertainment panel.\n\nNote: MAF: minor allele frequency; HO and HE: observed and expected heterozygosity. Several SNPs have the same MAF, HO, and HE due to the relatively small number of coyotes in the ascertainment panel. For example, HO = 0.765 if 13 of 17 individuals were heterozygous.\n\nI extracted total genomic DNA from muscle or hide using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, USA) according to the manufacturer’s instructions, and also used DNA samples that were extracted in the New York State Museum as described in Kays et al.5. I quantified DNA concentrations using a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, Wilmington, USA) and diluted the samples in water to attain concentrations of 5–30 ng/μl. I prepared four 96-well plates with template genomic DNA of 378 eastern coyotes, seven of which served as controls because they were already genotyped with the canine SNP microarray8. Two western coyotes from Washington and two western gray wolves (Canis lupus) from Yellowstone National Park served as additional controls; two wells containing only water served as negative controls.\n\nSNP genotyping was performed at the GenoSeq Core laboratory in the University of California, Los Angeles, using a high resolution melting curve quantitative PCR assay on a LightCycler 480 thermal cycler (Roche, Indianapolis, USA). Each of the 16 SNPs was amplified and genotyped separately. DNA was amplified in a total volume of 10 μl, including 1 μl (5–30 ng) of genomic DNA, 0.2 µM of each primer, 4.2 mM MgCl2, and 1× Roche High Resolution Melting Master kit mix. The latter contains FastStart Taq DNA polymerase, dNTP mix, and ResoLight, a high resolution melting dye that fluoresces when DNA is double-stranded. During the melt curve analysis, the temperature increases very slowly to denature double-stranded DNA. Samples with variations in DNA sequence, even in one base pair, are distinguished by discrepancies in the shape of the melt curve, thus discriminating each of the two homozygous and the heterozygous genotypes. I processed the raw data using the Gene Scanning module of the Roche LightCycler software and followed Roche’s recommendations for evaluating data quality. The software automatically generates genotypes from the raw melt curve data. All SNPs were biallelic, so there are three possible genotypes per locus–AA, AB and BB–each forming three distinct clusters when signal intensity is plotted against temperature. I visually inspected all software-generated genotype calls and manually removed the ambiguous ones that did not conform to any of the three possible genotype clusters,\n\nTo the 378 samples genotyped in this study, I added 7 more northeastern coyote samples genotyped by vonHoldt et al.8 making a total sample size of 385. I used PLINK to calculate average observed and expected heterozygosity, measures of genetic diversity, and deviations from Hardy-Weinberg equilibrium in the overall sample and in each of the three phylogeographic zones inferred by Kays et al.5 (Figure 1). In order to assess the ascertainment bias of using dog genome SNPs to study genetic diversity and population structure in coyotes, I compared genetic variation in five subsets of canids: northeastern coyotes, western coyotes, western gray wolves, Great Lakes wolves, and dogs (Canis familiaris)8,17. I calculated average observed and expected heterozygosity using all 61,435 SNPs from the microarray because the vast majority of the loci in the microarray were ascertained by dog-dog comparisons17, and again using only the 16 high-heterozygosity SNPs genotyped in this study.\n\nTo assess population genetic structure, I excluded all individuals with more than five missing genotypes, resulting in 247 coyotes (16 in Ohio, 118 in contact zone, 113 in northeast zone). I used the program STRUCTURE 2.321 to infer the most likely number of genetic populations. STRUCTURE implements a Bayesian algorithm to assign multilocus genotypes to genetic clusters by calculating the likelihood that a group of individuals constitutes a population. I tested whether finer population structure was detectable with the high-heterozygosity SNPs relative to the coarse structure detected with hypervariable mtDNA. I analyzed all 247 coyotes together to test whether more than three populations were detectable at the regional level, and then analyzed each zone separately to test whether more than one population was detectable within each zone. For all analyses, I used three replicate runs of 20,000 burn-in and 100,000 Markov chain Monte Carlo iterations, used the admixture ancestry model with correlated allele frequencies22, and set the number of populations from K = 1 to K = 8. I verified that alpha and likelihood statistics reached convergence during the burn-in period for each number of populations analyzed. I used STRUCTURE HARVESTER23 to evaluate the relative support for each value of K by plotting Ln P(D), the mean posterior probability of the data21, and ΔK, a quantity related to the second-order rate of change of the likelihood function with respect to K10. I used CLUMPP24 to align and average the three replicate cluster membership coefficient matrices, and ArcMap 10 (Esri, Redlands, USA) to visualize the spatial distribution of genetic structure. I considered individuals with ancestry coefficients q > 0.8 as belonging to a specific cluster and individuals with all q < 0.8 as being admixed, consistent with recent genetic investigations of Canis25,26. In order to corroborate inferences from the STRUCTURE analysis with a model-free approach, I conducted a principal components analysis (PCA) using the adegenet 1.3–4 package27 in R28. I also computed pairwise FST, the inbreeding coefficient within populations relative to the total, among the populations inferred by STRUCTURE and tested the significance of the differentiation by analysis of molecular variance (AMOVA) using 999 permutations in GenAlEx 6.529,30.\n\n\nResults\n\nI interrogated 16 SNP loci in 378 coyotes for a total of 6,048 expected genotypes (Data File). Genotyping efficacy varied by source of DNA, with fecal samples amplifying less effectively than tissue samples (Mann-Whitney test: U = 1771, P = 0.017). That is, the fecal samples had, on average, more missing or ambiguous genotypes than the tissue samples.\n\nThe overall sample of 385 eastern coyotes had lower genetic diversity than expected (Table 2), even though observed heterozygosity generally exceeded expected heterozygosity in the ascertainment panel of 17 northeastern and Ohio coyotes (Table 1). Ohio coyotes were the most genetically diverse in the region, but the eastward decay in genetic diversity observed with mtDNA was not replicated with nuclear SNPs. Although coyotes from the northeast zone had the lowest levels of mitochondrial genetic diversity, these same individuals had a level of nuclear genetic diversity comparable to Ohio coyotes (Table 2). The most pronounced differences between observed and expected heterozygosity occurred in the contact zone and in the overall regional analysis (Table 2). In the overall sample of 385 eastern coyotes, five loci were in Hardy-Weinberg equilibrium. The number of loci in Hardy-Weinberg equilibrium increased when each phylogeographic zone was analyzed separately: 13 in Ohio, nine in the contact zone, and nine in the northeast zone (Table 2).\n\nMost individuals genotyped at 16 nuclear SNPs represent a subset of those individuals sequenced.\n\nNote: mtDNA data and zone designations from Kays et al.5. N: sample size; HO and HE: observed and expected heterozygosity; HWE: number of loci in Hardy-Weinberg equilibrium.\n\nDogs appeared to be the most genetically diverse when the diversity of the five different canid groups was estimated using all 61,435 SNPs from the canine microarray. The genome-wide ascertainment bias was towards dogs: the expected heterozygosity of dogs was almost twice that of western coyotes. But the ascertainment bias reversed when heterozygosity was measured using only the 16 selected SNPs: coyotes appeared to be the most genetically diverse, with northeastern coyotes having a very high expected heterozygosity, whereas dogs appeared the least genetically diverse (Table 3).\n\nNote: Data for 61,435 SNPs from vonHoldt et al.8. N: sample size; HO and HE: observed and expected heterozygosity.\n\nIn the region-wide population structure analysis of 247 individuals with little missing data, the values of K with the strongest statistical support were K = 2 and K = 3 (Figure 2A, B). Pairwise FST among the three genetic clusters varied from 0.08 to 0.10 and all were significant in the AMOVA framework (P = 0.001). This indicates that there are three primary genetic subdivisions in the broad sampling area. The three groups did not correspond to the Ohio, contact, and northeast zones previously inferred by mtDNA (Figure 2C). There is some geographic structuring, but the three groups overlap extensively in space. Although the red cluster in Figure 2B includes most of the Ohio coyotes, it is more cosmopolitan, also including many coyotes from the contact and northeast zones. The green cluster is mostly restricted to the contact zone, but extends slightly into eastern New York and Vermont. The PCA corroborated the results from STRUCTURE (Figure 3). The first two PCA axes explained 24.2% of the total variance and clearly separated the three STRUCTURE-inferred clusters. Most admixed individuals with no clear membership in any STRUCTURE-inferred cluster also showed no clear association with any PCA cluster.\n\nA) Estimation of the number of genetic populations using two criteria: ΔK (solid line) and Ln P(D) (dashed line). The most probable number of populations is the value of K with the maximal value of ΔK and Ln P(D). The data strongly support K = 2 and K = 3. B) STRUCTURE bar plots of N = 247 coyotes subdivided into K = 2 and K = 3 genetic populations; each individual is represented by a vertical bar partitioned into two or three colored segments indicating that individual’s proportional membership in each of two or three genetic clusters. C) Spatial distribution of K = 3 genetic populations. Symbol colors represent the genetic cluster with > 80% assignment from panel B, or black if highly admixed (i.e., no assignment > 80%). Symbol shapes represent phylogeographic zones as in Figure 1. Symbol locations are slightly jittered to display each individual and to reduce clutter.\n\nSymbol colors correspond to K = 3 STRUCTURE-inferred clusters with > 80% assignment, or black if highly admixed (i.e., no assignment > 80%), as in Figure 2C. Inset shows scree plot of eigenvalues.\n\nAmple population genetic structure was detected in the contact zone; the values of K with the strongest statistical support were K = 3 and K = 5 (Figure 4A). However, the K = 5 structure seems biologically unrealistic, characterized by highly admixed individuals of the various “populations” (Figure 4B). Alternatively, the K = 3 structure identified for the contact zone was virtually identical to the regional K = 3 structure (Figure 4C). In contrast to the contact zone, no fine-scale genetic structure was detected in Ohio or in the northeast zone. In Ohio, the value of K with the highest explanatory power was K = 1 (Figure 5A); in the northeast zone, the value of Ln P(D) does not increase beyond K = 2 and the change in Ln P(D) between K = 1 and K = 2 is minimal, indicating weak support for genetic structure (Figure 5B).\n\nA) Estimation of the number of genetic populations using two criteria: ΔK (solid line) and Ln P(D) (dashed line). The data strongly support K = 3 and K = 5. B) STRUCTURE bar plots of N = 118 coyotes subdivided into K = 3 and K = 5 genetic clusters. C) Spatial distribution of K = 3 genetic clusters. Symbol colors represent the genetic cluster with > 80% assignment from panel B, or black if highly admixed (i.e., no assignment > 80%).\n\nAbsence of fine-scale population genetic structure in A) N = 16 coyotes from Ohio, and B) N = 113 coyotes from the northeast zone. See Figure 1 for zone designations.\n\n\n\n\nDiscussion\n\nThis study documents the presence of fine-scaled population genetic structure in eastern coyotes. Specifically, the contact zone exhibits a strong signal of population structure, the same signal detected in the regional analysis. This pattern may reflect the recent merging of two colonization fronts and the highly heterogeneous landscape of New York and Pennsylvania. A separate, spatially-explicit assessment with an independent set of SNPs demonstrated that northeastern coyotes exhibit a strong signal of population structure in Central New York1. Furthermore, the same investigation showed that population structure is partially explained by ecological factors, such as deer density and human land use1. Indeed, eastern coyotes living in areas of high deer density are genetically more wolf-like than those living in areas of low deer density31.\n\nIn contrast to the contact zone, coyotes in Ohio appear to make up a single panmictic population, as do coyotes in the northeast zone. Given the high mtDNA and nuclear diversity observed in Ohio by Kays et al.5 and in this study, it is unlikely that the lack of genetic structure stems from a founder effect. The failure to uncover more than one genetic population in Ohio may be due instead to its landscape homogeneity or to the lack of resolution afforded by 16 individuals to detect finer levels of structure. On the other hand, the failure to uncover more than one genetic population in the northeast zone is surprising given the vast geographic area with its ecological heterogeneity and the large number of individuals sampled. Coyotes in the northeast zone are likely the descendants of a few founders and therefore do not exhibit a strong signal of population genetic structure.\n\nThe perception of population structure, even in highly vagile animals where it was least expected, has been refined by steadily improving molecular data and geographic sampling. Initially, using mtDNA restriction site polymorphisms and nuclear microsatellites, no evidence of population structure or isolation by distance was found in coyotes, even at the continental scale32,33. Various behavioral and historical explanations have been invoked to explain these early genetic patterns. But a more likely explanation is that the patterns of weak differentiation were artifacts of sparse geographic sampling or poor resolution due to the use of few molecular markers. More recent studies employing advanced analyses of spatial and genetic data have revealed strong differentiation among parapatric populations of coyotes and wolves, even in the absence of physical barriers to movement1,34–38. These investigations used multiple loci and dense geographic sampling to uncover cryptic genetic subdivisions. Strong genetic differentiation between adjacent populations of coastal and inland wolves in British Columbia was shown with mtDNA39, demonstrating that fine-scale genetic differentiation can be detected with denser sampling alone, even using a single molecular mtDNA marker. Similar cryptic subdivisions have been discovered in several highly mobile groups, such as Lynx40,41, ungulates42–44, cetaceans45,46, and hawks47. In all these cases, genetic subdivisions appear to emerge from ecological factors and local foraging adaptations. Future studies should focus on the ecological mechanisms underlying the cryptic genetic structure in northeastern coyotes, especially because they have only inhabited the region for the last 30–80 years2. Confirming ecological determinants of population structure in the absence of obvious physical dispersal barriers would provide an interesting example of rapid ecological differentiation.\n\nThere are some important similarities and discrepancies between mtDNA and autosomal SNP patterns. The data indicate that coyotes in Ohio are the most genetically diverse in the region when surveyed with nuclear SNPs, as with mtDNA5. However, the gradual eastward decay in genetic diversity observed with mtDNA is not replicated with nuclear SNPs. The marked reduction of heterozygosity in the contact zone and in the overall region is very likely caused by population structure, i.e., the Wahlund effect. This interpretation is supported by the increase in the number of loci in Hardy-Weinberg equilibrium at smaller geographic scales and by the congruent signal of population structure in the overall region and in the contact zone. In addition, the three primary populations detected in this study do not correspond to the three subdivisions inferred with mtDNA5. Together, these results suggest that studies based solely on mtDNA should be interpreted cautiously. For example, mtDNA sequence similarity suggested that a small population of Scandinavian wolves was founded by individuals released from Swedish zoos, but nuclear polymorphic markers falsified the release hypothesis and instead supported a hypothesis of natural immigration or expansion from an unknown relict wolf pack48. Discrepancies between patterns observed with mtDNA and nuclear DNA may be caused by true organismal processes, such as sex-biased dispersal49. However, there is no evidence for sex-specific dispersal behaviors in eastern coyotes, consistent with their monogamous breeding system50,51. Alternatively, discrepancies may be caused by marker-specific phenomena such as effective population size, lineage sorting, mutation rate, and coalescent times52, or the violation of certain assumptions of mtDNA inheritance, such as recombination, paternal leakage, and heteroplasmy53. Future studies should further evaluate these sources of discrepancies.\n\nThe present study underscores three related methodological issues that are of broad interest, especially as SNPs continue to be in vogue in population and wildlife genomics. First, these data confirm that SNPs discovered in a model organism are an appropriate tool to address various questions regarding the ecology and evolution of non-model relatives. The sequencing of the dog genome20 quickly enabled SNP-based investigations into wild members of the family Canidae, including coyotes, wolves, jackals, and foxes8,54–57. Second, this study highlights the importance of evaluating the ascertainment bias of markers employed in a survey of genetic variation, especially in multi-species comparisons. Many SNP-based studies are not addressing the issue of ascertainment bias14. In the present study, the genome-wide analysis of variation is dog-biased because SNPs were ascertained primarily from comparisons of boxer and poodle genomes after the completion of the dog genome project8. On the other hand, the high-heterozygosity SNP analysis is coyote-biased because the 16 SNPs were chosen from an ascertainment panel of northeastern coyotes. The bias in the latter case is not problematic because the ascertainment panel from which the loci were selected is representative of the whole population of northeastern coyotes. However, the diversity measures reported in this study should not be compared to similar measures from other populations, unless the comparison corrects for ascertainment bias58. Lastly, this study emphasizes the necessity of selecting SNPs very carefully to match the research question of interest. Here, a set of high-heterozygosity SNPs was interrogated in order to examine geographic patterns of genetic diversity and population structure. But other research questions may require polymorphic markers with other properties. For example, in order to better understand the complex hybrid ancestry of the northeastern coyote, Monzón et al.31 used species-diagnostic SNPs to quantify the relative contributions of its parental populations. In addition, recent advances in the molecular genetics of phenotypic traits in dogs allow the use of SNPs linked to genes of known function to address long-standing questions about morphological, physiological, and behavioral adaptations in northeastern coyotes and other wild canids1,59.\n\n\nData availability\n\nfigshare: Data of genetic diversity in northeastern coyotes. http://dx.doi.org/10.6084/m9.figshare.94348360",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nResearch reported in this publication was supported by a Stony Brook University Turner Fellowship research grant to Javier Monzón, and by the National Institute of General Medical Science of the National Institutes of Health IRACDA grant K12GM102778 to Jorge Benach.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nRobert Wayne, John Pollinger, and Bridgett vonHoldt provided technical support to initiate this project and shared the 61K-SNP dataset. John True generously provided access to lab resources. Roland Kays kindly provided access to the coyote collection of the New York State Museum. Shian-Ren Liou and Nashwa Khalil assisted with laboratory procedures. Daniel Dykhuizen, Catherine Graham and four anonymous reviewers provided constructive comments to earlier drafts of this manuscript.\n\n\nReferences\n\nMonzón J: Rapid evolution of northeastern coyotes. PhD thesis, Stony Brook University, 2012. Reference Source\n\nParker G: Eastern Coyote: the Story of its Success. (Nimbus Publishing, 1995). Reference Source\n\nGompper ME: The ecology of northeast coyotes: current knowledge and priorities for future research. (Wildlife Conservation Society, Bronx, New York, 2002). Reference Source\n\nFener H, Ginsberg J, Sanderson E, et al.: Chronology of range expansion of the coyote, Canis latrans, in New York. Can Field Nat. 2005; 119: 1–5. Reference Source\n\nKays R, Curtis A, Kirchman J: Rapid adaptive evolution of northeastern coyotes via hybridization with wolves. Biol Lett. 2010; 6(1): 89–93. 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}
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[
{
"id": "3946",
"date": "18 Mar 2014",
"name": "Astrid Stronen",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review this manuscript. Javier Monzón presents a timely and relevant study of population structure in Northeastern coyotes, which complements earlier work in this area and highlights new and intriguing research questions. The author builds on previous genomic analyses of canids with a high-density SNP chip yet a limited sample of Northeastern coyotes, and a study of northeastern coyotes that used several hundred samples but a single genetic marker. The panel of selected SNPs is biased toward the study population of Northeastern coyotes, but the author makes this very clear and includes an evaluation of ascertainment bias across canid groups included in the initial study with the high-density SNP chip from which this panel of 16 loci was selected. The article is well written, and the author has done a good job explaining the main limitations of the work and outlining why findings should be interpreted with caution. I have one suggestion on supplementary data that could be included for an additional perspective on genetic structure, and some minor comments on the manuscript text:\n\nThe author describes the SNP results relative to the areas of Ohio, the contact zone and the northeast zone from Kays et al. 2010, and in Table 2 notes that “most individuals genotyped at 16 nuclear SNPs represent a subset of those individuals sequenced” for mtDNA. If individual mtDNA results are available for coyotes, it would be valuable to see the extent to which the mtDNA haplotypes classified as Great Lakes wolf (or eastern wolf), coyote and gray wolf from Kays et al. 2010 (Fig 2) correspond with genetic structure based on SNPs.This would provide important information to help understand how similarities/discrepancies in genetic structure are distributed across space.\n\nThe relatively low genetic diversity for coyotes in Table 3 and the results from vonHoldt et al. 2011 suggest a bias against coyotes, although coyotes may at least in some regions, have higher genetic diversity than wolves considering their range expansion and increase in population size. It might be worth noting that sequencing coyotes directly could provide a more in-depth view of genomic regions under selection. Although I agree with the author’s conclusion about the utility of using a closely related and well-studied species, alternate methods such as genotyping-by-sequencing could be recommended as possibilities for future research.\n\nIntroduction, 1st sentence “Historically restricted to the open deserts and plains...”. Though the coyote is widely regarded as a species of western and central North America, it is difficult to exclude the possibility that it could also have been present farther east prior to European colonization and landscape modifications. Introduction, 3rd paragraph:- The sentence “Population genetic structure plays considerable roles in evolution... the splitting of one species into two if the environments are markedly different”. In addition to adaptation, it may be important also to mention genetic drift, and subsequent development of incompatibilities between isolated populations.- The sentence starting with “The detection of genetic structure largely depends on the type and number of molecular markers examined”: Here you may want to mention explicitly the temporal resolution of markers with different mutation rates (as you have done in the Discussion).- “SNPs have become a popular and inexpensive tool”: perhaps “increasingly affordable” or similar would be better here?\n\nDiscussion, 3rd paragraph, sentence: “...cryptic genetic structure of Northeastern coyotes, especially as they have only inhabited the region for 30-80 years”. The author notes earlier in the text that coyotes in the northeast zone may descend from a few founders, which likely arrived via a northern route through Ontario, whereas a second colonization front occurred farther south through Ohio. Bierne et al. 2011 proposed that intrinsic (environment-independent) genetic incompatibilities may coincide with ecological boundaries, as these environmental transition zones often “trap” the tension zones resulting from e.g. assortative mating. Such processes might be influencing the contact zone between the southern and northern colonization fronts, and interact with the selective pressures associated with forested, agricultural and urban habitat, and deer density, noted in Monzón 2012 (PhD Dissertation).",
"responses": []
},
{
"id": "3944",
"date": "01 May 2014",
"name": "Linda Rutledge",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a very well-written, interesting article on utilizing highly-polymorphic SNPs to identify cryptic substructure in a population of eastern coyotes. The analyses are comprehensive (including Bayesian cluster analysis, principal components analysis, and Fst calculations) and the data do seem to support fine-scale population substructure. If it weren't for the extensive analyses I would find it very difficult to believe that 16 SNPs (even highly polymorphic ones) could find such fine-scale structure because it has been estimated that it take at least 15x more SNPs than microsatellites to identify structure in populations (Haasl & Payseur 2011, Heredity). Presumably the same clustering patterns would remain if the samples were analyzed at thousands of genome-wide SNPs that have not been selected for their polymorphism - i.e. presumably more, less polymorphic loci should show the same substructure.The biological/ecological reason for the clustering remains unclear (but that is clearly beyond the scope of the paper and is really quite difficult to tease apart). The author provides plausible explanations for the structure (e.g. \"merging of the two colonization fronts\"; possible deer or human density patters), but I wonder if social structure (i.e. family/pack groups) may contribute to the genetic patterns found. Overall, the manuscript reads very well and provides a rigorous analysis of a novel dataset that complements the authors other work.",
"responses": []
},
{
"id": "3945",
"date": "30 May 2014",
"name": "Mark Weckel",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFirst, thank you for the opportunity to review Javier Monzón’s article, “First regional evaluation of nuclear genetic diversity and population structure in northeastern coyotes (Canis latrans).”I found this to be a well-written, engaging, and thoughtful manuscript with important contributions to the use of SNPs in studies of population structure and in elucidating the genetic structure of the northeastern coyote. The author adeptly targets a need for using multiple markers across multiple individuals for proper inference. The author furthermore supports the use of the canine SNP array in studies of coyotes, following the recommendation of vonHoldt et al. (2011), while also asserting the need for addressing ascertainment bias prior to the use of SNPs in non-model organisms.I have no major reservations, rather a few minor comments regarding clarification of the methods along with some questions and considerations for future discussion and research.Methods:With such a large sample of high quality tissue available for the analysis, why were scats (n=6) included in the analysis? Were the scats important for adequate sampling across the study area? Fourteen of vonHoldt et al’s. (2011) coyote specimens were included in the initial selection of the 16 SNP panel used in subsequent analyses. Why were only 7 of vonHoldt et al.’s (2011) samples included in the analysis of diversity and structure? Results:Using the SNP panel, the author reports the observed and expected heterozygosity for the three putative population zones identified by Kays et al. (2010; Table 2). It would be interesting to see the Ho and He for the population clusters identify by the current STRUCTURE analysis (i.e., K =2 & K=3). What are the pairwise Fst values for two genetic clusters also supported by the STRUCTURE analysis? Discussion:There is ample evidence of genetic structure; however, I’m not sure that I would agree with the author’s suggestion that there is “some geographic structuring,” at least based on Figure 2c. The author does a thorough job of listing the factors and processes that may be involved in explaining a lack of geographic structure, as well as the discrepancy between the current nuclear analysis of population structure and the previous mtDNA analysis (Kays et al. 2010). A possible further explanation for the lack of geographic structuring could be that a SNP panel - with such high heterozygosity - may actually reflect older mutations that have had a longer time to move around the landscape via dispersal (see Morin et al. 2004). This might allow one to detect patterns of genetic structure without any obvious geographic patterns. The author also mentions, correctly, that based on the current state of knowledge, there is no sex-bias in coyote dispersal. I wonder if the author’s data set could be used to investigate this by factoring in sex and rerunning the analyses presented in this manuscript.",
"responses": []
}
] | 1
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https://f1000research.com/articles/3-66
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https://f1000research.com/articles/3-65/v1
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03 Mar 14
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{
"type": "Research Article",
"title": "Calcium content, in vitro digestibility, and bioaccessibility in leaves of spinach (Spinacia oleracea), sweet potato (Ipomea batatas), and drumstick tree (Moringa oleifera)",
"authors": [
"Jonathan C. Allen",
"Joseph Y. Issa",
"Weiting Cai",
"Joseph Y. Issa",
"Weiting Cai"
],
"abstract": "Low calcium intake, poor calcium absorption, excessive calcium losses, or some combination of these factors contribute to calcium deficiency diseases. Calcium insufficiency is associated with osteoporosis, hypertension and colon cancer among other diseases. For individuals who do not have access to milk and dairy products, do not tolerate them, or prefer not to consume them, plants may be alternative sources of calcium. However, calcium bioavailability may be low in plant foods because calcium forms complexes with oxalates, phytate and other competing minerals. The objective of this study was to compare the calcium content, digestibility and bioaccessibility in the leaves of spinach (Spinacia oleracea), sweet potato (Ipomea batatas), and drumstick tree (Moringa oleifera). Calcium content was analyzed in dry leaf powder by atomic absorption, followed by a two-stage in vitro digestion and dialysis against a mock serum solution to determine calcium digestibility and bioaccessibility. Moringa oleifera had higher calcium content than spinach and sweet potato leaves: 1.54±0.11% of dry matter for three Moringa samples (two African and one from India), and 0.99±0.001 and 1.06±0.001, respectively, in spinach and sweet potato leaves. The mean in vitro calcium digestibility was 1.62±0.08% in spinach, 3.4±0.68% in sweet potato leaves and 33.7±9.6% for Moringa. A dialysis system was designed to model bioaccessibility of calcium, revealing that bioaccessible calcium in sweet potato leaves was a non-significant 1.4 times higher and in Moringa was 9.2 to 19.4 times higher than in spinach. Therefore, the calcium contained in Moringa leaves does not appear to be associated with poorly bioavailable complexes such as oxalate. We confirmed previous reports that bioaccessibility of the calcium is low in spinach. These findings imply that increased utilization of Ipomea batatas and Moringa oleifera leaves might increase calcium intake in people in tropical and warm temperate regions where these plants grow, or these plants might become a valuable export crop.",
"keywords": [
"Calcium is an essential element responsible for bone strength and regulation of numerous functions in cells and tissues",
"such as muscle contraction and exocytosis. Approximately 1 to 2% of the human body is calcium. Perhaps 99% of that quantity is immobilized in bones and teeth. The remaining 1% is either ionized calcium or bound in soft tissues in a way that can more readily transfer among tissue compartments. Dietary calcium is absorbed by both active transport and passive diffusion across the intestinal mucosa. Active transport of calcium ions into enterocytes and out on the serosal side depends on the action of 1",
"25-dihydroxyvitamin D and its intestinal receptors. However",
"passive diffusion involves the movement of calcium between mucosal cells",
"is dependent on the luminal:serosal calcium concentration gradient",
"and may involve low-molecular weight calcium complexes in addition to calcium ions1."
],
"content": "Introduction\n\nCalcium is an essential element responsible for bone strength and regulation of numerous functions in cells and tissues, such as muscle contraction and exocytosis. Approximately 1 to 2% of the human body is calcium. Perhaps 99% of that quantity is immobilized in bones and teeth. The remaining 1% is either ionized calcium or bound in soft tissues in a way that can more readily transfer among tissue compartments. Dietary calcium is absorbed by both active transport and passive diffusion across the intestinal mucosa. Active transport of calcium ions into enterocytes and out on the serosal side depends on the action of 1,25-dihydroxyvitamin D and its intestinal receptors. However, passive diffusion involves the movement of calcium between mucosal cells, is dependent on the luminal:serosal calcium concentration gradient, and may involve low-molecular weight calcium complexes in addition to calcium ions1.\n\nCalcium deficiency diseases can result from low calcium intake (primary deficiency), poor calcium absorption (secondary deficiency), or excessive calcium losses. Calcium insufficiency in various life stages may be associated with osteoporosis, hypertension, colon cancer or other diseases. Individuals who do not have access, do not tolerate, or prefer not to consume milk and dairy products may prefer or require, plant sources of calcium. However, calcium bioavailability may be reduced in plant foods due to binding to oxalates or phytate, or to minerals competing for absorptive pathways. Benway and Weaver2 reported in vitro bioavailability for calcium of 0.3, 26 and 76% for spinach, wheat, and kale, respectively. Rapidly growing spinach forms high concentrations of insoluble calcium oxalate when the growth medium is high in available calcium3. High levels of oxalate were also observed in the leaves of sweet potato (Ipomea batatas)4. The formation of calcium oxalate crystals in numerous plant species may be beneficial to the plant but may be deleterious to human and animal consumers of plant tissues5. The leaves of the sweet potato are rarely consumed in Western countries but are a staple food or a delicacy in many African countries, including Zimbabwe, Zambia and Nigeria where the fresh leaves are cooked6. Different varieties are sometimes selected for leaf and root consumption. Purple and green sweet potato leaves in Tanzania were shown to contain significant quantities of nutritional and antinutritional compounds7. Purple sweet potato leaves that are commonly consumed in Asian countries have been investigated for the bioactivity of their polyphenol content8.\n\nMoringa oleifera (drumstick tree) grows in tropical and sub-tropical climates. In the tropical regions of Asia, Africa and South America, leaves from both naturally occurring and cultivated Moringa are eaten fresh. The fruits, the flowers and the immature pods of this tree are also edible and they are used to varying extents in traditional diets in many tropical and sub-tropical regions9. In addition, M. oleifera is used as a medicinal plant10. In traditional herbal medicine different ethnic groups use parts of the Moringa tree differently11. As a traditional food plant in Africa, M. oleifera has the potential to improve nutrition, boost food security, foster rural development, and support sustainable land care. In some parts of the world such as Senegal and Haiti, health workers have been treating malnutrition in small children, pregnant and nursing women with Moringa leaf powder12. The leaves of M. oleifera are a good source of protein, vitamin A, B and C and minerals such as calcium and iron13.\n\nOduro et al.14 found the calcium content in M. oleifera to average 2009 mg per 100 g sample. According to Price12, M. oleifera fresh pods and leaves and dried leaf powder had 30 mg, 440 mg and 2003 mg per 100 g of edible portion, respectively. A study on the nutritional and functional properties of M. oleifera leaves found that fresh mature leaves and fresh young shoots had 454 ± 63 mg and 82 ± 31 mg of calcium per 100 g of sample, respectively15. A previous study on in vitro iron bioavailability found that boiling the fresh leaves and dried powder of M. oleifera enhanced the in vitro iron bioavailability by 3.5 and 3 times, respectively16. The USDA Nutrient Database lists 152 mg of calcium per 100 g of boiled drumstick leaves, 136 mg of calcium per 100 g of boiled spinach, and 33 mg of calcium per 100 g of boiled sweet potato leaves (http://ndb.nal.usda.gov/ndb/search/list). Although the literature shows high calcium content in these plant materials, there are fewer data on the calcium bioavailability. The study reported here was therefore designed to investigate the potential calcium bioavailability in spinach (Spinacia oleracea), sweet potato (Ipomea batatas), and drumstick tree (Moringa oleifera) leaves by measuring bioaccessibility using an in vitro simulated digestive system. True bioavailability cannot be determined from an in vitro system because it does not distinguish between forms of calcium that can be absorbed by regulated active transport processes and passive absorption, nor between retention and rapid renal excretion.\n\n\nMaterials and methods\n\nGreen and mature Moringa leaf samples were collected from several trees (about 3 trees in each area) in low-lying hot areas of Malawi where it grows naturally and the conditions are favorable for its growth. In these areas, average temperatures are 30°C. The temperature can reach up to 40°C in the dry season. The sample collection areas, Lilongwe and Karonga, are along the lake shore of Lake Malawi and Shire River. These areas are generally hot and flat with sandy loam soils. Another Moringa oleifera sample was bought in USA from an Internet distributor (MoringaSource.com). This commercial product was originally imported from India and sold in the United States as powdered Moringa oleifera leaf packed in aluminum foil bags. Fresh sweet potato leaves (Covington variety) were harvested at the North Carolina State University Central Crops Research Station, Clayton, NC, washed and stored at -20°C prior to use in this study. Fresh spinach leaves were bought from a local grocery store in Raleigh, NC, USA. All leaf samples were air dried, powdered in a mortar, and packaged in plastic bags. All samples were stored at room temperature and analyzed together at North Carolina State University.\n\nTotal calcium in the samples was determined by atomic absorption spectrophotometry (AAS). Powdered dry leaf samples were subjected to dry-ashing before the determination of calcium. A 0.1 g sample of each material was weighed and ashed at 525°C for 8 hours. The ash was dissolved with 0.1% HCl and 0.5% lanthanum oxide (La2O3) followed by AAS analysis to determine calcium content.\n\nIn vitro digestion was used to determine calcium digestibility. The leaf powders were subjected to a simulated gastric phase followed by intestinal phase digestion, modified from the method of Fogleman et al.17. Pepsin enzyme from porcine gastric mucosa (Pcode 1000848645 Sigma-Aldrich, St. Louis, MO, USA) and hydrochloric acid were used in the gastric phase digestion. A 0.1 g sample of leaf powder was combined with 0.25 mL of pepsin solution (40 mg pepsin/mL of 0.1 M HCl), and pH was then adjusted to 2.0 with additional HCl. The mixture was incubated in a shaking water bath at 37°C for 120 strokes per minute for 2 hours, and then placed on ice for 10 minutes. pH was raised to 7 by adding 1 M NaHCO3. This gastric digestion was followed by pancreatic enzymes (pancreatin from porcine pancreas, P1750, Sigma-Aldrich) and porcine bile extract (B8631, Sigma Aldrich) solution in the intestinal phase digestion. The enzyme stock contained 0.05 g pancreatin and 0.3 g bile extract in 25 mL 0.1 M NaHCO3. A 1.25 mL portion of the pancreatin-bile extract stock solution was added to each digested leaf sample, with additional incubation in a shaking water bath at 37°C for 120 strokes per minute for 2 hours. The mixtures were placed on ice for 10 minutes and adjusted to pH 7.2 by addition of 0.5 M NaOH. All samples were brought to a common volume with distilled water and filtered through Whatman No.4 filter paper. The solution was diluted appropriately with 0.1 M HCl and 0.5% lanthanum oxide (La2O3) followed by AAS analysis to determine calcium content.\n\nThe bioaccessibility of calcium in the samples was determined by simulated in vitro absorption of calcium by using dialysis tubes. This involved the passive trans-membrane diffusion of calcium ions from one medium to another. A 10 mL portion of each sample (previously digested) was loaded inside of the dialysis tubing in a Floatalyzer™ G2 apparatus (Spectrum Laboratories, Inc., Rancho Dominquez, CA) by pipettes. The outside compartment of the dialysis tubing container for each sample was loaded with 25 mL of 0.9% NaCl solution with 0.1% bovine serum albumin (fraction V, Sigma-Aldrich). The samples were then dialyzed at room temperature for 24 hours. The volumes of the solution outside and inside the dialysis tubing were measured and emptied in separate test tubes for calcium analysis. Calcium was analyzed by atomic absorption, and the calcium that was able to pass to the outside of the dialysis tubing corresponded to the bioaccessible calcium.\n\nBioaccessible calcium was calculated as the product of the calcium concentration in the external fluid and the combined fluid volume inside and outside the Floatalyzer™. The bioaccessibility of calcium was the amount of dialyzable calcium expressed as a percentage of total calcium in the sample. Calcium digestibility was calculated as the quantity of calcium in the filtrate divided by the calcium content in the original powdered leaf sample.\n\nSAS and JMP software (SAS, Inc., Cary, NC) were used to statistically analyze the data. The Fit X by Y one-way analysis of variance subroutine was used to see the relationship among the two Moringa oleifera samples from Malawi, the Moringa oleifera sample from India, sweet potato leaves, and spinach in terms of calcium content, digestibility and bioaccessibility. The entire digestion procedure was replicated three times, using duplicate or triplicate samples in each replicate. Two samples of spinach, three samples of sweet potato leaves, and one sample from Moringa #4 could not be included in the data analysis, because the atomic absorption spectrophotometer did not report a meaningful calcium concentration possibly due to consumption of the sample in the analyzer. Tukey’s Studentized Range (TSR) test was used to determine whether or not significant differences existed in the mean values of the sample calcium content, digestibility and bioaccessibility at P ≤ 0.05.\n\n\nResults\n\nCalcium content and digestibility of Moringa oleifera, spinach and sweet potato leaves are provided in Table 1.\n\n1Samples: M3 = Moringa sample from India, M4 = first Moringa sample from Karonga, Malawi, M5 = second Moringa sample from Lilongwe, Malawi, S = spinach, SP = sweet potato leaves.\n\nabcFor the dependent variable within each column, the calcium sources (sample) in rows not connected by the same letter are significantly different from each other as determined by ANOVA and the Tukey-Kramer HSD test with α=0.05.\n\nCalcium from the samples was expressed as percentage of the dry matter in the three Moringa oleifera and the spinach and sweet potato leaves. The results indicated that Moringa oleifera leaves had a higher percentage of calcium than spinach and sweet potato leaves. However, among the three Moringa oleifera samples, there was a significant difference in the percentage calcium content (P<0.05) using Tukey’s Studentized Range Test at α=0.05. The mean percentage calcium ± SEM was 1.67 ± 0.001 1.44 ± 0.018 and 1.55 ± 0.00 for Moringa oleifera samples from India (M3), from Karonga, Malawi (M4) and another sample from Lilongwe, Malawi (M5), respectively. The mean percentage calcium in spinach and sweet potato leaves was 0.99 ± 0.0003 and 1.06 ± 0.001, respectively.\n\nThe digestible calcium from Moringa oleifera samples and the control samples, spinach and sweet potato leaves are shown in Table 1.\n\nDigestibility of calcium from the leaf powder was expressed as percentage of the calcium in dry matter that was solubilized by the digestion procedure. The digestibility results indicated that the three Moringa oleifera samples had higher digestible calcium than spinach and sweet potato leaves. Using Tukey’s Studentized Range Test at α=0.05, there was no significant difference in total digestible calcium between the two samples from Malawi, M4 and M5. However, there was a significant difference in percentage of digestible calcium between Moringa oleifera samples from India and Malawi (P<0.05). The mean (± SEM) percentage of digestible calcium was 22.77 ± 1.04, 46.57 ± 8.34 and 37.69 ± 0.76% for Moringa oleifera samples from India (M3), from Malawi (M4) and the second sample from Malawi (M5), respectively. The mean percentage digestible calcium in spinach and sweet potato leaves were 1.35 ± 0.21, and 3.79 ± 0.21%, respectively.\n\nThe bioaccessibility of calcium from Moringa oleifera samples and the control samples, spinach and sweet potato leaves, are provided in Table 2.\n\nValues represent means ± SEM of the number of replicates shown in parentheses.\n\n1Samples: M3 = Moringa sample from India, M4 = first Moringa sample from Karonga, Malawi, M5 = second Moringa sample from Lilongwe, Malawi, S = spinach, SP = sweet potato leaves.\n\nabcFor the dependent variable within each column, the calcium sources (sample) in rows not connected by the same letter are significantly different from each other as determined by ANOVA and the Tukey-Kramer HSD test with α=0.05.\n\nBioaccessibility of calcium from the samples was expressed as percentage of the dry matter. Bioaccessible calcium was equivalent to dialyzable calcium. The bioaccessibility results from spinach and sweet potato leaves and the three Moringa oleifera samples indicated that Moringa oleifera had higher dialyzed calcium than spinach and sweet potato leaves. Using Tukey’s Studentized Range Test at α=0.05, there was no significant difference in total bioaccessible calcium between the two Moringa oleifera samples from Malawi, but these had more bioaccessible calcium than the Moringa oleifera sample from India. Also, the percentage bioaccessible calcium in all Moringa oleifera samples was greater than that in spinach and sweet potato (P<0.05).\n\nTable 2 also shows the percentage of the digestible calcium that was dialyzable. Despite the substantial variation in the data, the table shows that a greater amount of the digestible calcium from spinach was dialyzable compared with the Moringa samples or sweet potato leaves, suggesting that the calcium oxalate in spinach is largely indigestible and remains in the initial filtrate, whereas the complexes that can be filtered can also pass through the dialysis membranes. The filtration step in the digestibility assay retained particles greater than 20–25 µm and allowed calcium in smaller particles to pass through the fluid that was used in the dialysis procedure. The dialysis membrane only allows molecular complexes from 500 to 1000 Da to go through and be counted in the “bioaccessible” fraction. The bioaccessible fraction was 45% of the digestible calcium fraction for spinach, but only 22 to 32% for sweet potato and Moringa leaves (Table 2).\n\n\nDiscussion\n\nIn vitro methods for assessing the bioaccessibility of essential minerals are widely used because they are simple, fast, and inexpensive. Teixeira et al.18 recently measured in vitro digestibility of protein fractions in Moringa oleifera with different protein extraction pretreatments. The protein digestibility was relatively low, in contrast to the calcium bioaccessibility data we present here. Kamchan et al.19 used an in vitro method to study the bioavailability of calcium in vegetables, legumes and seeds; in their study, fresh vegetables were prepared by blanching in boiling deionized water for three minutes followed by homogenization in a food processor whereas seed and pod samples were cooked and homogenized in an electric blender and all the samples were stored in polyethylene bottles at -20°C before analysis. In contrast, in our study Moringa oleifera leaf samples were air dried, pounded into powder and stored in polyethylene bags before being subjected to chemical analysis. We assessed calcium content, digestibility and bioaccessibility in Moringa oleifera leaf powder from Malawi and India, along with spinach and sweet potato leaf powder. There was a statistically significant difference in the percentage calcium content from the Moringa oleifera samples from India and the two samples from Malawi. This may be due to differences in the growing conditions because some soils have higher amounts of calcium. The amount of digestible calcium in the two Moringa oleifera samples from Malawi was statistically similar, and the samples from Malawi differed in this respect from the sample from India. This may be due to similar growing conditions for the samples from Malawi, which may differ from the growing conditions in India, or it may be due to genetic drift in the Moringa genome20. Dialyzed calcium was significantly different between the two samples from Malawi and one sample from India when a Tukey’s Studentized Range Test with α=0.05 was used. The dialyzed calcium from all the Moringa oleifera samples was significantly higher than spinach and sweet potato leaves. The bioaccessible calcium in spinach in this study was similar to the calcium bioavailability of spinach reported by Benway and Weaver2. In vitro methods used to study calcium dialyzability from different vegetables revealed percentage dialyzable calcium in kale, celery and Chinese cabbage to be 38.9 ± 2.1, 36.2 ± 4.1 and 32.2 ± 4.6, respectively19. This may be due to different chemical and biological compositions of these plants that inhibit absorption of calcium, such as phytate, oxalate, and dietary fiber, or due to different calculation methods. Ezeike et al.21 were able to extract 0.28 mg of oxalate per gram of dry matter from Moringa oleifera leaves, and cited data to calculate that spinach contains 100 mg oxalate per gram of dry matter. Oxalic acid in fresh sweet potato leaves was 3–5 mg per gram fresh weight, or about 16–27 mg per gram of dry matter22. The results from the study presented here are different from those found by Kamchan et al.19 probably because different species of plants and different sample preparatory methods were used. An in vitro iron bioavailability study16 found that boiling the fresh leaves and dried powder of Moringa oleifera enhanced the in vitro iron bioavailability by 3.5 and 3 times, respectively. Whether a similar preparatory method would enhance the calcium bioaccessibility requires further study.\n\nPrice12 found calcium content in Moringa oleifera pods, leaves and leaf powder to be 30 mg, 440 mg and 2003 mg per 100 g of edible portion, respectively. We found in our study that the percentage calcium content in powdered Moringa oleifera leaves is 1.67 ± 0.001 for samples from India (M3), 1.44 ± 0.018 for samples from Karonga, Malawi (M4), and 1.55 ± 0.00, for samples from Lilongwe, Malawi (M5). If we translate these results per 100 g edible sample, it yields 1.67 g (1670 mg), 1.44 g (1440 mg) and 1.55 g (1550 mg) for M3, M4, and M5, respectively, averaging 1553 mg/100 g. These results are not very different from that of Price12 for the leaf powder, which was 2003 mg per 100 g sample. The current calcium RDA for pregnant and lactating women is 1300 mg/day for women aged 14–18 years and 1000 mg/day for women aged 19–50 years23. A dietary intake study undertaken in Malawi, Africa, showed that calcium intake from women was lower than the recommended intake with average intake of 620 mg/day for non-lactating women and 622 mg/day for lactating women24. Another nutrient intake study conducted in South Africa in three ethnic groups (black, white and mixed ancestry subjects) found that mean dietary calcium intake was higher in white subjects than in black and mixed ancestry subjects; however, calcium intake was low in all the groups with about half of the RDA of 1000 mg/day25. If the dietary intake of Moringa oleifera is increased, calcium intake could also be tremendously increased, especially in developing countries such as Malawi, where growing these trees requires little care. This can be achieved by promoting plantation of these trees in areas where they grow easily.\n\nAccording to our study, only 64 g of dried Moringa leaf powder would supply the 1000 mg/day RDA. From this intake, 357 mg of calcium are estimated to be digestible, and 93 mg of calcium are estimated to be bioaccessible, or available for absorption, based on the average of the Moringa oleifera plant samples tested in our study. This study also shows sweet potato leaves to be a good source of calcium but more similar to spinach than to Moringa oleifera leaves.\n\n\nConclusion\n\nThe percentage dialyzed or bioaccessible calcium is higher in Moringa oleifera leaves than other vegetable sources such as spinach and sweet potato leaves. Percentage dialyzed calcium of Moringa oleifera grown in Malawi was slightly higher than the sample grown in India. Because there is evidence from related research that percentage calcium dialyzability differs in different foods, especially vegetable foods because different inhibitory components may be present, additional research can be conducted to find out the inhibitory components in Moringa oleifera leaves. Such inhibitory components may be the contributing factors for the differences in percentage bioaccessible calcium in different plant leaf samples.\n\n\nData availability\n\nfigshare: Data of calcium content, digestibility and bioaccessibility of Moringa oleifera, spinach and sweet potato leaves http://dx.doi.org/10.6084/m9.figshare.94348226",
"appendix": "Author contributions\n\n\n\nJCA was responsible for editing the manuscript, experimental design and final data analysis. JYI proposed the research area, developed methods and wrote the first draft of the manuscript. YC conducted the laboratory analyses and prepared the data for analysis.\n\n\nCompeting interests\n\n\n\nThe authors have no competing interests in the publication of this paper.\n\n\nGrant information\n\nThis research was supported by grants from USAID – UILTCB-Malawi in a subcontract from Michigan State University, and by the North Carolina Sweet Potato Commission to author JCA.\n\n\nAcknowledgements\n\nThanks to Ruth Watkins, the laboratory manager of the Nutrition Technical Services Lab, Food, Bioprocessing and Nutrition Sciences Department at NC State University and Dr April D. Fogleman for their assistance and guidance in conducting the laboratory analysis, and to Lusungu G. Mwaungulu for collection of Moringa leaf. Thanks to Dr Van-Den Truong and Katie Maloney for collection and preparation of sweet potato leaves. This research was conducted and submitted in a thesis for partial fulfillment of the Master of Science degree for author JYI at North Carolina State University.\n\n\nReferences\n\nBronner F, Pansu D: Nutritional aspects of calcium absorption. J Nutr. 1999; 129(1): 9–12. PubMed Abstract\n\nBenway DA, Weaver CM: Assessing chemical form of calcium in wheat, spinach, and kale. J Food Sci. 1993; 58(3): 605–608. Publisher Full Text\n\nZhang Y, Li Y, Wei J, et al.: Effects of nitrogen and calcium nutrition on oxalate contents, forms, and distribution in spinach. J Plant Nutr. 2009; 32(12): 2123–2139. Publisher Full Text\n\nMbaeyi-Nwaoha IE, Emejulu VN: Evaluation of phytochemical composition and antimicrobial activity of sweet potato (Ipomoea batatas) leaf. Pakistan J Nutr. 2013; 12(6): 575–586. Reference Source\n\nFranceschi VR, Nakata PA: Calcium oxalate in plants: formation and function. 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Reference Source\n\nYang RY, Chang LC, Hsu JC: Nutritional and Functional Properties of Moringa Leaves-From Germplasm, to Plant, to Food, to Health. Accra, Ghana, November 16–18, 2006. Reference Source\n\nYang RY, Tsou STS, Lee TC: Effect of cooking on in vitro iron bioavailability of various vegetables. In TC Lee and CT Ho (eds.), Bioactive compoundsin foods: effect of processing and storage. American Chemical Society, Washington, D.C. 2002; p130–142. Publisher Full Text\n\nFogleman AD, Cohen RS, Sakamoto P, et al.: Effect of added calcium, phosphorus, and infant formula on calcium and phosphorus dialyzability in preterm donor human milk. J Ped Gastroenterol Nutr. 2012; 55(4): 390–397. PubMed Abstract | Publisher Full Text\n\nTeixeira EM, Barbieri Carvalho MR, Neves VA, et al.: Chemical characteristics and fractionation of proteins from Moringa oleifera Lam. leaves. Food Chem. 2014; 147: 51–54. Publisher Full Text\n\nKamchan A, Puwastien P, Sirichakwal PP, et al.: In vitro calcium bioavailability of vegetables legumes and seeds. J Food Comp Anal. 2004; 17(3-4): 311–320. Publisher Full Text\n\nMuluvi GM, Sprent JI, Soranzo N, et al.: Amplified fragment length polymorphism (AFLP) analysis of genetic variation in Moringa oleifera Lam. Mol Ecol. 1999; 8(3): 463–470. PubMed Abstract | Publisher Full Text\n\nEzeike CO, Aguzue OC, Thomas SA: Effect of brewing time and temperature on the release of manganese and oxalate from Lipton tea and Azadirachta indica (Neem), Phyllanthus amarus and Moringa oleifera blended leaves. J Appl Sci Environ Management. 2011; 15(1): 175–177. Publisher Full Text\n\nAlmazan AM: Antinutritional factors in sweet potato greens. J Food Comp Analysis. 1995; 8(4): 363–368. Publisher Full Text\n\nIOM (Institute of Medicine). Dietary Reference Intakes for Calcium and Vitamin D. Washington DC: National Academy Press. 2011. PubMed Abstract\n\nHullund J, Hatloy A, Benesi I, et al.: Snacks are important for fat and vitamin intakes among rural African women: a cross-sectional study from Malawi. Eur J Clin Nutr. 2008; 62(7): 866–871. PubMed Abstract | Publisher Full Text\n\nCharlton KE, Steyn K, Levitt NS, et al.: Diet and blood pressure in South Africa: intake of foods containing sodium, potassium, and magnesium in three ethnic groups. Nutrition. 2005; 21(1): 39–50. PubMed Abstract | Publisher Full Text\n\nAllen JC, Issa JY, Cai W: Data of calcium content, digestibility and bioaccessibility of Moringa oleifera spinach and sweet potato leaves. Figshare. 2014. Data Source"
}
|
[
{
"id": "4129",
"date": "14 Mar 2014",
"name": "Mark L Failla",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article by Prof. Allen and associates addresses the important issue of inadequate intake of calcium by those who do not have access to, or the ability/willingness to consume milk and dairy products. The possibility that leaves from three common plant foods in rural areas or developing countries can serve as an alternative source of calcium merits investigation. The results provide novel information regarding calcium content and dialyzability in dried powders of leaves from spinach, sweet potato and drumstick. These results will be of interest to nutrition and food scientists, horticulturists and food processors. I have several specific suggestions for enhancing the presentation: Abstract: Use of the term “mock serum solution” is a stretch for the presence of BSA outside the dialysis bag. Plasma contains numerous proteins and low molecular weight compounds that can bind a portion of plasma calcium. The rationale for use of BSA should be added to the manuscript e.g., serves as a “sink” for calcium diffusing into the outer solution, some calcium in plasma and some of the divalent metal in plasma complexes with albumin. I suggest that albumin be substituted for mock serum solution. Abstract: middle of paragraph 2. “Moringa oleifera had higher calcium content…..”. Add % after 0.99 and 1.06. Abstract: paragraph 2. “A dialysis system was designed...” “1.4 times higher” - than what? The term bioaccessibility generally refers to availability for uptake by, or paracellular transfer across the epithelial cell barrier. Passive diffusion across the barrier represents a portion of calcium absorption; particularly when the lumen concentration of soluble calcium is relatively high. As the investigators determined the amount of dialyzable calcium I suggest that it is not appropriate to use the term bioaccessibility in the title and throughout the manuscript. The great majority of papers reporting results for electrolytes and low molecular weight organic compounds obtained using in vitro digestion coupled with dialysis refer to dialyzability; not bioaccessibility.\n\nResults: The text and tabular data are quite redundant with means and SD from the table stated in text. The text can be less specific, i.e., delete the numerical details. Discussion: The translation of the in vitro results to the amount of calcium provided by powdered leaves is excellent. However, individuals in poor rural areas of developing countries generally consume boiled leaves from the three plants. How much calcium would 1 serving of leaves from each plant provide? Might a distinction of use of these leaves in a dried form as a supplement vs as an actual food, provide a more balanced viewpoint?",
"responses": []
},
{
"id": "4973",
"date": "06 Jun 2014",
"name": "Antonio Cilla",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript of Prof. Allen and colleagues highlights the potential use of leaves of spinach, sweet potato and drumstick tree as an alternative to other calcium dietary sources such as milk and dairy products. In addition they measure the bioaccessibility, as a prior step of bioavailability, to check if these leaves can be a potential source of available calcium in the geographic areas in which they are grown or cultivated easily and are a cost-effective approach for people in these zones. Research in this field is very important in food science and nutrition since results of this kind provide a scientific rationale, as mentioned by the authors, to improve nutrition, boost food security, foster rural development and support sustainable land care. However, I suggest some modifications to improve the study.1. Bioaccessibility (defined as a fraction of a compound that is released from its food matrix within the gastrointestinal tract and that becomes available for absorption) is a term which comprises both solubility and dialyzability (Fernández-García et al. Nutr. Res. 2009, 29, 751-760). The results, as demonstrated in many other publications, should be expressed as % bioaccessibility (understood as % dialysis or % solubility). See examples (Perales et al. J. Agric. Food Chem. 2005, 53, 3721-3726 or Galán et al. J. Agric. Food Sci. 2014, 94, 515-521). This should be applied to the entire text.Abstract:2. Second Paragraph - “revealing that bioaccessible calcium in sweet potato leaves was a non-significant 1.4 times higher ...” – what is this in comparison to?Introduction:3. Polyphenols can also bind calcium and other divalent minerals and thus reduce their bioavailability. – This should be included in the introduction.4. You need to specify that there is fewer data on calcium bioavailability and bioaccessibility.Materials and methods:5. Evaluation of calcium bioaccessibility: the authors should indicate in the last sentence that this \"...corresponded to the bioaccessible (dialyzable) calcium.\"Results:6. As reported by the previous reviewer (Dr. Failla) please do not duplicate table results in the text.7. In table 2 the nomenclature in the 2nd column needs to be changed to Ca bioaccessibility (% of total Ca).8. How are the authors sure that the complexes formed are with oxalate and not with phytate and/or polyphenols? Discussion:9. I do not understand why authors compare bioaccessibility of calcium with that of proteins from the study of Teixeira et al.10. The authors introduce the study of Kamchan et al. but do not include anything about this study’s findings or compare them with their own results.11. Sometimes the terms bioavailability and bioaccessibility are used indistinctly, but they are not the same. The authors are comparing bioaccessibility with bioaccessibility (solubility) and not bioavailability in the work of Benway and Weaver. This must be modified.",
"responses": []
},
{
"id": "4972",
"date": "07 Jul 2014",
"name": "Linda-Maria Dimitrova Mårtensson",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article “Calcium content, in vitro digestibility, and bioaccessibility in leaves of spinach (Spinacia oleracea), sweet potato (Ipomea batatas), and drumstick tree (Moringa oleifera)” by Allen et al. concerns important issues of Calcium intake when dairy products are replaced, irrespective of the cause behind the need or desire to the substitution. The findings are valuable for global nutritional security as well as for further research. However, I find numerous ways to improve the publication in its current format. Overall suggestions for improvement:The use of Latin name and common name should be done in a more consistent style throughout the entire article. Latin should preferably be used as the standard throughout the article and for all species/crops in the study. The number of significant digits should also be consistent throughout the article: e.g. 0.99 has two but 1.54 has three. Use of abbreviations: the first time a term is used is should be spelled out and then can be written as an abbreviation in brackets after. In the continued text use the abbreviation only, e.g. HCl is used before the spelled out hydrochloric acid. Specific suggestions for improvement:AbstractThe sentence “… revealing that bioaccessible calcium in sweet potato leaves was a non-significant 1.4 times higher and in Moringa was 9.2 to 19.4 times higher than in spinach.” must be clarified. The results provide more information than this and can be summarized in the abstract.Add % after results in second paragraph.Second paragraph: “…1.4 times higher than…” – what are you comparing to?Introduction – no commentsMaterials and methods Page 4, line 18: “common volume” - The authors are advised to give the actual volume here.Title “Calculation of calcium bioaccessibility” should include digestibility.Keep the order in presentation - e.g. digestibility should be presented first in “Calculation of calcium bioaccessibility” to follow earlier flow.Page 4, second column: “because the atomic absorption spectrophotometer…” - Clarify this.P value and alfa are mixed throughout the results section, which needs to be either clarified or changed. ResultsBoth starting sentences in the “Results” section are misplaced. They would perhaps be better off in the materials and methods section?Table 1: hyperlinks (and explanations?) are missing for indications d and e.Results need not be repeated in result text if already presented in tables.Results need not to be repeated in the discussion. DiscussionSoil chemistry is reflected upon but information/data is lacking – Is it possible to supplement?Results should not be repeated here.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-65
|
https://f1000research.com/articles/2-240/v1
|
12 Nov 13
|
{
"type": "Short Research Article",
"title": "Accumulation of oocytes and/or embryos by vitrification: a new strategy for managing poor responder patients undergoing pre implantation diagnosis",
"authors": [
"Alexia Chatziparasidou",
"Martine Nijs",
"Martha Moisidou",
"Oraiopoulou Chara",
"Christina Ioakeimidou",
"Christos Pappas",
"Nicos Christoforidis",
"Alexia Chatziparasidou",
"Martha Moisidou",
"Oraiopoulou Chara",
"Christina Ioakeimidou",
"Christos Pappas",
"Nicos Christoforidis"
],
"abstract": "Background: Low (or poor) responder patients are women who require large doses of stimulation medications and produce less than an optimal number of oocytes during IVF cycles. Low responder patients produce few oocytes and embryos, which significantly reduces their chances for success in a preimplantation genetic diagnosis (PGD) cycle. Accumulation of vitrified oocytes or embryos before the actual PGD cycle is a possible strategy that might increase patient’s chances for a healthy pregnancy.Aim of the study: This retrospective study evaluates the efficacy of a PGD program in low responder patients after repeated ovarian stimulation cycles with cumulative vitrification of oocytes and embryos.Methods: Over a period of 30 months, 13 patients entering the PGD program were identified as poor responders after their first ovarian stimulation. These patients started a PGD cycle for one of the following indications: history of recurrent implantation failure (n=1), cystic fibrosis (n=1), X-linked microtubular myopathy (n=1), recurrent miscarriages (n=5), Duchene muscular dystrophy (n=1), chromosomal translocation (n=1) and high sperm aneuploidy (n=1). After multiple ovarian hormonal stimulations patients had either all mature oocytes (Group A; 3 patients) or all of their day 2 embryos vitrified (group B; 10 patients). Mean total number of oocyte collections per patient was 2.3 (range: 2 - 5 cycles).Results: In the actual PGD cycle, all vitrified oocytes from group A patients were warmed and underwent intra cytoplasmic sperm injection (ICSI) followed by culture up to day 3. For group B patients all vitrified day 2 embryos were warmed and cultured overnight. On day 3 of culture, all embryos from Group A and B had blastomere biopsy followed by genetic analysis. In group A, 20 embryos were found suitable for biopsy and genetic analysis; at least one healthy embryo was available for transfer for each patient. For group B, 72 embryos in total were available for biopsy and PGD. All patients, except one, had at least one healthy day 5 embryo for transfer (mean number of 2.1 embryos per transfer). Nine patients had a clinical pregnancy; 7 patients delivered a healthy baby.Conclusion: Low responder patients entering a PGD program might increase their chances for a healthy pregnancy by repeat ovarian stimulation in combination with cumulative oocyte or embryo vitrification.",
"keywords": [
"Low responder patients undergoing hormonal stimulation for an IVF or ICSI treatment have a reduced potential to produce an adequate number of oocytes and hence also embryos1",
"2. Especially for patients seeking a healthy pregnancy through preimplantation genetic diagnosis (PGD)",
"this low production of oocytes and embryo(s) in one cycle will significantly reduce their chances of success. Multiple consecutive ovarian stimulation cycles combined with serial vitrification of oocytes and embryos obtained before the actual PGD could be an option to increase the chances for these patients. Until now",
"only one successful case report has been presented by Chung et al.3 where a normal birth was obtained after serial vitrification of oocytes from 5 consecutive ovarian stimulation cycles for a patient carrying reciprocal translocations."
],
"content": "Introduction\n\nLow responder patients undergoing hormonal stimulation for an IVF or ICSI treatment have a reduced potential to produce an adequate number of oocytes and hence also embryos1,2. Especially for patients seeking a healthy pregnancy through preimplantation genetic diagnosis (PGD), this low production of oocytes and embryo(s) in one cycle will significantly reduce their chances of success. Multiple consecutive ovarian stimulation cycles combined with serial vitrification of oocytes and embryos obtained before the actual PGD could be an option to increase the chances for these patients. Until now, only one successful case report has been presented by Chung et al.3 where a normal birth was obtained after serial vitrification of oocytes from 5 consecutive ovarian stimulation cycles for a patient carrying reciprocal translocations.\n\nThis retrospective cohort study evaluates the efficacy of a PGD program in low responder patients after repeated ovarian stimulation and accumulation of vitrified oocytes or embryos before genetic analysis, in combination with PGD on embryos obtained from a fresh ICSI cycle.\n\n\nMethods\n\nThis retrospective cohort study was performed over a 30 month-period (2011–2013) at Embryolab, a private fertility treatment centre in Thessaloniki, Greece.\n\nDuring the 30 month period 13 patients of those entering the PGD program showed to be poor responders. PGD patients with more than 6 oocytes or 5 embryos from their first fresh PGD cycle were excluded from the study. Study patients started a PGD cycle for one of the following indications: history of recurrent implantation failure (n=1), cystic fibrosis (n=1), X-linked microtubular myopathy (n=1), recurrent miscarriages (n=5), Duchene muscular dystrophy (n=1), chromosomal translocation (n=1) and high sperm aneuploidy (n=1).\n\nPatient’s ovarian stimulation protocol consisted of a standard down-regulation protocol or antagonist protocol4. Hormonal stimulation treatment showed these patients to be poor responders and very few oocytes could be harvested at the time of the first oocyte collection. Following counseling, couples opted for serial vitrification of oocytes (group A) or embryos (group B) from repeat ovarian stimulation cycles. Allocation to either group was based on the outcome of a medical counseling session with the patient. One to two extra hormonal stimulation cycles were initiated to obtain an accumulated minimum of 6 oocytes (group A) or alternatively of 5 embryos (group B) for each patient.\n\nOocyte collection was carried out 36 hours post-hCG administration. Fresh semen samples were prepared by density gradient centrifugation and one wash step (Quinn's Advantage Sperm Washing Medium, Sage). ICSI was performed according to standard procedures5. Oocytes were checked for presence of 2 pronuclei 18–22 hours post oocyte collection. Fertilised oocytes were group-cultured in 0.7 ml droplets (Cleavage medium, Sage) and embryo quality was checked daily under a microscope using a standard protocol10. Oocytes and day 2 embryos were vitrified and warmed using the methods described by Kuwayama et al. (Cryotop, Cryotec, http://cryotech-japan.jp/method/warming_Protocol.htm)6 and stored in liquid nitrogen.\n\nEmbryos were biopsied on day 3 of development. Three different genetic techniques were applied, depending on the indication: fluorescent in situ hybridization (FISH)7 was used for patients suffering from X-linked microtubular myopathy, Duchene muscular dystrophy, high sperm aneuploidy or recurrent implantation failure; polymerase chain reaction (PCR)8 was the technique used for patients at risk for offspring with cystic fibrosis. Array complete genome hybridisation techniques (aCGH)9 were applied for patients at risk for recurrent miscarriage or for reciprocal translocations. Biopsied embryos were cultured individually in 50 μl droplets under oil (washed sterile oil, Sage, USA) until day 5 for transfer (Cleavage medium, Sage, USA). Embryo quality was checked daily under the microscope according to a standard protocol10.\n\nEmbryo(s) were transferred under abdominal ultrasound guidance (Logic 400 MD) to the patient in 0.1 ml of medium (Cleavage medium, Sage) using a Wallace- (Smithsor Labotect soft catheter (Genetec). Clinical pregnancy was defined as the presence of a gestational sac with fetal heartbeat by ultrasound imaging at 8–10 weeks after embryo transfer.\n\nThe IVF laboratory at Embryolab has ISO 9001:2000 accreditation (2007) and has been assessed in accordance to ISO 15189-2007.\n\nGiven the retrospective nature and lack of identifiable health data used in the study, no institutional review board approval was needed. Patients signed an informed consent before the start of the treatment.\n\n\nResults\n\nDuring the 30 month study period, 13 patients were shown to be poor responders because of failure to produce a sufficient number of oocytes or embryos to continue their PGD analysis (< 6 mature oocytes or < 5 embryos on day 2). Mean age of the patients was 35.2 years (range: 31–41 years, SD: 3.4). After medical counseling all 13 patients agreed to accumulate their oocytes or embryos by vitrification, and hence underwent repeat hormonal stimulations and oocyte collections (mean: 2.3; range 2–5 stimulations) until a sufficient number was stored (< 6 mature oocytes or < 5 embryos on day 2). Mean total number of oocyte collections per patient (cycles) was 2.3 (range: 2–5 cycles). Details on laboratory and clinical outcomes are listed in Table 1. On day 3 of culture, a total of 92 embryos were biopsied and diagnosed genetically. In total, 33 embryos were diagnosed as being healthy (35,8%), and a mean average number of 2.1 embryos per patient were transferred. Eleven remaining normal embryos were vitrified post-biopsy. One patient with a history of repeated failure of implantation had no healthy embryos available for transfer. Twelve out of 13 patients had an embryo transfer of a healthy embryo (92,3%) and 9 patients had a clinical pregnancy (75% clinical pregnancy rate in patients with embryo transfer). In total, 2 patients miscarried and 7 patients delivered a healthy baby (7/12; 58.3% delivery rate).\n\n\nDiscussion\n\nLow responder patients undergoing IVF are characterised by a low number of oocytes retrieved because of suboptimal oocyte maturation, poor embryo quality, hormonal stimulation cycle or embryo transfer cancellation2. Cobo et al.11 demonstrated in a prospective study that accumulation of oocytes by vitrification is a successful strategy for managing low responder patients in ‘classical’ IVF/ICSI treatments: delivery and cumulative delivery rates per patient were statistically higher in the low responder group (36.4%) than the low responder fresh group (23.7%). Our study could demonstrate, although on a limited number of patients, that this accumulation strategy can also be applied for a specific patient population, namely patients undergoing PGD for specific genetic diseases. Although we did not compare our outcomes to those of a control group of low responder fresh PGD patients from our center, we could demonstrate that the strategy to accumulate vitrified oocytes or embryos from consecutive hormonal stimulation cycles resulted in a sufficient number of embryos available for genetic diagnosis. As a consequence, a high percentage of patients had an embryo transfer of a healthy embryo (92,3%). It is evident that in order to accumulate oocytes and embryos by vitrification for the management of low responder patients, an efficient and well-established oocyte vitrification system needs to be in place. Survival rates after warming of these oocytes and embryos need to be optimal (between 80 and 100%); if this is not the case, this approach should not be offered to low responder patients. Our laboratory has high survival rates for oocytes and embryos (up to 100%) with the Cryotop and Cryotec vitrification method6,11.\n\nAlthough the treatment costs can be double or triple compared to one single hormonal stimulation for ICSI with PGD, the total costs of the accumulated cycles are lower because patients have to pay for only one ICSI procedure (in case of accumulation of oocytes) and only one genetic analysis combined with one embryo transfer.\n\nMoreover, this accumulation strategy resulted in higher outcomes (58.3% delivery rate per transfer) as compared to the 24% delivery rate per fresh embryo transfer presented by the ESHRE PGD consortium for 200812.\n\nThis retrospective cohort study demonstrates, although on a limited number of patients, that low responder patients in need of PGD can benefit from serial vitrification of oocytes and/or embryos after repeated ovarian stimulation cycles to improve their chances of a successful pregnancy. Future studies should address the ideal number of vitrified oocytes and/or embryos necessary in order to increase success in low responder patients undergoing PGD.",
"appendix": "Author contributions\n\n\n\nAC, NC, IC and CP conceived the study. AC, MN and NC designed the research. AC, MM and OC carried out the research. MM and OC provided expertise in vitrification. MN and AC prepared the first draft of the manuscript. NC and IC contributed to the preparation of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nAcknowledgements\n\nThese data were partially presented in a poster at the 12th International Conference on Preimplantation Genetic Diagnosis, Istanbul, Turkey, 2013.\n\nWe are grateful to the staff of Embryolab and EUROGENETICA SA Genetic Laboratories for assisting in all daily aspects of the treatment of these patients.\n\n\nReferences\n\nSurrey ES, Schoolcraft WB: Evaluating strategies for improving ovarian response of the poor responder undergoing assisted reproductive techniques. Fertil Steril. 2000; 73(4): 667–676. PubMed Abstract | Publisher Full Text\n\nKarande V, Gleicher N: A rational approach to the management of low responders in in-vitro fertilization. Hum Reprod. 1999; 14(7): 1744–1748. PubMed Abstract | Publisher Full Text\n\nChung JT, Son WY, Zhang XY, et al.: Normal birth following PGD for reciprocal translocation after serial vitrification of oocytes from a poor responder: a case report. RBMonline. 2012; 25(5): 521–6. PubMed Abstract | Publisher Full Text\n\nOlivennes F, Cunha-Filho J, Fanchin R, et al.: The use of GnRH antagonists in ovarian stimulation. Hum Reprod Update. 2002; 8(3): 279–290. PubMed Abstract | Publisher Full Text\n\nPalermo G, Joris H, Devroey P, et al.: Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet. 1992; 2(8810): 17–18. PubMed Abstract | Publisher Full Text\n\nKuwayama M, Vajta G, Kato O, et al.: Highly efficient vitrification method for cryopreservation of human oocytes. RBMOnline. 2005; 11(3): 300–308. PubMed Abstract | Publisher Full Text\n\nPrice CM: Fluorescence in situ hybridization. Blood Rev. 1993; 7(2): 127–134. PubMed Abstract | Publisher Full Text\n\nBeverly C, John PL, John JP, et al.: Polymerase Chain Reaction. Methods Mol Biol. 1993; 15: 1–29. Publisher Full Text\n\nWells D, Alfarawati S, Fragouli E: Use of comprehensive chromosomal screening for embryo assessment: microarrays and CGH. Molec Hum Reprod. 2008; 14(12): 703–710. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology. The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Hum Reprod. 2011; 26(6): 1270–1283. PubMed Abstract | Publisher Full Text\n\nCobo A, Garrido N, Crespo J, et al.: Accumulation of oocytes: a new strategy for managing low-responder patients. RBMOnline. 2012; 24(4): 424–432. PubMed Abstract | Publisher Full Text\n\nGoossens V, Traeger-Synodinos J, Coonen E, et al.: ESHRE PGD Consortium data collection XI cycles from January to December 2008 with pregnancy follow-up to October 2009. Hum Reprod. 2012; 27(7): 1887–1911. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "2412",
"date": "20 Nov 2013",
"name": "Pedro Barri",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting paper that addresses an important issue, but the sample size is small (13 patients) and there is relevant information lacking. I believe that this article should have the following recommendations taken into consideration:Provide information concerning patients’ baseline characteristics (as well as age, AFC and hormonal profile). Could the authors specify how allocation was done? (Please explain “based on the outcome of a medical counselling session with the patient”.) In Methods, Ovarian stimulation of patients, in the phrase “to obtain an accumulated minimum of 6 oocytes” the word “mature” should be added after oocytes. How was the cut-off of 6 mature oocytes established? Taking into account the average rates of fertilization and development to D2 embryos, isn’t this cut-off too low? (It is true that with this cut-off the obtained results are good but the sample size is small…) In Results, the phrase “until a sufficient number was stored (< 6 mature oocytes or < 5 embryos on day 2)” : the < should be a >. One patient did not have any healthy embryos for transfer: could you please explain how many embryos were biopsied in this patient? The total number of embryos available for PGD is given for each group, but could you provide the mean number of biopsied embryos ± SD per patient? If multiple pregnancy rate is zero it should be specified better, if it is not zero the rate should be given. I agree with the conclusion of the study but, in order to firmly state that vitrification for accumulation purposes in PGD cycles increases the chances of success, the next 2 points should be taken into account:Was the rate of development to day 3 embryos the same in fresh and vitrified and warmed cycles?It could be interesting to analyze the euploidy rate between embryos coming from fresh oocytes vs. vitrified + warmed oocytes; the same for fresh vs. vitrified + warmed embryos.",
"responses": [
{
"c_id": "639",
"date": "09 Dec 2013",
"name": "Martine Nijs",
"role": "Author Response F1000Research Advisory Board Member",
"response": "We would like to thank Dr. Pedro Barri for his useful comments concerning our study on the evaluation of the efficacy of a PGD program in low responder patients after repeated ovarian stimulation and accumulation of vitrified oocytes or embryos. Indeed our sample size was small, but still indicative for the usefulness of vitrification as a tool for poor responders in a PGD/PGS program. Hence we opted to describe our observations in a ‘short’ research article.The following baseline characteristics will be included in the revised article: Mean Age: 35.2 years; Mean AFC:7; Mean BMI: 24.6; Mean D2 FSH: 7.43. This retrospective cohort study was performed over a 30 month-period (2011–2013). Patients were counseled on both options (serial oocyte or embryo vitrification) with clear explanations on the pro and cons of each option. Patients selected themselves for serial oocyte or embryo vitrification. ‘Mature’ will be added to the specific sentence. Our study population consisted of patients with a low number of eggs retrieved and embryos produced. Our cut off was the minimum number that was possible to be obtained by this patient population. A higher cut off would require additional stimulation cycles which was not an option, as it required more repetitive cycles and a higher treatment cost. The < will be changed to >. This patient had 2 embryos from her first cycle and decided to vitrify them in order to proceed to another stimulated cycle. The aim was to increase the number of the available embryos for biopsy, and hence increase the number of having at least one healthy embryo to transfer after the screening. The next stimulation cycle resulted in 3 fresh embryos. In total 5 embryos were biopsied (2 thawed and 3 fresh). None of the embryos tested was genetically healthy and the transfer was cancelled. The mean number of biopsied embryos per patient: 7.2 (+SD: 2.1). Out of the 9 pregnancies obtained, two twin pregnancies were noted; both patients had delivery of healthy babies. According to our in house data there is no difference in development or implantation rate of fresh versus vitrified embryos, hereby confirming results of Rienzi et al. (2009) and Ku et al. (2012). This observation can be included in the discussion part. Unfortunately, we do not have in house data on the euploidy status of vitrified oocytes. Forman et al. (2012) however, did not observe an increase in aneuploidy rates after vitrification and warming of embryos."
}
]
},
{
"id": "3264",
"date": "30 Jan 2014",
"name": "Joep PM Geraedts",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe ability to accumulate oocytes or embryos from multiple cycles before PGD is done, could be an interesting development as is suggested in this article. However, the numbers are too small to be conclusive.I agree with the remarks of the first referee, furthermore I would like to add the following points for revision:Table 1:a. The numbers of normal and abnormal embryos should be specified.b. What are the definitions of clinical pregnancy rate per patient with transfer and the pregnancy rate per patient with transfer?c. How many embryos were frozen?d. How many FETs (Frozen Embryo Transfers) were done?e. All percentages should be given using decimal points.In the discussion it is stated that a high percentage of patients had an embryo transfer of healthy embryos. In my opinion this cannot be concluded from the material presented in the manuscript. In a number of cases FISH was used on day 3 embryos, which means that not PGS, not PGD was done. This screening can only give the results for the chromosomes included in the FISH analysis, while all other chromosomes can still be aneuploid. Furthermore it is known that mosaicism can complicate analysis at day 3.Finally the information given with respect to the costs is not detailed enough. The prices of an ICSI cycle and the PGD analysis should be given in order to come to conclusions about the financial aspects of this approach.",
"responses": [
{
"c_id": "698",
"date": "10 Feb 2014",
"name": "Martine Nijs",
"role": "Author Response F1000Research Advisory Board Member",
"response": "We thank Prof. Dr. Geraedts for his questions, comments and suggestions on our manuscript. We have listed our replies below. They will be implemented in in the final version of the manuscript.a. In total 40 embryos were diagnosed as normal, 43 as abnormal and for 9 embryos no result was obtained.b. Clinical pregnancy is defined as the presence of a gestational sac with foetal heartbeat by ultrasound at 8-10 weeks after embryo transfer.c. Eleven supernumerary embryos, diagnosed as being normal, were frozen post-embryo transfer.d. No Frozen embryo transfer was done for these patients.e. All percentages will be listed using decimal points.The discussion will be rephrased and will state the following: ‘As a consequence, a high percentage of patients had transfer of an embryo diagnosed to be negative for the specific genetic test (92,3%).’Our cost calculation of the treatments was based on the following:Traditional strategy involves repeat hormonal stimulation, repeat oocyte collection, repeat ICSI and repeat genetic diagnosis tests, each step associated with low chance for embryo transfer because of low number of oocytes collected. Cost of the accumulation strategy includes repeat hormonal stimulation, repeat oocyte collection, repeat vitrification and storage, one warming cycle, one ICSI and one genetic diagnosis test with very good chance for an embryo transfer."
}
]
}
] | 1
|
https://f1000research.com/articles/2-240
|
https://f1000research.com/articles/3-64/v1
|
27 Feb 14
|
{
"type": "Short Research Article",
"title": "Error evaluation of ventricular mechanics measurement in patients with discoordinate ventricular contraction",
"authors": [
"Borut Kirn"
],
"abstract": "In patients with discoordinate ventricular contraction, accurate identification of local mechanics from all heart regions is essential. Current measurement techniques are imperfect because a large part of the ventricular wall may be excluded, resulting in non-physiological average strain. In order to evaluate this error, we propose to compare the measured average strain to a reference strain obtained by averaging through a series of measurements.We assessed magnetic resonance (MR)-tagged images and determined circumferential strain in 160 regions of the ventricular wall in 10 patients with idiopathic dilated cardiomyopathy and left bundle branch block and 9 healthy volunteers. For each subject a global strain was calculated as the average of all measured strains. Then a reference strain was determined as the average of global strains for both experimental groups.The reference strains of a patient group and healthy controls both had a physiological pattern, with a peak shortening of -0.034 and -0.15, respectively.A large difference between the measured global strain and the reference strain indicates measurements which have large regions of ventricular wall which are excluded in the measurements.",
"keywords": [
"error evaluation",
"ventricular contraction"
],
"content": "Introduction\n\nA cardiac strain pattern explains how a part of the ventricular wall is shortening or lengthening during the cardiac cycle. In ventricles of healthy subjects, all parts of the ventricular wall shorten fairly uniformly during systole1,2. This is different in patients whose patterns of myocardial electrical activation and/or regional distribution of myocardial contractility are irregular3,4.\n\nIn these patients, the contraction is discoordinate and the uniformity of strain shortening is replaced by a complex interplay of simultaneous shortening and stretching in different regions. The regions that are activated earlier shorten, and those that are activated later and have not developed enough tension, stretch.\n\nIn studies evaluating ventricular mechanics, the accurate identification of local mechanics from all regions is essential. The existing measurement techniques such as magnetic resonance (MR)-tagging and ultrasound speckle tracking only measure a limited number of regions within the left ventricular wall5–7. Some parts of the ventricular wall that are important in determining the global ventricular mechanics may be excluded from the regions measured8–10. Therefore, the resulting average strain may appear non-physiological, for example, as lengthening during systole, which indicates cardiac filling. But we know that ventricle is contracting during ejection and therefore the average strain must always have negative slope during this period. We suggest that this characteristic could be used to evaluate the quality of the measurements.\n\nIn this study, a reference strain was determined from a series of measurements by averaging. Separate reference strains were determined for patients with discoordinate contraction and healthy controls.\n\n\nMethods\n\nThe study included 10 patients (age 67±8) with idiopathic dilated cardiomyopathy (DCM) and left bundle branch block (LBBB) who were selected for cardiac resynchronization therapy (DCM+LBBB group) and 9 healthy volunteers (control group). The ethics committee of the Catharina Hospital approved the study protocol, and all subjects gave informed consent.\n\nThe methods for acquiring the MR-tagged images, the measurement parameters, the technique of strain extraction from the recorded MR-tagged images and the partial data analyses of the measured data have been described previously11,12. Briefly, images were acquired using a conventional 1.5-T scanner (Philips Gyroscan T5-II, Philips Medical Systems, Best, The Netherlands). A 500-ms acquisition period was started 20 ms after the electrocardiogram trigger, and the time interval between two consecutive images was 20 ms. MR-tagged images were recorded in five parallel short-axis cross-sections, each slice was further divided into 32 sectors. For each of 160 sectors, we determined the pattern of circumferential mid-wall strain (εcc).\n\nA global strain was obtained as an average of 160 strain measurements. A reference strain was obtained as an average of global strains separately for patients and controls. The ejection phase based on the reference strain was determined as described previously11. Briefly, we located the peak negative slope during systole on the strain curve and fitted a linear curve to it. The beginning and the end of the ejection phase were determined where the linear curve crossed the zero strain and maximum strain points, respectively.\n\n\nResults\n\nFigures 1A and 1B show the measured strain patterns from 160 regions of a patient with DCM+LBBB and a healthy control, respectively. The patient with DCM+LBBB showed an exchange of negative and positive slopes during the initial systole phase in the average strain pattern. In contrast, the control subject showed an increasingly negative slope during the same period. The strain patterns in the patient with DCM+LBBB were highly heterogeneous compared to the control subject, and the curve band patterns were similar. The peak shortening for the average strain was -0.06 and -0.17 for the patient and control, respectively.\n\nLeft ventricular mid-wall circumferential strain patterns from 160 regions in (A) a patient with idiopathic dilated cardiomyopathy (DCM) and left bundle branch block (LBBB) and (B) a healthy control. Note the large variability seen in the patient and relative uniformity in healthy control. The average value of the cardiac strains (global strain) is represented by a red line.\n\nThe average strains for the patients with DCM+LBBB and the controls are shown in Figures 2A and 2B, respectively. The average strain patterns in five cases showed a period of positive slope during the first half of systole. The average strains were more homogenous in the controls, where they decreased smoothly during the entire early systole phase. The obtained reference strain decreased in patients and controls during the entire initial systole phase. The peak shortening for the reference strain was -0.034 and -0.15 for the patient and control, respectively (red lines, Figures 2A and 2B).\n\nThe global strain from 10 patients with idiopathic dilated cardiomyopathy (DCM) and left bundle branch block (LBBB) (A) and from 9 healthy controls (B). The group average, that is, the reference average strain, is identified by a red line.\n\nOn the basis of the reference strain, the ejection phase was from 1 to 16 of the MR-tagged images in patients with DCM+LBBB and from frames 1 to 14 in controls.\n\n\nDiscussion\n\nWe constructed a reference strain for patients with cardiac activation disorder and healthy controls. As expected, the resulting reference strain presented a negative slope during the entire early systole phase. A large difference between the measured global strain of each subject and the reference strain is indicative of insufficient measurements.\n\nThe shortcomings of this measurement technique are reflected in patient individual average strains. The first measured point in the strain pattern is during systole, that is, 20 ms after the electrocardiogram QRS trigger has started the measurements. In five of the patients assessed, the average strain pattern (Figure 2A) had a positive slope during the first half of systole. A positive slope on the curve indicates ventricular filling, but ventricular filling does not occur after the onset of tension generation in the ventricular wall. This measurement technique does not assess the mechanics of the entire ventricular wall, just a part of it. It relies on the coincidence of individual cardiac mechanics and selection of regions, which are measured irrespective of whether this artifact is pronounced. Because of the stochastic nature of this event, the averaging of cardiac strains throughout the group yields a better estimate of the actual cardiac strain than individual measurements.\n\nA standard procedure to determine the ejection phase on the basis of the average cardiac strain is based on identifying the peak negative slope during systole, fitting a linear curve to it, and identifying the time points where the linear curve crosses the zero strain and maximum strain points, which represent the beginning and end of the ejection phases, respectively. Without considering if the patients had positive slopes during systole, this procedure would be associated with several errors in case of our measured strains. The reference strain averages out the measurement errors in patients and thus facilitates more accurate detection of cardiac phases.\n\nCurrently, ventricular function is frequently evaluated on the basis of patterns of a regional strain. A simple test of global ventricular strain could additionally verify whether the measurements included all the relevant regions of the ventricle.\n\n\nData availability\n\nfigshare: Data of ventricular mechanistic measurements evaluation. http://dx.doi.org/10.6084/m9.figshare.94097313\n\n\nConsent\n\nWritten informed consent for publication of clinical details and clinical images was obtained from all the participants involved.",
"appendix": "Competing interests\n\n\n\nThere are no conflicts of interest to declare.\n\n\nGrant information\n\nResearch was supported by Slovenian Ministry for Education, Science and Sport, Slovenia, Grant No.: PO-510-381.\n\n\nAcknowledgements\n\nThe author wishes to thank Theo Arts and Frits Prinzen for their advices and guidance in MR-tagged images processing and interpretation, and Annemieke Jansen and Berry van Gelder for data acquisition.\n\n\nReferences\n\nLeitman M, Lysiansky M, Lysyansky P, et al.: Circumferential and longitudinal strain in 3 myocardial layers in normal subjects and in patients with regional left ventricular dysfunction. J Am Soc Echocardiogr. 2010; 23(1): 64–70. PubMed Abstract | Publisher Full Text\n\nKang Y, Sun MM, Cui J, et al.: Three-dimensional speckle tracking echocardiography for the assessment of left ventricular function and mechanical dyssynchrony. Acta Cardiol. 2012; 67(4): 423–430. PubMed Abstract\n\nTrayanova NA, Constantino J, Gurev V: Electromechanical models of the ventricles. Am J Physiol Heart Circ Physiol. 2011; 301(2): H279–H286. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChao PK, Chan HL, Wang CL, et al.: Patterns of left ventricular contraction in strain vector space related to bundle branch block with heart failure by speckle-tracking echocardiography. Ultrasound Med Biol. 2011; 37(4): 595–604. PubMed Abstract | Publisher Full Text\n\nAdamu U, Schmitz F, Becker M, et al.: Advanced speckle tracking echocardiography allowing a three-myocardial layer-specific analysis of deformation parameters. Eur J Echocardiogr. 2009; 10(2): 303–308. PubMed Abstract | Publisher Full Text\n\nTee M, Noble JA, Bluemke DA: Imaging techniques for cardiac strain and deformation: comparison of echocardiography, cardiac magnetic resonance and cardiac computed tomography. Expert Rev Cardiovasc Ther. 2013; 11(2): 221–231. PubMed Abstract | Publisher Full Text\n\nDewey M, Müller M, Eddicks S, et al.: Evaluation of global and regional left ventricular function with 16-slice computed tomography, biplane cineventriculography, and two-dimensional transthoracic echocardiography: comparison with magnetic resonance imaging. J Am Coll Cardiol. 2006; 48(10): 2034–2044. PubMed Abstract | Publisher Full Text\n\nAltiok E, Neizel M, Tiemann S, et al.: Quantitative analysis of endocardial and epicardial left ventricular myocardial deformation-comparison of strain-encoded cardiac magnetic resonance imaging with two-dimensional speckle-tracking echocardiography. J Am Soc Echocardiogr. 2012; 25(11): 1179–1188. PubMed Abstract | Publisher Full Text\n\nRutz AK, Manka R, Kozerke S, et al.: Left ventricular dyssynchrony in patients with left bundle branch block and patients after myocardial infarction: integration of mechanics and viability by cardiac magnetic resonance. Eur Heart J. 2009; 30(17): 2117–2127. PubMed Abstract | Publisher Full Text\n\nSonne C, Sugeng L, Takeuchi M, et al.: Real-time 3-dimensional echocardiographic assessment of left ventricular dyssynchrony: pitfalls in patients with dilated cardiomyopathy. JACC Cardiovasc Imaging. 2009; 2(7): 802–812. PubMed Abstract | Publisher Full Text\n\nKirn B, Jansen A, Bracke F, et al.: Mechanical discoordination rather than dyssynchrony predicts reverse remodeling upon cardiac resynchronization. Am J Physiol Heart Circ Physiol. 2008; 295(2): H640–H646. PubMed Abstract | Publisher Full Text\n\nDelhaas T, Kotte J, van der Toorn A, et al.: Increase in left ventricular torsion-to-shortening ratio in children with valvular aortic stenosis. Magn Reson Med. 2004; 51(1): 135–139. PubMed Abstract | Publisher Full Text\n\nKirn B: Data of ventricular mechanistic measurements evaluation. Figshare. 2014. Data Source"
}
|
[
{
"id": "5198",
"date": "23 Jun 2014",
"name": "Roman Leischik",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nStrain is a novel method for objective quantification of systolic function using 2-D echocardiography 1,2, 3D-Echocardiography 3-5 or MRI 6-9. Cardiac MRI (cMRI) is usually considered the reference standard for measurement of myocardial strain 9,10. The most common cMRI method, termed tagged cMRI, allows full 3D assessment of regional strain. However, due to its complexity and lengthy times for analysis, tagged cMRI is not usually used outside of academic centers. Tagged cMRI is also primarily used only in research studies 9.Strain echocardiography (StE) promises to be a new tool for quantitative assessment of cardiac function. Clinical application in daily routine has begun at the moment 2. Analysis of intra- and interobserver reliability is an important aspect in the process of developing these novel techniques from theory to the implementation into daily routine diagnostics 11,12. This is also true for MRI. A reproducibility study should be done in this study/report. I miss the definition of a concrete aim of the study and clinical conclusions. I think in the application to the ethics committee the aim of the study and the number of patients have to be mentioned. I miss a statistical analysis. Does the study have a descriptive character? All findings can be open discussed, but they should be discussed and so I am missing an open, positive-negative, hypothetical and critical discussion about MRI-strain. This study is in principle well conducted and promising, but can be “indexed” only after some changes.It is an interesting idea that the segmental split/deviation of the strain values discredit this method for clinical use. Otherwise medicine works with “deviations” (e.g. colour doppler findings). Is this what the author wants to tell us?Generally there is a need for critical discussion regarding the decision-making process in individual patient and specific diagnosis or the diagnostic consequences. I believe that it is important issue to discuss controversial findings and to analyze the clinical relevance of novel methods. I am sure, regarding the strain measurements, that there are a lot of open questions. We have to be aware of all possible pitfalls and ambiguities concerning strain-technology with the knowledge of asynchronicity, poor echo-window pitfalls (echocardiography), and reproducibility problems. The author has to increase the number of patients (a minimum of 30 participants with DCM and 20 healthy participants) and there is a need for statistical comparison. 20 Patients with preserved function and without left bundle branch block would be very important too. The author should compare the research tool MRI with 2D Strain- or 3D Strain-echocardiography. We have to learn more about the comparability of MRI-Strain values and echocardiography values. In the results section the times of the investigation of the single patients and the time for the analysis must be specified (for MRI and for Echocardiography). The number of-non-examined segments in both techniques should be examined/counted. There is a need for a comparison of strain values measured by MRI and echocardiography. Author have to improve the discussion of the clinical importance of strain measured by MRI, in comparison to echocardiography (when MRI and when echocardiography?) and should discuss advantages and disadvantages. The error/problems of MRI and the applicability in daily routine and the costs have to be discussed. An inter-observer reproducibility of the analysis should be carried out (if possible, in comparison to echocardiography). The impact of left bundle brunch block on the results of strain values in comparison to normal synchronisation should be discussed (for example Sonne et al., 200913). The final message should be emphasized. Is MRI-Strain not ready for use in clinical routine? Is the error/deviation of the values so big, that a diagnosis is not possible? The “split” or width range of segmental strain-values are known (the relevance of these mechanism for human health is still unclear), otherwise the clinical use of global strain and “averaged values” is accepted.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-64
|
https://f1000research.com/articles/2-233/v1
|
05 Nov 13
|
{
"type": "Study Protocol",
"title": "Study protocol for examining job strain as a risk factor for severe unipolar depression in an individual participant meta-analysis of 14 European cohorts",
"authors": [
"IPD-Work Consortium",
"Ida E. H. Madsen",
"Harald Hannerz",
"Solja T. Nyberg",
"Linda L. Magnusson Hanson",
"Kirsi Ahola",
"Lars Alfredsson",
"G. David Batty",
"Jakob B. Bjorner",
"Marianne Borritz",
"Hermann Burr",
"Nico Dragano",
"Jane E. Ferrie",
"Mark Hamer",
"Markus Jokela",
"Anders Knutsson",
"Markku Koskenvuo",
"Aki Koskinen",
"Constanze Leineweber",
"Martin L. Nielsen",
"Maria Nordin",
"Tuula Oksanen",
"Jan H. Pejtersen",
"Jaana Pentti",
"Paula Salo",
"Archana Singh-Manoux",
"Sakari Suominen",
"Töres Theorell",
"Salla Toppinen-Tanner",
"Jussi Vahtera",
"Ari Väänänen",
"Peter J. M Westerholm",
"Hugo Westerlund",
"Eleonor Fransson",
"Katriina Heikkilä",
"Marianna Virtanen",
"Reiner Rugulies",
"Mika Kivimäki",
"Harald Hannerz",
"Solja T. Nyberg",
"Linda L. Magnusson Hanson",
"Kirsi Ahola",
"Lars Alfredsson",
"G. David Batty",
"Jakob B. Bjorner",
"Marianne Borritz",
"Hermann Burr",
"Nico Dragano",
"Jane E. Ferrie",
"Mark Hamer",
"Markus Jokela",
"Anders Knutsson",
"Markku Koskenvuo",
"Aki Koskinen",
"Constanze Leineweber",
"Martin L. Nielsen",
"Maria Nordin",
"Tuula Oksanen",
"Jan H. Pejtersen",
"Jaana Pentti",
"Paula Salo",
"Archana Singh-Manoux",
"Sakari Suominen",
"Töres Theorell",
"Salla Toppinen-Tanner",
"Jussi Vahtera",
"Ari Väänänen",
"Peter J. M Westerholm",
"Hugo Westerlund",
"Eleonor Fransson",
"Katriina Heikkilä",
"Marianna Virtanen"
],
"abstract": "Background: Previous studies have shown that gainfully employed individuals with high work demands and low control at work (denoted “job strain”) are at increased risk of common mental disorders, including depression. Most existing studies have, however, measured depression using self-rated symptom scales that do not necessarily correspond to clinically diagnosed depression. In addition, a meta-analysis from 2008 indicated publication bias in the field. Methods: This study protocol describes the planned design and analyses of an individual participant data meta-analysis, to examine whether job strain is associated with an increased risk of clinically diagnosed unipolar depression based on hospital treatment registers. The study will be based on data from approximately 120,000 individuals who participated in 14 studies on work environment and health in 4 European countries. The self-reported working conditions data will be merged with national registers on psychiatric hospital treatment, primarily hospital admissions. Study-specific risk estimates for the association between job strain and depression will be calculated using Cox regressions. The study-specific risk estimates will be pooled using random effects meta-analysis. Discussion: The planned analyses will help clarify whether job strain is associated with an increased risk of clinically diagnosed unipolar depression. As the analysis is based on pre-planned study protocols and an individual participant data meta-analysis, the pooled risk estimates will not be influenced by selective reporting and publication bias. However, the results of the planned study may only pertain to severe cases of unipolar depression, because of the outcome measure applied.",
"keywords": [
"Unipolar depression is prevalent and incurs substantial costs for the individuals affected and society at large1",
"2. The disorder is thought to develop in a complex interplay of biological",
"psychological and social factors3–5. Following the diathesis-stress framework6",
"etiological determinants throughout the life-course may affect vulnerability to depression or act as triggering factors."
],
"content": "Introduction\n\nUnipolar depression is prevalent and incurs substantial costs for the individuals affected and society at large1,2. The disorder is thought to develop in a complex interplay of biological, psychological and social factors3–5. Following the diathesis-stress framework6, etiological determinants throughout the life-course may affect vulnerability to depression or act as triggering factors.\n\nAccording to the job strain model7, a psychosocial work environment characterized by high psychological demands and low control may result in stress-reactions and lead to adverse health outcomes. Accordingly, job strain has been linked to several health conditions, including coronary heart disease8 and unipolar depression9,10. There are, however, indications of publication bias in the field, suggesting that the published literature may be biased towards studies showing stronger associations between job strain and depression10. In addition, many previous studies on job strain and depression have applied outcome measures with uncertain diagnostic validity, for example self-reported symptoms9. Hence, the applicability of these findings to clinically diagnosed depression is uncertain9. In this study protocol, we set out our plan to obtain data from 14 European cohort studies on work environment and health, and to examine the association between job strain and subsequent first hospitalisation due to a diagnosis of unipolar depression. The purpose of this planned project is to examine whether stressful working conditions characterized by high psychological demands and low control, i.e. job strain7, are a risk factor for the development of unipolar depressive disorder. We hypothesize that individuals experiencing job strain are more likely to become hospitalized with a diagnosis of unipolar depression than individuals without job strain. Furthermore we aim to explore whether the association between job strain and depression is similar across strata of sex, age and socioeconomic status (SES).\n\n\nData\n\nThe IPD-Work Consortium combines data from a number of European work environment studies. For the analyses on job strain and depression we include data from 14 studies that are linked with hospital admission registers including psychiatric admissions. These studies encompass a total of approximately 120,000 individuals. Table 1 gives an overview of the included studies.\n\naStudy acronyms: COPSOQ: Copenhagen Psychosocial Questionnaire Study, DWECS: Danish Work Environment Cohort Study, FPS: Finnish Public Sector study, HeSSUP: Health and Social support Study, IPAW: Intervention Project on Absence and Well-being, PUMA: Burnout, Motivation and Job Satisfaction study, SLOSH: Swedish Longitudinal Occupational Survey of Health, WOLF: Work, Lipids, Fibrinogen (N = Norrland, S = Stockholm).\n\nbEstimates based on previous analyses or baseline data.\n\nIn each study, data on psychiatric hospital treatment are available through national registers11–14. Most registers include both inpatient and outpatient treatments. Outpatient data are available since 1995 in the Danish register, since 2001 in the Swedish register, since 1998 in the Finnish register and since 2003 in the UK register. To maximize the number of cases, these data are included when available.\n\n\nStudy population & design\n\nThe study is designed as a prospective cohort study. Participants will be included if they are employed at baseline of the respective study and have provided data on job strain, sex, age, cohabitation and socioeconomic status (SES). To ensure a prospective design we exclude all individuals with a hospital-based diagnosis of unipolar depression before or at baseline. Data are analysed using a two-step individual participant data meta-analysis; i.e. we first obtain study-specific risk estimates using harmonised exposure and outcome data and then combine these estimates using meta analytic techniques15. The study-specific risk estimates are calculated using Cox regressions analysis and the pooled estimates by random effects meta-analysis (see section “main analyses” for details).\n\nJob strain is self-reported in each of the studies listed in Table 1. The measure of job strain, i.e. the combination of high demands and low control, has previously been developed and harmonized, as documented by Fransson et al.16, and applied in previous analyses on job strain and other outcomes, including health behaviours, cardiovascular disease and cancer8,17–20. Briefly, study-specific measures for high demands and low control are defined dichotomously by the study-specific standardized median for each dimension (demands and control). Individuals with high demands and low control are considered exposed to job strain. The reference group is all other combinations of demands and control, i.e. individuals with low demands and high control, low demands and low control, high demands and high control.\n\nData on sex, age, cohabitation and SES will be included from each study to control for potential confounding influences. These covariates are chosen as potential confounders because they have been associated with depression21–23 and may be associated with job strain.\n\nDiagnoses in the included hospital records are coded according to the International Classification of Disease (ICD) system24 following versions 8, 9 or 10. Table 2 shows the diagnostic codes from each ICD-version we use to define unipolar depression. We include only principal diagnoses in the outcome definition, as auxiliary diagnoses may be underreported12 and it is uncertain whether such underreporting is related to patient characteristics.\n\n\nMain analyses\n\nAll study-specific analyses will be conducted using Cox proportional hazards regression with the occurrence of the first hospital record of unipolar depression as the failure-date, and censoring for migration (where available), death and end of follow up. All summary risk estimates will be calculated by pooling study-specific risk estimates and standard errors using inverse variance weighted random effects meta-analysis. Pooling will be conducted in R (www.rproject.org) using the meta package25 and the degree of heterogeneity between the study-specific estimates will be assessed by I226. Table 3 gives a ghost table for the main results.\n\naStudy acronyms: COPSOQ: Copenhagen Psychosocial Questionnaire Study, DWECS: Danish Work Environment Cohort Study, FPS: Finnish Public Sector study, HeSSUP: Health and Social support Study, IPAW: Intervention Project on Absence and Well-being, PUMA: Burnout, Motivation and Job Satisfaction study, SLOSH: Swedish Longitudinal Occupational Survey of Health, WOLF: Work, Lipids, Fibrinogen (N = Norrland, S = Stockholm).\n\nbHazard ratios are adjusted for sex, age and cohabitation.\n\nThe main analysis will be adjusted for sex, age and cohabitation. We will consider our hypothesis confirmed if the pooled adjusted hazard ratio is statistically significantly greater than 1 (p<0.05). We will not adjust for SES in the main analysis as this construct is conceptually intertwined with job strain27, and consequently this model could then be considered overadjusted. However, analyses concerning whether the estimated risk is independent from SES will be included as a sensitivity analysis. Apart from cohabitation (self-reported, living with a partner/spouse, yes/no) the covariate measurements and categorizations have been documented previously8,17–19. Briefly, SES will be based on occupation, except in data from the HeSSup study, where it will be based on highest educational qualification, and categorized as low (routine and manual occupations or comprehensive education), intermediate (non-manual intermediate occupations or vocational education), high (higher managerial, administrative and professional occupations or university-level education) or other (missing data on job title).\n\n\nStatistical power calculations\n\nThe expected numbers of cases of hospital-treated unipolar depression in each of the cohorts are presented in Table 4. The estimates are based on observations in the Danish register data concerning the incidence of hospital treatment for unipolar depression in gainfully employed Danes. These numbers were applied to the studies to estimate the expected number of cases, and the observed numbers of cases in the databases may diverge from this estimation. If there are no observed cases amongst individuals exposed to job strain in a study, this study will not be included in the analyses, as a risk estimate cannot be obtained.\n\naStudy acronyms: COPSOQ: Copenhagen Psychosocial Questionnaire Study, DWECS: Danish Work Environment Cohort Study, FPS: Finnish Public Sector study, HeSSUP: Health and Social support, IPAW: Intervention Project on Absence and Well-being, PUMA: Burnout, Motivation and Job Satisfaction study, SLOSH: Swedish Longitudinal Occupational Survey of Health, WOLF: Work, Lipids, Fibrinogen (N = Norrland, S = Stockholm).\n\nThe expected statistical power as a function of the hazard ratio is shown in Figure 1. The planned analysis is expected to be powered to show an association of 1.23 with >90% power. This is under the assumption that the actual number of cases will match the expected number of cases in Table 4 and that all studies will provide cases and thus are included in the analysis.\n\n\nSensitivity analyses\n\nThe following section specifies the planned sensitivity analyses which will examine the robustness of the results. All statistical tests for the sensitivity analyses will be two-sided with a significance threshold of P<0.05. We will conduct a series of sensitivity analyses which may lead to concerns of mass significance due to multiple testing. To avoid inflating the type I error due to multiple testing, we will consider the sensitivity analyses nested within the main hypothesis test. Thus, their interpretation will depend on the results of the main analysis: if our main hypothesis is confirmed, we will consider the significance tests of the nested hypotheses valid and the tests which yield p-values <0.05 statistically significant. The sensitivity analyses may in this case be considered as an examination of the conditions under which the average population effect found in the main analysis holds. However, if the main hypothesis is not confirmed, we will not consider the tests of the sensitivity analyses (nested hypotheses) with p<0.05 confirmatory, i.e. the results of the sensitivity analyses will be considered exploratory and hypothesis generating. This strategy will retain the overall probability of a type I error under 0.05, whenever the main null hypothesis is true.\n\nOur first set of sensitivity analyses examines whether the association between job strain and depression is modified by sex, age (≤35, 36–49, 50+ years) or SES (low, intermediate, high8) following indications from previous studies28–30. If there are too few cases to obtain estimates for 3 categories of age and SES in more than half of the eligible studies, we will use the categories ≤49/50+ years and low SES/other instead. Following the STROBE recommendations31 we will present results on effect-modification so that both departure from additivity and multiplicativity may be assessed32. However, our conclusions on effect-modification will be based on departure from additivity, as such information is particularly important from clinical and public health perspectives31,33,34. The statistical test will be based on the Central Limit Theorem, and Gauss propagation of error formulas.\n\nIf any statistically significant associations are found in the main analyses, we will conduct a second set of sensitivity analyses to examine how these associations are affected by accounting for SES, self-reported baseline mental health, and chronic physical disorders. Regarding mental health, we will a) adjust for mental health (continuous score, see Table 5 for measures) and b) exclude individuals with poor mental health (the poorest quintile, based on the measures presented in Table 5). Regarding chronic physical disorders we will exclude individuals with baseline coronary heart disease, stroke, cancer, chronic obstructive pulmonary disease, musculoskeletal disorders or diabetes, where data are available, as chronic physical disorders are associated with increased risk of mental disorder35. In a third and final sensitivity analysis we will examine how unipolar depression is related to the separate dimensions of demands and control (standardized continuous scores and the job strain model quadrants, i.e. comparing the three other combinations of demands and control to individuals with low demands and high control (cf.8).\n\naStudy acronyms: COPSOQ: Copenhagen Psychosocial Questionnaire Study, DWECS: Danish Work Environment Cohort Study, FPS: Finnish Public Sector study, HeSSUP: Health and Social support Study, IPAW: Intervention Project on Absence and Well-being, NWCS: The Netherlands Working Conditions Survey, POLS: Permanent Onderzoek Leefsituatie, PUMA: Burnout, Motivation and Job Satisfaction study, SLOSH: Swedish Longitudinal Occupational Survey of Health, WOLF: Work, Lipids, Fibrinogen (N = Norrland, S = Stockholm).\n\n\nDiscussion\n\nA major strength of the planned analyses is the register-based outcome with a specific diagnosis. The diagnoses are based on clinical assessments, which are independent of job strain assessments, and have high validity42. The use of such outcomes has not been possible in most previous analyses on work environment and depression9,10, as the relatively low incidence of hospital-treated unipolar depression necessitates an exceptionally large dataset. Some exclusively register-based analyses (e.g.43) have been conducted previously using hospital discharge registers. Such analyses have, however, not examined work environment variables measured at the individual level but exposures approximated using job exposure matrices. Although such approximation is useful when exposure measures are unavailable, the lack of individual level measurement hampers the interpretation of the findings as they are open to the ecological fallacy44.\n\nMeasuring depression incidence exclusively through hospital treatment registers also has limitations. It is likely that only the most severe cases of depression are treated in a hospital. Consequently, the results of the planned analyses will not be extendable to mild and moderate depression, and severe depression not treated in a hospital, if the aetiology of depression varies with severity and treatment. Furthermore, the exclusion of individuals with depression before or at baseline, to ensure a prospective study design, is also based on hospital-treated depressive episodes. Hence, it is possible that participants are suffering from, or have suffered from, untreated depression before baseline. However, in this consortium study, the only way to obtain accurate retrospective diagnoses is based on hospital treatment. Although data on treatment with antidepressants for example may also be obtained from registers, these medications are used to treat a range of conditions, other than depression, such as anxiety, pain and incontinence45, and consequently do not provide any accurate diagnostic information.\n\nAnother potential limitation of the planned analyses is the self-reported exposure data which could be sensitive to reporting biases due to negative affect at baseline; a bias which may result in inflated risk estimates, if individuals with reduced mental wellbeing report their exposure more negatively and are at increased risk of developing depression46. We address this limitation via the sensitivity analyses by adjusting for baseline mental health. Although this analysis may be overadjusted, at least if negative affect at baseline is a consequence of the working environment, any remaining association between job strain and depression will provide a strong argument that the results are not explained by reporting bias.\n\nThe planned analysis uses data from 14 studies conducted in 4 European countries. The studies differ in design, timing, and study population. Whereas some studies (e.g. DWECS, SLOSH) include the general working population, others are restricted to employees of specific organisations or occupations (e.g. FPS, Whitehall). Although this combination of different study populations means great gains in statistical power, the generalizability of the findings must be considered. Ideally, we may generalize our findings to the working population of (Northern) Europe. Such generalization would be supported by low degrees of heterogeneity in the pooled estimates, as we found for the association between job strain and coronary heart disease8. If there are greater levels of heterogeneity in the findings, however, the generalizability of the associations outside the examined cohorts is less clear. In that case we may need additional post hoc sensitivity analyses to examine the reasons for heterogeneity, for example the length of follow up, the period with hospital data available before baseline, and whether or not outpatient hospitalisation data are included throughout the follow up period.\n\nAll participating studies have been approved separately by the relevant national ethical committees (see appendices of8,17 for details). The results of this planned study will be published in an article in a scientific peer-reviewed journal. This planned study will constitute the largest in the field to date and as such is likely to set the parameters of research in this field for some time to come.\n\n\nProject organization\n\nThe project is organized as part of the IPD-Work (\"Individual-participant-data meta-analysis of working populations\") Consortium47. IPD-Work was funded by the NEW OSH ERA48, and is led by Professors Mika Kivimäki (Finnish Institute of Occupational Health and University College London, UK), Töres Theorell (Stress Research Institute, University of Stockholm, Sweden), Reiner Rugulies (National Research Centre for the Working Environment, Denmark) and Nico Dragano (Department of Medical Sociology, University of Düsseldorf, Germany). The IPD-Work Consortium is coordinated by Prof. Mika Kivimäki. The principal investigator in the analyses described in the present protocol is Dr. Ida E. H. Madsen, researcher at the National Research Centre for the Working Environment, Denmark.",
"appendix": "Author contributions\n\n\n\nThis IPD-Work study and this study protocol were conceptualized by IEHM, RR, TT, ND, HA, and MK. All authors participated in designing the protocol, generating hypotheses, and writing and critically revising the manuscript. IEHM wrote the first draft of the protocol in collaboration with HA and RR. All authors have agreed to the final content of this protocol.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe IPD-Work Consortium is supported by the EU New OSH ERA research programme (funded by the Finnish Work Environment Fund, Finland, the Swedish Research Council for Working Life and Social Research, Sweden, the Danish National Research Centre for the Working Environment, Denmark, the Academy of Finland (grant #132944), the BUPA Foundation (grant 22094477), the Economic and Social Research Council, UK, and a renewal grant from the Finnish Work Environment Fund, Finland. The work of IEHM, HH and RR on this manuscript was further funded by The Danish Working Environment Research Fund (grant #9-2011-03).\n\n\nReferences\n\nWittchen HU, Jacobi F: Size and burden of mental disorders in Europe--a critical review and appraisal of 27 studies. Eur Neuropsychopharmacol. 2005; 15(4): 357–76. PubMed Abstract | Publisher Full Text\n\nAlonso J, Angermeyer MC, Bernert S, et al.: Disability and quality of life impact of mental disorders in Europe: results from the European Study of the Epidemiology of Mental Disorders (ESEMeD) project. Acta Psychiatr Scand Suppl. 2004; 109(420): 38–46. PubMed Abstract | Publisher Full Text\n\nKendler KS, Gardner CO, Prescott CA: Toward a comprehensive developmental model for major depression in men. Am J Psychiatry. 2006; 163(1): 115–24. PubMed Abstract | Publisher Full Text\n\nKendler KS, Gardner CO, Prescott CA: Toward a comprehensive developmental model for major depression in women. Am J Psychiatry. 2002; 159(7): 1133–45. PubMed Abstract | Publisher Full Text\n\nLichtenstein P, Yip BH, Bjork C, et al.: Common genetic determinants of schizophrenia and bipolar disorder in Swedish families: a population-based study. Lancet. 2009; 373(9659): 234–9. PubMed Abstract | Publisher Full Text\n\nMonroe SM, Simons AD: Diathesis-Stress Theories in the Context of Life Stress Research: Implications for the Depressive-Disorders. Psychol Bull. 1991; 110(3): 406–25. PubMed Abstract | Publisher Full Text\n\nKarasek R, Theorell T: Healthy work: stress, productivity, and the reconstruction of working life. New York: Basic Books. 1990. Reference Source\n\nKivimäki M, Nyberg ST, Batty GD, et al.: Job strain as a risk factor for coronary heart disease: a collaborative meta-analysis of individual participant data. Lancet. 2012; 380(9852): 1491–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBonde JP: Psychosocial factors at work and risk of depression: a systematic review of the epidemiological evidence. Occup Environ Med. 2008; 65(7): 438–45. PubMed Abstract | Publisher Full Text\n\nNetterstrøm B, Conrad N, Bech P, et al.: The relation between work-related psychosocial factors and the development of depression. Epidemiol Rev. 2008; 30(1): 118–32. PubMed Abstract | Publisher Full Text\n\nMors O, Perto GP, Mortensen PB: The Danish Psychiatric Central Research Register. Scand J Public Health. 2011; 39(7 Suppl): 54–7. PubMed Abstract | Publisher Full Text\n\nSund R: Quality of the Finnish Hospital Discharge Register: A systematic review. Scand J Public Health. 2012; 40(6): 505–15. PubMed Abstract | Publisher Full Text\n\nLudvigsson J, Andersson E, Ekbom A, et al.: External review and validation of the Swedish national inpatient register. BMC Public Health. 2011; 11(1): 450. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSinha S, Peach G, Poloniecki JD, et al.: Studies using English administrative data (Hospital Episode Statistics) to assess health-care outcomes--systematic review and recommendations for reporting. Eur J Public Health. 2013; 23(1): 86–92. PubMed Abstract | Publisher Full Text\n\nThompson S, Kaptoge S, White I, et al.: Statistical methods for the time-to-event analysis of individual participant data from multiple epidemiological studies. Int J Epidemiol. 2010; 39(5): 1345–59. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFransson EI, Nyberg ST, Heikkila K, et al.: Comparison of alternative versions of the job demand-control scales in 17 European cohort studies: the IPD-Work consortium. BMC Public Health. 2012; 12(1): 62. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNyberg ST, Heikkilä K, Fransson EI, et al.: Job strain in relation to body mass index: pooled analysis of 160,000 adults from 13 cohort studies. J Intern Med. 2012; 272(1): 65–73. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHeikkilä K, Nyberg ST, Fransson EI, et al.: Job Strain and Alcohol Intake: A Collaborative Meta-Analysis of Individual-Participant Data from 140,000 Men and Women. PLoS One. 2012; 7(7): e40101. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHeikkilä K, Nyberg ST, Fransson EI, et al.: Job Strain and Tobacco Smoking: An Individual-Participant Data Meta-Analysis of 166,130 Adults in 15 European Studies. PLoS One. 2012; 7(7): e35463. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHeikkilä K, Nyberg ST, Theorell T, et al.: Work stress and risk of cancer: meta-analysis of 5700 incident cancer events in 116,000 European men and women. BMJ. 2013; 346: f165. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlonso J, Angermeyer MC, Bernert S, et al.: Prevalence of mental disorders in Europe: results from the European Study of the Epidemiology of Mental Disorders (ESEMeD) project. Acta Psychiatr Scand Suppl. 2004; 109(420): 21–7. PubMed Abstract | Publisher Full Text\n\nSimon RW: Revisiting the relationships among gender, marital status, and mental health. AJS. 2002; 107(4): 1065–96. PubMed Abstract | Publisher Full Text\n\nHarris T: Recent developments in understanding the psychosocial aspects of depression. Br Med Bull. 2001; 57(1): 17–32. PubMed Abstract | Publisher Full Text\n\nWorld Health Organization. International statistical classification of diseases and related health problems - 10th Revision. World Health Organization 2007. Reference Source\n\nSchwarzer G: Package 'meta'. The R Foundation for Statistical Computing 2012. Reference Source\n\nHiggins JP, Thompson SG, Deeks JJ, et al.: Measuring inconsistency in meta-analyses. BMJ. 2003; 327(7414): 557–60. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJohnson JV, Hall EM: Class, work, and health. In: Amick B Levine S Tarlov A Chapman Walsh Deditors. Society and health. New York / London: Oxford University Press. 1995; p. 247–71. Reference Source\n\nWang J, Patten SB, Currie S, et al.: A Population-based Longitudinal Study on Work Environmental Factors and the Risk of Major Depressive Disorder. Am J Epidemiol. 2012; 176(1): 52–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWege N, Dragano N, Erbel R, et al.: When does work stress hurt? Testing the interaction with socioeconomic position in the Heinz Nixdorf Recall Study. J Epidemiol Community Health. 2008; 62(4): 338–41. PubMed Abstract | Publisher Full Text\n\nChandola T, Britton A, Brunner E, et al.: Work stress and coronary heart disease: what are the mechanisms? Eur Heart J. 2008; 29(5): 640–8. PubMed Abstract | Publisher Full Text\n\nVandenbroucke JP, Von Elm E, Altman DG, et al.: Strengthening the reporting of observational studies in epidemiology (STROBE): Explanation and elaboration. PLoS Med. 2007; 4(10): e297. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKnol MJ, VanderWeele TJ: Recommendations for presenting analyses of effect modification and interaction. Int J Epidemiol. 2012; 41(2): 514–20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGreenland S, Lash TL, Rothman KJ: Concepts of interaction. Modern Epidemiology 3. edition ed. Philadelphia, Pa.: Wolters Kluwer 2008; p. 71–83. Reference Source\n\nGreenland S: Interactions in Epidemiology: Relevance, Identification, and Estimation. Epidemiology. 2009; 20(1): 14–7. PubMed Abstract | Publisher Full Text\n\nBarnett K, Mercer SW, Norbury M, et al.: Epidemiology of multimorbidity and implications for health care, research, and medical education: a cross-sectional study. Lancet. 2012; 380(9836): 37–43. PubMed Abstract | Publisher Full Text\n\nBjorner JB, Damsgaard MT, Watt T, et al.: Tests of data quality, scaling assumptions, and reliability of the Danish SF-36. J Clin Epidemiol. 1998; 51(11): 1001–11. PubMed Abstract | Publisher Full Text\n\nPejtersen JH, Kristensen TS, Borg V, et al.: The second version of the Copenhagen Psychosocial Questionnaire. Scand J Public Health. 2010; 38(3 Suppl): 8–24. PubMed Abstract | Publisher Full Text\n\nGoldberg DP, Hillier VF: A scaled version of the General Health Questionnaire. Psychol Med. 1979; 9(1): 139–45. PubMed Abstract | Publisher Full Text\n\nBeck AT, Ward CH, Mendelson M, et al.: An inventory for measuring depression. Arch Gen Psychiatry. 1961; 4: 561–71. PubMed Abstract | Publisher Full Text\n\nLipmann R: Depression scales derived from the Hopkins Symptom Checklist. In: Sartorius N Bann T editors. Assessment of depression. Berlin: Springer. 1986; p. 232–48. Publisher Full Text\n\nMagnusson Hanson L, Theorell T, Bech P, et al.: Psychosocial working conditions and depressive symptoms among Swedish employees. Int Arch Occup Environ Health. 2009; 82(8): 951–60. PubMed Abstract | Publisher Full Text\n\nKessing L: Validity of diagnoses and other clinical register data in patients with affective disorder. Eur psychiat. 1998; 13(8): 392–8. PubMed Abstract | Publisher Full Text\n\nWieclaw J, Agerbo E, Mortensen PB, et al.: Psychosocial working conditions and the risk of depression and anxiety disorders in the Danish workforce. BMC Public Health. 2008; 8: 280. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRothman KJ, Greenland S, Lash TL: Modern epidemiology. 3. ed. Philadelphia: Lippincott Williams & Wilkins 2008. Reference Source\n\nGardarsdottir H, Heerdink ER, van Dijk L, et al.: Indications for antidepressant drug prescribing in general practice in the Netherlands. J Affect Disord. 2007; 98(1–2): 109–15. PubMed Abstract | Publisher Full Text\n\nKolstad HA, Hansen Å, Kærgaard A, et al.: Job strain and the risk of depression: is reporting biased? Am J Epidemiol. 2011; 173(1): 94–102. PubMed Abstract | Publisher Full Text\n\nNEW OSH ERA. The IPD-Work (\"Individual-participant-data meta-analysis of working populations\") Consortium. NEW OSH ERA 2011. Reference Source\n\nNEW OSH ERA. NEW OSH ERA - What is NEW OSH ERA. NEW OSH ERA 2011. Reference Source"
}
|
[
{
"id": "2772",
"date": "16 Dec 2013",
"name": "Joanna Wieclaw",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nRegarding the titleI find it a bit misleading that the title says “14 European cohorts” as the study utilizes the Scandinavian cohort and only one other cohort, the UK's Whitehall study. Article contentAs the authors themselves point out, the studies included differ in design, timing and study population, which consist a challenge for a meta-analyses. The authors propose a number of sophisticated statistical methods to meet this challenge. However I have some reservations, particularly about including the Whitehall study - which is otherwise very good - as it is the only non Scandinavian study, and includes a selected population of white collars employees in a specific environmental setting. Also, to my knowledge, the UK registers differ from the Scandinavian practices, which renders comparisons problematic.Regarding measure of job strain it is not quite clear to me if additive, multiple, or both conceptualisation will be used. Also I find using a dichotomous measure of variables involved (low/high) rather crude. I suggest underlining more clearly that the risk of depression will be calculated in relation to other possible combinations of job strain model variables, which I find important as there is some evidence of diverging results.The measure of SES based only on occupation is a bit limited. Is it not possible to include fx. income and urbanity?The description of the statistics is detailed and lengthy. For the readers with scientific background this is very satisfactory, but I believe that the readers with less research training will be lost here. I suggest that the purpose of this sophisticated statistical analyses be more clear, or may be also plainly explained.Is it necessary to go as far back as ICD-8 in obtaining cases? And is “manic-depressive psychosis” included? I believe it would make data unnecessary heterogeneous. ConclusionIn all I find the study interesting and well designed. Its strength are the large population and register-based outcome. At the same time the limitation is that the outcome is restricted to a rather severe case of depression, and only ones treated in psychiatric hospitals, which are less prevalent.",
"responses": []
},
{
"id": "3029",
"date": "27 Jan 2014",
"name": "Pamela L. Perrewé",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI find the proposal examining the association between high strain jobs and unipolar depression to be fascinating. Examining clinically diagnosed depression as opposed to self-reported depression is an important step in job strain research. My only suggestion is to be cautious about arguing that individuals with high strain jobs leads to depression. This is not a longitudinal study, and it is just as likely that individuals who suffer from clinical depression are more likely to hold jobs that are characterized by high demands and low control. Regardless, this is an important piece of research and I look forward to reading the findings.",
"responses": []
}
] | 1
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https://f1000research.com/articles/2-233
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https://f1000research.com/articles/2-281/v1
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20 Dec 13
|
{
"type": "Research Article",
"title": "Rapid and inexpensive body fluid identification by RNA profiling-based multiplex High Resolution Melt (HRM) analysis",
"authors": [
"Erin K. Hanson",
"Jack Ballantyne",
"Erin K. Hanson"
],
"abstract": "Positive identification of the nature of biological material present on evidentiary items can be crucial for understanding the circumstances surrounding a crime. However, traditional protein-based methods do not permit the identification of all body fluids and tissues, and thus molecular based strategies for the conclusive identification of all forensically relevant biological fluids and tissues need to be developed. Messenger RNA (mRNA) profiling is an example of such a molecular-based approach. Current mRNA body fluid identification assays involve capillary electrophoresis (CE) or quantitative RT-PCR (qRT-PCR) platforms, each with its own limitations. Both platforms require the use of expensive fluorescently labeled primers or probes. CE-based assays require separate amplification and detection steps thus increasing the analysis time. For qRT-PCR assays, only 3-4 markers can be included in a single reaction since each requires a different fluorescent dye.To simplify mRNA profiling assays, and reduce the time and cost of analysis, we have developed single- and multiplex body fluid High Resolution Melt (HRM) assays for the identification of common forensically relevant biological fluids and tissues. The incorporated biomarkers include IL19 (vaginal secretions), IL1F7 (skin), ALAS2 (blood), MMP10 (menstrual blood), HTN3 (saliva) and TGM4 (semen). The HRM assays require only unlabeled PCR primers and a single saturating intercalating fluorescent dye (Eva Green). Each body-fluid-specific marker can easily be identified by the presence of a distinct melt peak. Usually, HRM assays are used to detect variants or isoforms for a single gene target. However, we have uniquely developed duplex and triplex HRM assays to permit the simultaneous detection of multiple targets per reaction. Here we describe the development and initial performance evaluation of the developed HRM assays. The results demonstrate the potential use of HRM assays for rapid, and relatively inexpensive, screening of biological evidence.",
"keywords": [
"Forensic Science",
"Body Fluid Identification",
"Messenger RNA (mRNA) Profiling",
"High Resolution Melt (HRM) Analysis"
],
"content": "Introduction\n\nIdentification of the tissue source of biological material present on individuals, evidentiary items and at crime scenes can be crucial to a fuller understanding of the circumstances pertaining to a crime. However traditional protein-based body fluid identification methods use a variety of labor intensive and technologically diverse techniques that do not permit the identification of all body fluids and tissues. Thus there remains a need to develop definitive molecular based strategies for the conclusive identification of all forensically relevant biological fluids and tissues. Although protein1–3, epigenetic DNA markers4–10 and microRNAs11–17 are promising examples of such a molecular based approach, messenger RNA (mRNA) profiling represents the current gold standard in this area due to the identification and development of a number of specific and sensitive mRNA assays for the identification of all of the forensically-relevant body fluids and tissues, namely blood, semen, saliva, vaginal secretions, menstrual blood and skin13,18–36. Importantly, messenger RNA markers are surprisingly stable in the dried state in that they can be successfully detected in environmentally impacted and partially degraded samples20,22,25,26,37–40.\n\nCurrent mRNA body fluid identification assays typically involve capillary electrophoresis (CE) or quantitative RT-PCR (qRT-PCR) platforms13,20,24,28,29,31,32,34,35, each with its own limitations. Both platforms require the use of expensive fluorescently labeled primers or probes. CE-based assays require separate amplification and detection steps thus increasing the time required for analysis. For qRT-PCR assays, only 3 or 4 markers can be included in a single reaction since each marker requires a different fluorescent dye.\n\nIn an attempt to simplify mRNA profiling assays, and to reduce the time and cost of analysis, we have investigated alternative technology solutions for mRNA profiling, particularly High Resolution Melt analysis (HRM). HRM is a rapid and relatively cheap bio-analytical method that was initially developed for sequence variant screening (i.e. mutation analysis) but which has also found utility in single nuclear polymorphism (SNP) typing41–44, methylation analysis45,46, copy number variant confirmation47–49, clone characterization50 and as an alternative for gel electrophoresis51. As its name suggests, HRM is a technique that permits the identification of specific PCR products (i.e. amplicons) by their melting temperature (Tm). An amplicon’s precise melting temperature is dependent on its sequence, length and the ionic strength of its environment and can be measured post-PCR to within 0.1°C with specialized software and hardware. HRM assays require only the use of unlabeled PCR primers and a single saturating intercalating fluorescent dye (e.g. Eva Green). The latter provides enhanced sensitivity by ensuring that all available double stranded DNA binding sites are saturated during the melting process. After amplification the amplicon is melted slowly by increasing the temperature and a loss of fluorescence occurs as the DNA strands separate and release the bound saturating dye into solution. By measuring the negative first derivative of fluorescence (F) with respect to temperature (T) (-dF/dT) a distinct and characteristic melt curve is obtained with the peak maximum representing the Tm. Importantly, due to the high resolution nature of the amplicon melt analysis, it is possible to perform multiplex analysis of several amplicons in one tube52.\n\nHere we describe the development and initial performance evaluation of singleplex and multiplex RNA-based HRM assays for the identification of the commonly encountered forensically relevant body fluids and tissues.\n\n\nMethods\n\nBody fluids were collected from volunteers using procedures approved by the University’s Institutional Review Board. Informed written consent was obtained from each donor. Blood samples (10mL, Bioreclamation, Westbury, NY) (16 donors; male and female; 21–56 yrs old) were collected by venipuncture into additive-free vacutainers and 50 μl aliquots were placed onto cotton cloth and dried at room temperature. Freshly ejaculated semen (12 donors; 30–55 yrs old) was provided in sealed plastic tubes and stored frozen. After thawing, the semen was absorbed onto sterile cotton swabs and allowed to dry. Buccal samples (saliva) (18 donors; male and female; 26–60 yrs old) were collected from donors using sterile swabs by swabbing the inside of the donor’s mouth. Semen-free vaginal secretions (18 donors; 20–60 yrs old) and menstrual blood (5 donors; 20–33 yrs old) were collected using sterile cotton swabs. Human skin total RNA was obtained from commercial sources: Stratagene/Agilent Technologies (Santa Clara, CA), Biochain® (Hayward, CA), Zenbio (Research Triangle Park, NC), and Zyagen (San Diego, CA). Cellular skin samples were collected by swabbing human skin or a touched object surface with a sterile water pre-moistened sterile swab. All samples were stored at -20°C or at room temperature until needed. A 50 μl stain or a single cotton swab was used for RNA isolation.\n\nTotal RNA was extracted from blood, semen, saliva, vaginal secretions, menstrual blood and skin using a manual organic RNA extraction (guanidine isothiocyanate-phenol-chloroform mixture)28–32. Briefly, 500 μl of pre-heated (56°C for 10 minutes) denaturing solution (4 M guanidine isothiocyanate, 0.02 M sodium citrate, 0.5% sarkosyl, 0.1 M β-mercaptoethanol) was added to a 1.5 mL Safe Lock tube extraction tube (Eppendorf, Westbury, NY) containing the stain or swab. The samples were incubated at 56°C for 30 minutes. The swab or stain pieces were then placed into a DNA IQTM spin basket (Promega, Madison, WI), re-inserted back into the original extraction tube, and centrifuged at 14,000 rpm (16,000 × g) for 5 minutes. After centrifugation, the basket with swab/stain pieces was discarded. To each extract the following was added: 50 μl 2 M sodium acetate and 600 μl acid phenol:chloroform (5:1), pH 4.5 (Ambion by Life Technologies). The samples were placed at 4°C for 30 minutes to separate the layers and then centrifuged for 20 minutes at 14,000 rpm (16,000 × g). The RNA-containing top aqueous layer was transferred to a new 1.5 ml microcentrifuge tube, to which 2 μl of GlycoBlue™ glycogen carrier (Ambion by Life Technologies) and 500 μl of isopropanol were added. RNA was precipitated for 1 hour at -20°C. The extracts were then centrifuged at 14,000 rpm (16,000 × g). The supernatant was removed and the pellet was washed with 900 μl of 75% ethanol/25% DEPC-treated water. Following a centrifugation for 10 minutes at 14,000 rpm (16,000 × g), the supernatant was removed and the pellet dried using vacuum centrifugation (56°C) for 3 minutes. Twenty microliters of pre-heated (60°C for 5 minutes) nuclease free water (Ambion by Life Technologies) was added to each sample followed by an incubation at 60°C for 10 minutes. Samples were used immediately or stored at -20°C until needed. All extracts were DNase treated to remove residual DNA using the Turbo DNA-free™ kit (Applied Biosystems (AB) by Life Technologies, Carlsbad, CA) according to the manufacturer’s protocol. RNA extracts were quantitated with Quant-iT™ RiboGreen® RNA Kit (Invitrogen by Life Technologies, Carlsbad, CA) as previously described28–32. Fluorescence was determined using a Synergy™ 2 Multi-Mode microplate reader (BioTek® Instruments, Inc., Winooski, VT).\n\nAll samples were reverse transcribed using the High Capacity cDNA Reverse Transcription kit (AB by Life Technologies) according to manufacturer’s protocols. The desired total RNA input was reverse transcribed in a 20 μl RT reaction volume (standard input – 25 ng total RNA). If no quantitative value was obtained or quantitation was not performed, an aliquot of the total RNA extract was used (14.2 μl). A reverse transcription negative reaction (containing total RNA and reaction buffer but no reverse transcriptase enzyme) was performed for each sample.\n\nSingleplex, Duplex, Triplex Assays: HRM assays were performed using the Type-It® HRM™ PCR kit (QIAGEN, Germantown, MD). Each 25 μl reaction included: 1 × HRM master mix (contains HotStar Taq Plus DNA Polymerase, EvaGreen dye, and manufacturer-designated ‘optimized concentrations’ of Q-solution (commercial reagent, exact concentrations not specified by the manufacturer), dNTPs and MgCl2), 2.8 μM primers (Table 1), 2 μl of RT reaction (cDNA), and RNase-free water. All assays were run on the Rotor-Gene® Q real time PCR instrument (QIAGEN), using the following cycling conditions: 95°C 5 min, followed by 45 cycles of 94°C 10 sec, 55°C 30 sec, 72°C 10 sec. HRM analysis was performed using a 65–90°C temperature range, with +0.1°C increments (90 sec of pre-melt conditioning on first step; and 2 sec for each step afterwards).\n\n‘All Body Fluids’ Hexaplex Assay: HRM assays were performed using the Type-It® HRM™ PCR kit (QIAGEN, Germantown, MD). Each 25 μl reaction included: 1X HRM master mix (contains HotStar Taq Plus DNA Polymerase, EvaGreen dye, and optimized concentrations (commercial reagent, exact concentrations not specified by the manufacturer) of Q-solution, dNTPs and MgCl2), 0.56 – 1.4 μM primers (Table 3) (ALAS2 – 0.56 μM, TGM4 – 0.56 μM, HTN3 – 1.4 μM, IL19 – 0.84 μM, MMP10 – 0.56 μM, CCL27 – 0.56 μM), 2 μl of RT reaction (cDNA), and RNase-free water. All assays were run on the Rotor-Gene® Q real time PCR instrument (QIAGEN), using the following cycling conditions: 95°C 5 min, followed by 45 cycles of 94°C 10 sec, 57°C 40 sec, 72°C 20 sec. HRM analysis was performed using a 73–90°C temperature range, with +0.1°C increments (90 sec of pre-melt conditioning on first step; 2 seconds for each step afterwards).\n\n\nResults\n\nDevelopment of the HRM assay. Based on our previous work with mRNA profiling13,24,28,29,31,32, we selected one marker for each body fluid/tissue based on amplification efficiency, specificity and/or sensitivity. The fluid- and tissue-specific markers selected were ALAS2 (blood)21,22,32, MMP10 (menstrual blood)32, HTN3 (saliva)26,30–32, TGM4 (semen)26,53, IL19 (vaginal secretions)29 and IL1F7 (skin)27,28 (Table 1). We performed singleplex HRM assays for each of these markers to ensure that they could be uniquely identified based on their different Tms. Samples from two donors of each body fluid of interest were used to estimate the Tm of each of the selected markers. The observed Tms are listed in Table 1 and the melt curves for each marker are displayed in Figure 1. Derivative plots are shown, with the temperature range (70–90°C) used in the HRM assay is displayed along the x-axis and the -dF/dT value (negative first derivative of fluorescence (F) with respect to temperature (T)) is displayed along the y-axis. An analysis threshold can be set for each axis, which is represented by a horizontal (blue) line on each plot. As can be seen in Figure 1, a single melt curve was observed for most markers with the exception of MMP10 in which two products are observed (81.8°C and 83.2°C). The same primer sequences for MMP10 used in these experiments are also used in our CE-based mRNA profiling multiplexes, where only one product is observed. Upon evaluation of the amplified sequence and comparison to different MMP genes, it was determined that the MMP10 primer set was capable of also amplifying the MMP3 gene. There are four mismatched bases in the forward primer, three of which are in the 3´end of the primer (i.e. 3 mismatches in the last 9 bases of the primer). This likely therefore causes amplification inefficiencies and results in a failure to detect the MMP3 product in some assays. The standard HRM protocol (QIAGEN Type-It® HRM™ kit) utilizes a 55°C annealing temperature which may explain why this second product is detected using this platform. Additional work would be needed to conclusively determine if this second peak is in fact MMP3 and to determine if the primer sequences can be modified to be MMP10-specific. MMP10 is also the only marker in the body fluid set in which HRM DNA products were observed, one at 78.5°C and another at 83.1°C (Supplementary Figure 1). These were clearly distinguishable from a positive mRNA MMP10 result (82.2°C and 83.5°C). The presence of two DNA products further supports the hypothesis that MMP3 is being co-amplified, one DNA product resulting for MMP3 and MMP10 (expected DNA product size is 319 and 369 bp, respectively).\n\nHigh resolution derivative melt curve plots for individual body fluid specific markers: A) ALAS2 (blood); B) MMP10 (menstrual blood); C) HTN3 (saliva); D) TGM4 (semen); E) IL19 (vaginal secretions); F) IL1F7 (skin). The plots from two different donors are shown for each marker. The x-axis represents the temperature and the y-axis indicates the first derivative of the change of fluorescence with temperature (-dF/dT). The average Tm (°C) for each marker is displayed. The horizontal line on each plot represents the analysis threshold.\n\nWhile some of the observed Tms were similar or overlapping (Table 1), there was sufficient resolution to incorporate all markers into three separate duplex reactions: blood/menstrual blood, semen/saliva and vaginal secretions/skin. For the development of the duplex HRM assays, the primer sets for each of the two markers were incorporated into a single reaction. No other parameters of the HRM assay were modified from the original singleplex assays. For each duplex HRM assay, known samples (total n = 56-65) of each of the targeted body fluids of interest were detected (Figure 2, Table 2). The melt curves obtained during the initial testing of the duplex HRM assays are shown in Figure 2. The melt curves for each marker are shown overlaid in order to indicate their relative locations within the duplex. For each of these single source samples, only the expected Tm(s) for the body fluid of interest was observed, with the exception of the menstrual blood samples (Figure 2A). With respect to the latter, as can be seen from the “pink” melt curves (representing the known menstrual blood samples), both MMP10 and ALAS2 were detected in the majority of samples (4/5). This is expected, as menstrual blood samples will contain varying amounts of peripheral blood, which is demonstrated by the presence of ALAS2. We observe the same co-detection in CE based mRNA profiling assays31. The simultaneous identification of both MMP10 and ALAS2 in individual menstrual blood samples also serves to confirm the functionality of this particular duplex assay.\n\nThe number of samples from individuals in which the marker was successfully detected is displayed (numerator) out of the total number of individuals tested (denominator). The shading reflects the number of positive samples out of the total number tested: white – no detection; light grey 1–59%; dark grey ≥ 60%.\n\nDuplex HRM assays incorporate a body fluid specific marker for each of two body fluids or tissues into a single reaction. High resolution derivative melt curve plots for the duplex assays are shown (where n = number of biological replicates (i.e. different individuals)): A) blood/menstrual blood (respectively: ALAS2, red, n = 8; MMP10, pink, n = 5); B) saliva/semen (respectively: HTN3, blue, n = 18; TGM4, yellow, n = 5); C) vaginal secretions/skin (respectively: IL19, green, n = 10; IL1F7, orange, n = 10). For interpretation of the reference to color, the reader is directed to the online version of the article.\n\nDuring the development of the duplex assays, we observed the appearance of broad peaks (“humps”) in the lower temperature ranges for the TGM4 and IL19 melt curves in the semen/saliva and vaginal/skin duplex assays, respectively (Figure 2B and 2C). The artifacts were observed in the amplification blanks (data not shown) and are likely due to primer dimers or non-specific primer interactions. For the vaginal/skin assay, this does not interfere with data interpretation as the Tms of these markers are at a higher temperature. Since the HRM temperature range was kept consistent for all duplex assays, the x-axis threshold can simply be set to exclude the affected temperatures from analysis. However, for the semen/saliva assay, the artifacts originating from the TGM4 melt curves are located within the HTN3 Tm region. This could affect the interpretation of the data on this assay as it could appear as a positive HTN3 result, although the peaks are abnormally broad and are distinguishable from true peaks. The intensity of the saliva sample melt curves permits us to use a suitably high x-axis threshold in order to eliminate these “humps” from analysis.\n\nA number of different donors (4–18 per body fluid or tissue, Table 2) of each body fluid were tested using all three duplex assays (total n = 56 (vaginal/skin), n = 63 (blood/menstrual blood, n = 65 (saliva/semen), Table 2). For the semen/saliva duplex, semen and saliva were correctly identified in a majority of samples and no cross-reactivity was observed with any of the other body fluids. For the vaginal/skin assay, vaginal secretions and skin were correctly identified in a majority of samples, and no cross-reactivity was observed for blood, semen or saliva (Table 2). As can be seen from Table 2, 3/5 menstrual blood samples evaluated were positive for IL19 (vaginal secretions) which is not surprising since menstrual blood samples are expected to contain varying amounts of vaginal secretions. However, it does indicate the need for additional interpretation guidelines for menstrual blood. For the blood/menstrual blood assay, blood and menstrual blood was correctly identified in a majority of blood and menstrual blood samples and no cross-reactivity was observed with skin, saliva and semen. However, MMP10 (menstrual blood marker) was detected in ~20% of vaginal secretion samples (4/18). While early work with MMP10 demonstrated a high degree of specificity for menstrual blood, other studies have also reported detection of MMP10 in vaginal samples20. Enzymes of the matrix metalloproteinase (MMP) family that play a role in the tissue remodeling that takes place during menstruation are spatiotemporally expressed in endometrial tissue54,55. Therefore, it is possible that increased levels of MMP10 may be present just prior to menses. The time during the reproductive cycle in which the blood samples in this study were collected was not known. Further work will be needed to evaluate MMP10 expression levels throughout the female reproductive cycle to determine if any trends can be identified amongst numerous donors.\n\nA number of false negative results (9/56, 16%) were observed for the body fluids or tissues of interest for the various duplex assays (Table 2). However, no false negatives were observed for skin (IL1F7 detected in all 10 donors tested) and menstrual blood (MMP10 detected in all 5 donors tested) (Table 2). The precise reason for the absence of the body fluid specific markers in these known samples is unclear. The false negative results for vaginal secretions and saliva may be explained by possible differences in input quantity. All body fluid sample extracts are quantitated to allow for standard amounts of total RNA (i.e. 25 ng) to be added to each reverse transcription reaction. However, since no validated human specific RNA quantitation method is currently available, the quantitation values obtained for body fluids with commensal bacteria such as vaginal secretions and saliva may be inaccurate with respect to estimates of the amount of human RNA in such samples. While attempts were made to normalize total RNA input into these body fluid samples, the actual amount of total human RNA may be somewhat less. This could lead to false negative results since sufficient amounts of human total RNA are not added. However, this would not be expected for blood or semen, where one false negative result was observed for each (Table 2). Further optimization of assay conditions and/or the development of a human specific RNA quantitation method might serve to reduce the occurrence of false negative results.\n\nEvidentiary items of unknown body fluid/tissue origin would be analyzed using each of the three duplexes in order to determine which body fluids or tissues, or combination thereof, are present. We performed such a process during our evaluation of the body fluid duplexes by taking single source samples of known tissue provenance and testing them against the three duplex HRM assays. Interestingly the duplex HRM assays were able to successfully identify contaminated single source samples. A set of reportedly single source vaginal secretions samples were evaluated using each of the three duplex assays to check that no other cross reacting body fluids would be detected with the assay. As can seen from Figure 3A, TGM4 was detected in two of the vaginal samples (melt curves shown in green) indicating the presence of semen. We performed additional testing of these samples using our CE-based mRNA body fluid identification multiplex and were able to confirm the presence of contaminating semen (detection of PRM2 and TGM4) in these two putative single source samples (Supplementary Figure 2).\n\nA) Saliva/Semen Assay. Detection of semen in two different vaginal samples is indicated by the presence of the TGM4 peak. A composite image of known semen and saliva samples (yellow and blue, respectively) and the two vaginal samples (green) in which semen was detected is shown. B) Vaginal Secretions/Skin Duplex Assay. Samples were collected from a male finger after digital penetration and from male underwear ~3 hours after intercourse. The detection of the presence of both vaginal secretions and skin is indicated by the presence of the IL19 and IL1F7 peaks in each sample.\n\nThe performance of the duplex assays with a limited number of simulated casework samples was evaluated. The mock casework samples were designed to represent possible casework scenarios including digital penetration and sexual assault (vaginal intercourse). The simulated casework samples included (i) a swab of the surface of male fingers after digital vaginal penetration of a female participant and (ii) a swab of the inside of male underwear worn 3 hours after sexual intercourse in order to attempt to detect possible vaginal secretions that might have been transferred from the penis after a sexual assault. An evaluation of potential transfer of vaginal secretions to male underwear worn after intercourse was selected to represent a sexual assault case rather than vaginal swabs since we had previously demonstrated the ability to detect semen in vaginal swabs (described above). For both samples (male finger after digital penetration and the male underwear worn after intercourse) both vaginal secretions (IL19) and skin (IL1F7) were detected (Figure 3B). These results were consistent with the activity (i.e. behavior) of the participants prior to the collection of samples56.\n\nWe evaluated the ability to detect blood and semen in two person body fluid mixtures as an example of a sample admixture type encountered in casework. We prepared blood-semen mixture samples using a constant amount of blood (50 μl) and decreasing amounts of semen (50 μl, 25 μl, 10 μl, and 5 μl). Each admixed sample was evaluated with the three duplex HRM assays (Figure 4). Blood (ALAS2) and semen (TGM4) were successfully detected in all four admixed samples (Figure 4). The intensity of the TGM4 peaks varied among the four mixtures samples but did not seem to correlate with the known proportion of semen in the mixture. Thus further work using a more comprehensive sample set is needed to determine whether a quantitative assessment of peak heights correlate to the true marker proportions comprising a mixture.\n\nA two-body fluid mixture sample set containing different proportions of the two body fluids was created using a constant amount of blood (50 μl) and decreasing volumes of semen (50 μl, 25 μl, 10 μl, and 5 μl). The admixed samples were co-extracted and the isolated RNA (25 ng) was analyzed using each of the three duplex assays (A – blood/menstrual blood; B – saliva/semen; C – vaginal secretions/skin). Blood and semen were successfully detected, as indicated by the presence of ALAS2 (A) and TGM4 (B) in all four mixture samples.\n\n\nEpithelial cell triplex HRM assay\n\nSince epithelial cells have common functions, namely secretion, selective absorption, protection, trans-cellular transport and detection of sensation57, finding biomarkers capable of unequivocally differentiating and identifying each of the three cell types commonly found in casework (buccal, vaginal and skin epithelia) is challenging. We have recently had success in the identification of highly specific RNA biomarkers for vaginal secretions and skin28,29 that, in combination with well-characterized saliva markers such as HTN3 or STATH, are capable of differentiating epithelial cells. Encouraged by the success in developing duplex HRM assays, we conceived and formulated a triplex assay for the identification of epithelial cell containing fluids and tissues (i.e. saliva, vaginal secretions and skin). The assay employs the HTN3 (saliva), IL19 (vaginal secretions) and skin (IL1F7) RNA biomarkers (Figure 5A). The melt curves from single source samples (2 donors for each fluid; for vaginal secretions, one of the two samples is a menstrual blood sample in which vaginal secretions was detected) are shown overlaid in order to demonstrate the non-overlapping location of each marker in the triplex assay. To initially test the performance of this triplex assay with forensic casework type samples, we swabbed the surface of a computer mouse and analyzed it using the triplex assay. The presence of skin was detected on the computer mouse sample as indicated by the presence of IL1F7 (Figure 5B). This preliminary indication that the sensitivity of the assay might permit the identification of touched objects augurs well for its potential use in casework.\n\nA Triplex HRM assay was created to permit identification of epithelial-cell containing tissues and fluids (saliva, vaginal secretions and skin). A) A body fluid specific marker for each of the three fluids/tissues was incorporated into a single reaction (HTN3 – saliva, IL19 – vaginal secretions, IL1F7 – skin). Each individual sample was analyzed using a single triplex reaction. Two saliva donors (blue), one vaginal secretions (green) and one menstrual blood donor (pink) and two skin donors (orange) are shown overlaid to show the relative locations of each marker in the triplex. B) Successful detection of skin from a swab of a computer mouse (black) using the triplex HRM assay is indicated by the presence of IL1F7. A composite image of known saliva, vaginal and skin samples (blue, green and orange, respectively) is shown as a reference.\n\n\n‘All body fluids’ hexaplex HRM assay\n\nThe availability of a hexaplex assay that would permit a single tube identification of all of the common forensically relevant biological fluids and tissues (blood, semen, saliva, vaginal secretions, menstrual blood and skin) is desirable. The development of such an HRM assay is challenging, however, since the number of biomarkers, or sometimes the choice of biomarker, is dictated by the need to assure non-overlapping Tm values. Despite differences in amplicon size, the associated Tm values may be similar or the same depending on the particular amplicon sequences. For example, the inclusion of all biomarkers used in the singleplex, duplex and triplex assays described above into a single hexaplex system would not be possible. The Tm values of MMP10 overlap with IL19 (vaginal secretions), TGM4 (semen) and IL1F7 (skin). Nevertheless we sought to develop an ‘all body fluids’ hexaplex system using alternative highly specific biomarkers or primer modifications to intentionally alter the Tm value of the RNA amplicon.\n\nThe above described singleplex, duplex and triplex assays involved the use of the following markers: ALAS2 (blood), TGM4 (semen), HTN3 (saliva), IL19 (vaginal secretions), MMP10 (menstrual blood) and IL1F7 (skin). Our initial efforts were focused on attempting to include all of these biomarkers in the hexaplex system. However attempts at suitably modifying the IL1F7 primers were unsuccessful and resulted in the requirement for an alternative biomarker for skin. We selected CCL27 as a replacement for IL1F7 based on its Tm value, sensitivity and specificity28. A CCL27 Tm value of 84.8°C no longer presented a conflict with MMP10 (81.9°C). However, this was close to the Tm value of ALAS2 (85.8°C). Sequence modification of the reverse primer for ALAS2 permitted a shift of the observed ALAS2 Tm value to 86.8°C and therefore resolved any potential overlap with CCL27. The use of an alternative reverse primer sequence for IL19 also resulted in a new observed average Tm value of 78.6°C and therefore resolved the conflict between MMP10 and IL19. The remaining Tm conflict was between MMP10 and TGM4. The use of a modified MMP10 forward primer sequence and the inclusion of a “GGGGG” non-template addition to the MMP10 primer set resulted in a sufficient separation of MMP10 and TGM4 Tm values (~1°C shift of the MMP10 Tm value). With these modifications a single Tm value for MMP10 was also observed. With the success of these loci and primer modifications, together with alterations to the PCR cycling parameters, we were able to develop a prototype hexaplex HRM system that was suitable for undergoing a series of preliminary developmental validation studies. The hexaplex system is shown in Figure 6A with single source samples (two donors per body fluid or tissue) overlaid to indicate the location of each biomarker within the hexaplex. All of the incorporated markers are resolvable without any Tm overlap as indicated by measurement of the average Tm value and Tm value range (+ 3 standard deviations) (Table 3, Figure 6B). A DNA-specific product resulting from amplification with the new IL19 primer set was detected (Figure 6A, Table 3) but does not interfere with any of the included biomarkers (Tm of 80.6°C, between IL19 and MMP10 ranges). The presence of this DNA product can actually be useful in identifying samples that contain significant amounts of contaminating DNA and could therefore be used as an RNA sample quality control check for all sample types, irrespective of body fluid source. Interestingly, similar previously observed DNA products for MMP10 in the duplex assay were not apparent with the hexaplex system.\n\n*underlined text indicates non-template sequence addition\n\nA) Derivative melt curve plots for the hexaplex assay is shown: saliva (HTN3, blue), vaginal secretions (IL19, green), menstrual blood (MMP10, pink), semen (TGM4, yellow), skin (CCL27, orange), and blood (ALAS2, red). Two donors of each body fluid or tissue are shown. The melt plots shown are overlaid single source melt plots with all individual samples analyzed using the respective multiplex HRM assays (shown overlaid to demonstrate marker positions within the multiplex assay). The location of a peak originating from contaminating DNA (IL19) is shown in black. B) Hexaplex biomarker resolution as represented by the Tm (°C) range (average Tm + 3 standard deviations). C) Analysis of a saliva-vaginal admixture using the hexaplex. Tm values of 76.5°C (HTN3) and 78.6°C (IL19) were observed indicating the presence of saliva and vaginal secretions respectively.\n\nWe carried out initial performance checks on the hexaplex’s specificity. Single source body fluid and tissue samples (n total = 63; saliva, n = 13; vaginal secretions, n = 9; menstrual blood, n = 4; semen, n = 12; skin, n = 9; blood, n = 16) were tested. The correct biomarker was identified, except for a small number of false negative results with semen (one sample) and saliva (two samples) (Table 4). Similar to the results of specificity testing for the duplex assays, MMP10 was detected in some of the vaginal secretion samples (3/9, ~33%). Peripheral blood was detected in three of the four menstrual blood samples (Table 4), which is expected since menstrual blood samples are complex mixtures including peripheral blood and vaginal secretions. Forensic evidentiary items are frequently not single source samples and can contain admixed biological fluids. The results of hexaplex analysis of a saliva-vaginal secretion admixed sample (artificially created mixture in which 25 μl of neat saliva was added to half of a vaginal swab) are shown in Figure 6C. Both saliva (HTN3) and vaginal secretions (IL19) were correctly identified using the hexaplex system.\n\nThe number of single source samples from different individuals in which the marker was successfully detected (numerator) is displayed out of the total number of individuals tested (denominator). The shading reflects the number of positive samples out of the total number tested: white – no detection, light grey 1–74%; dark grey ≥ 75%.\n\nN = 63\n\nData Set 1. Tm Values for HRM duplex assay specificity.\n\nThis data set contains the individual Tm values for the specificity testing of the vaginal/skin, blood/menstrual and saliva/semen HRM duplex assay specificity presented in Table 2. Individual donors are listed according to body fluid or tissue type with the Tm values (°C) recorded under each biomarker. IL19 = vaginal biomarker, ALAS2 = blood, HTN3 = saliva biomarker, IL1F7 = skin biomarker, MMP10 = menstrual blood, TGM4 = semen biomarker, n.d. = not detected in body fluid of interest, empty cell = no detection.\n\nData Set 2. Tm Values for ‘All Body Fluids’ Hexaplex Assay Specificity.\n\nThis data set contains the individual Tm values for specificity testing of the ‘all body fluids’ hexaplex HRM assay presented in Table 4. Individual donors are listed according to body fluid or tissue type with the observed Tm values recorded under each biomarker. n.d. = not detected in body fluid of interest.\n\n\nDiscussion\n\nMessenger RNA profiling with a battery of highly specific biomarkers can be used to positively identify all of the commonly found forensically relevant body fluids and tissues. In this work, in order to simplify mRNA profiling assays and to reduce the time and cost of analysis, we have developed a number of prototype multiplex high resolution melt (HRM) assays for the identification of blood, semen, saliva, vaginal secretions, menstrual blood and skin. With respect to critical post-cDNA reagents, the HRM assays require only the use of unlabeled PCR primers and a single intercalating saturating fluorescent dye (Eva Green). In terms of hardware and software a real time instrument with HRM capabilities is required and a number of manufacturers make such instruments. Each body-fluid specific marker can easily be identified by the presence of a distinct melt peak.\n\nThe ability to multiplex different combinations of RNA biomarkers as evidenced by the compatibility of the markers in duplex, triplex and hexaplex assays indicates that there exists an opportunity for laboratories to customize HRM assays to suit their specific needs. For example, if the intended use of the HRM assays is to rapidly screen sexual assault evidence, it may be desirable to only incorporate biomarkers for the identification of semen and vaginal secretions or, alternatively, semen, vaginal secretions and saliva. Some may want an epithelial marker assay such as the triplex that can identify and differentiate saliva, vaginal secretions and skin. However, some laboratories may want to utilize HRM assays for the identification of all forensically relevant body fluids.\n\nWe recognize that the prototype HRM assays may need to be further optimized (using modified primers, cycling conditions or loci) using a larger and more varied sample set. For example, the semen/saliva duplex assay should be further optimized in order to reduce or eliminate artifacts such as the broad peaks/humps observed in the HTN3 region arising from the TGM4 melt curve. If these artifacts cannot be eliminated it may be necessary to replace TGM4 with another suitable semen marker with a melt curve profile that does not interfere with the interpretation of HTN3 data or to replace HTN3 with another saliva marker that has a higher Tm. It may also be possible to alter the Tm of the existing markers in the assay by changing the primer location. However, any changes to primer sequences would require additional studies to evaluate primer efficiency and to ensure that no changes to sensitivity or specificity are observed.\n\nThe principal advantages of HRM over current mRNA body fluid identification assays that employ capillary electrophoresis (CE) or quantitative RT-PCR (qRT-PCR) platforms are the not insignificant ones of cost and time. Both CE and qRT-PCR platforms require the use of expensive fluorescently labeled primers or probes whereas HRM uses unlabeled primers. CE-based assays require separate amplification and detection steps thus increasing the cost and time required for analysis. Post RNA extraction and cDNA formation, the closed tube HRM assay takes ~ 2 hours to perform which is similar to qRT-PCR assays. However for qRT-PCR assays, only 2 or 3 markers can be included in a single reaction since each marker and internal control requires a different fluorescent dye whereas HRM can multiplex at least 6 markers.\n\nIn summary, this proof of principle work describes the design and testing of a number of mRNA HRM assays that, after further validation and optimization, might prove useful in an operational casework setting. The principal advantages of HRM in terms of timeliness and cost may facilitate the technology transfer of mRNA profiling methodology into forensic casework.\n\n\nConsent\n\nBody fluids were collected from volunteers using procedures approved by the University’s Institutional Review Board. Informed written consent was obtained from each donor.",
"appendix": "Author contributions\n\n\n\nAuthor Jack Ballantyne conceived the study, designed the experiments, reviewed and analyzed results. Author Erin Hanson assisted with the design of experiments, performed the laboratory work, and reviewed and analyzed results. Both authors prepared and edited the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nSupport for this project was provided by the State of Florida through the National Center for Forensic Science at the University of Central Florida. The opinions, findings and conclusions or recommendations expressed in this publication are those of the authors and do not necessarily reflect those of the State of Florida. No grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe authors would like to acknowledge all of the anonymous donors who provided samples for this study. This work has been presented as a poster at the 25th World Congress of the International Society of Forensic Genetics, Melbourne, Australia (2013).\n\n\nSupplementary material\n\nHigh resolution derivative melt curve plot for a DNA sample for the MMP10 singleplex HRM assay. The presence of two products (78.5 and 83.1°C) was observed. The x-axis represents temperature (°C) and the y-axis represents the first derivative of the change of fluorescence with temperature (-dF/dT). The horizontal line on each plot represents the analysis threshold.\n\nRT-PCR products from RNA extracted from previously reported single source vaginal secretions samples were amplified in a multiplex reaction using a mRNA profiling assay for body fluid/tissue identification. The multiplex contains mRNA biomarkers for blood, semen, saliva, vaginal secretions, menstrual blood and skin. Shown here are two vaginal samples (A and B) in which semen was detected as indicated by the presence of the PRM2 and TGM4 (semen-specific biomarkers). This data is consistent with the detection of semen using the semen-saliva duplex HRM assay. The presence of vaginal secretions is indicated by the presence of CYP2B7P1. The x-axis indicates size in base pairs and the y-axis indicates relative fluorescence units (note: the y-axis scales are different for some panels due to varying signal intensities of the observed products). The grey areas represent the bins for biomarker identification. The biomarker name and relative fluorescence units are displayed.\n\n\nReferences\n\nSimons JL, Vintiner SK: Efficacy of several candidate protein biomarkers in the differentiation of vaginal from buccal epithelial cells. J Forensic Sci. 2012; 57(6): 1585–1590. 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PubMed Abstract | Publisher Full Text\n\nLaRue BL, King JL, Budowle B: A validation study of the Nucleix DSI-Semen kit--a methylation-based assay for semen identification. Int J Legal Med. 2013; 127(2): 299–308. PubMed Abstract | Publisher Full Text\n\nLee HY, Park MJ, Choi A, et al.: Potential forensic application of DNA methylation profiling to body fluid identification. Int J Legal Med. 2012; 126(1): 55–62. PubMed Abstract | Publisher Full Text\n\nMadi T, Balamurugan K, Bombardi R, et al.: The determination of tissue-specific DNA methylation patterns in forensic biofluids using bisulfite modification and pyrosequencing. Electrophoresis. 2012; 33(12): 1736–1745. PubMed Abstract | Publisher Full Text\n\nWasserstrom A, Frumkin D, Davidson A, et al.: Demonstration of DSI-semen--A novel DNA methylation-based forensic semen identification assay. Forensic Sci Int Genet. 2013; 7(1): 136–142. PubMed Abstract | Publisher Full Text\n\nCourts C, Madea B: Specific micro-RNA signatures for the detection of saliva and blood in forensic body-fluid identification. J Forensic Sci. 2011; 56(6): 1464–1470. PubMed Abstract | Publisher Full Text\n\nHanson E, Lubenow H, Ballantyne J: Identification of forensically relevant body fluids using a panel of differentially expressed microRNAs. Forensic Sci Int Genet. 2009; 2(1): 503–504. Publisher Full Text\n\nHanson E, Ballantyne J: RNA Profiling for the Identification of the Tissue Origin of Dried Stains in Forenic Biology. Forensic Sci Rev. 2010; 22(2): 145–157. Publisher Full Text\n\nHanson EK, Lubenow H, Ballantyne J: Identification of Forensically Relevant Body Fluids Using a Panel of Differentially Expressed microRNAs. Anal Biochem. 2009; 387(2): 303–314. PubMed Abstract | Publisher Full Text\n\nWang Z, Luo H, Pan X, et al.: A model for data analysis of microRNA expression in forensic body fluid identification. Forensic Sci Int Genet. 2012; 6(3): 419–423. PubMed Abstract | Publisher Full Text\n\nWang Z, Zhang J, Luo H, et al.: Screening and confirmation of microRNA markers for forensic body fluid identification. Forensic Sci Int Genet. 2013; 7(1): 116–123. PubMed Abstract | Publisher Full Text\n\nZubakov D, Boersma AW, Choi Y, et al.: MicroRNA markers for forensic body fluid identification obtained from microarray screening and quantitative RT-PCR confirmation. Int J Legal Med. 2010; 124(3): 217–226. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBauer M, Patzelt D: Evaluation of mRNA markers for the identification of menstrual blood. J Forensic Sci. 2002; 47(6): 1278–1282. PubMed Abstract\n\nBauer M, Patzelt D: Protamine mRNA as molecular marker for spermatozoa in semen stains. Int J Legal Med. 2003; 117(3): 175–179. PubMed Abstract | Publisher Full Text\n\nHaas C, Klesser B, Maake C, et al.: mRNA profiling for body fluid identification by reverse transcription endpoint PCR and realtime PCR. Forensic Sci Int Genet. 2009; 3(2): 80–88. PubMed Abstract | Publisher Full Text\n\nHaas C, Hanson E, Kratzer A, et al.: Selection of highly specific and sensitive mRNA biomarkers for the identification of blood. Forensic Sci Int Genet. 2011; 5(5): 449–458. PubMed Abstract | Publisher Full Text\n\nHaas C, Hanson E, Bar W, et al.: mRNA profiling for the identification of blood--results of a collaborative EDNAP exercise. Forensic Sci Int Genet. 2011; 5(1): 21–26. PubMed Abstract | Publisher Full Text\n\nHaas C, Hanson E, Kratzer A, et al.: Selection of highly specific and sensitive mRNA biomarkers for the identification of blood. Forensic Sci. Int Genet. 2011; 5(5): 449–458. PubMed Abstract | Publisher Full Text\n\nHaas C, Hanson E, Ballantyne J: Capillary electrophoresis of a multiplex reverse transcription-polymerase chain reaction to target messenger RNA markers for body fluid identification. Methods Mol Biol. 2012; 830: 169–183. PubMed Abstract | Publisher Full Text\n\nHaas C, Hanson E, Anjos MJ, et al.: RNA/DNA co-analysis from blood stains--results of a second collaborative EDNAP exercise. Forensic Sci Int Genet. 2012; 6(1): 70–80. PubMed Abstract | Publisher Full Text\n\nHaas C, Hanson E, Anjos MJ, et al.: RNA/DNA co-analysis from human saliva and semen stains--results of a third collaborative EDNAP exercise. Forensic Sci Int Genet. 2013; 7(2): 230–239. PubMed Abstract | Publisher Full Text\n\nHanson E, Haas C, Jucker R, et al.: Identification of skin in touch/contact forensic samples by messenger RNA profiling. Forensic Sci Int Genet. 2011; 3(1): e305–e306. Publisher Full Text\n\nHanson E, Haas C, Jucker R, et al.: Specific and sensitive mRNA biomarkers for the identification of skin in 'touch DNA' evidence. Forensic Sci Int Genet. 2012; 6(5): 548–558. PubMed Abstract | Publisher Full Text\n\nHanson EK, Ballantyne J: Highly specific mRNA biomarkers for the identification of vaginal secretions in sexual assault investigations. Sci Justice. 2013; 53(1): 14–22. PubMed Abstract | Publisher Full Text\n\nJuusola J, Ballantyne J: Messenger RNA profiling: a prototype method to supplant conventional methods for body fluid identification. Forensic Sci Int. 2003; 135(2): 85–96. PubMed Abstract | Publisher Full Text\n\nJuusola J, Ballantyne J: Multiplex mRNA profiling for the identification of body fluids. Forensic Sci Int. 2005; 152(1): 1–12. PubMed Abstract | Publisher Full Text\n\nJuusola J, Ballantyne J: mRNA profiling for body fluid identification by multiplex quantitative RT-PCR. J Forensic Sci. 2007; 52(6): 1252–1262. PubMed Abstract | Publisher Full Text\n\nNussbaumer C, Gharehbaghi-Schnell E, Korschineck I: Messenger RNA profiling: a novel method for body fluid identification by real-time PCR. Forensic Sci Int. 2006; 157(2–3): 181–186. PubMed Abstract | Publisher Full Text\n\nRichard ML, Harper KA, Craig RL, et al.: Evaluation of mRNA marker specificity for the identification of five human body fluids by capillary electrophoresis. Forensic Sci Int Genet. 2012; 6(4): 452–460. PubMed Abstract | Publisher Full Text\n\nRoeder AD, Haas C: mRNA profiling using a minimum of five mRNA markers per body fluid and a novel scoring method for body fluid identification. Int J Legal Med. 2013; 127(4): 707–721. PubMed Abstract | Publisher Full Text\n\nVisser M, Zubakov D, Ballantyne KN, et al.: mRNA-based skin identification for forensic applications. Int J Legal Med. 2011; 125(2): 253–263. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKohlmeier F, Schneider PM: Successful mRNA profiling of 23 years old blood stains. Forensic Sci Int Genet. 2012; 6(2): 274–276. PubMed Abstract | Publisher Full Text\n\nSetzer M, Juusola J, Ballantyne J: Recovery and stability of RNA in vaginal swabs and blood, semen, and saliva stains. J Forensic Sci. 2008; 53(2): 296–305. PubMed Abstract | Publisher Full Text\n\nZubakov D, Hanekamp E, Kokshoorn M, et al.: Stable RNA markers for identification of blood and saliva stains revealed from whole genome expression analysis of time-wise degraded samples. Int J Legal Med. 2008; 122(2): 135–142. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZubakov D, Kokshoorn M, Kloosterman A, et al.: New markers for old stains: stable mRNA markers for blood and saliva identification from up to 16-year-old stains. Int J Legal Med. 2009; 123(1): 71–74. PubMed Abstract | Publisher Full Text\n\nIntemann C, Thye T, Sieversten J, et al.: Genotyping of IRGM tetranucleotide promoter oligorepeats by fluorescence resonance energy transfer. Biotechniques. 2009; 46(1): 58–60. PubMed Abstract\n\nNguyen-Dumont T, Calvez-Kelm FL, Forey N, et al.: Description and validation of high-throughput simultaneous genotyping and mutation scanning by high-resolution melting curve analysis. Hum Mutat. 2009; 30(6): 884–890. PubMed Abstract | Publisher Full Text | Free Full Text\n\nReed GH, Wittwer CT: Sensitivity and specificity of single-nucleotide polymorphism scanning by high-resolution melting analysis. Clin Chem. 2004; 50(10): 1748–1754. PubMed Abstract | Publisher Full Text\n\nZhou L, Errigo RJ, Lu H, et al.: Snapback primer genotyping with saturating DNA dye and melting analysis. Clin Chem. 2008; 54(10): 1648–1656. PubMed Abstract | Publisher Full Text\n\nMaat W, van der Velden PA, Out-Luiting C, et al.: Epigenetic inactivation of RASSF1a in uveal melanoma. Invest Ophthalmol Vis Sci. 2007; 48(2): 486–490. PubMed Abstract | Publisher Full Text\n\nWorm J, Aggerholm A, Guldberg P: In-tube DNA methylation profiling by fluorescence melting curve analysis. Clin Chem. 2001; 47(7): 1183–1189. PubMed Abstract\n\nAten E, White SJ, Kalf ME, et al.: Methods to detect CNVs in the human genome. Cytogenet Genome Res. 2008; 123(1–4): 313–321. PubMed Abstract | Publisher Full Text\n\nBruder CE, Piotrowski A, Gijsbers AA, et al.: Phenotypically concordant and discordant monozygotic twins display different DNA copy-number-variation profiles. Am J Hum Genet. 2008; 82(3): 763–771. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDobrowolski SF, Hendrickx AT, van den Bosch, et al.: Identifying sequence variants in the human mitochondrial genome using high-resolution melt (HRM) profiling. Hum Mutat. 2009; 30(6): 891–898. PubMed Abstract | Publisher Full Text\n\nPepers BA, Schut MH, Vossen RH, et al.: Cost-effective HRMA pre-sequence typing of clone libraries; application to phage display selection. BMC Biotechnol. 2009; 9(50): 1–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVossen RH, Aten E, Roos A, et al.: High-resolution melting analysis (HRMA): more than just sequence variant screening. Hum Mutat. 2009; 30(6): 860–866. PubMed Abstract | Publisher Full Text\n\nSeipp MT, Durtschi JD, Voelkerding KV, et al.: Multiplex amplicon genotyping by high-resolution melting. J Biomol Tech. 2009; 20(3): 160–164. PubMed Abstract | Free Full Text\n\nFleming RI, Harbison S: The development of a mRNA multiplex RT-PCR assay for the definitive identification of body fluids. Forensic Sci Int Genet. 2010; 4(4): 244–256. PubMed Abstract | Publisher Full Text\n\nDong JC, Dong H, Campana A, et al.: Matrix metalloproteinases and their specific tissue inhibitors in menstruation. Reproduction. 2002; 123(5): 621–631. PubMed Abstract | Publisher Full Text\n\nSalamonsen LA, Woolley DE: Matrix metalloproteinases in normal menstruation. Hum Reprod. 1996; 11(Suppl 2): 124–133. PubMed Abstract\n\nCook R, Evett I, Jackson G, et al.: A hierarchy of propositions: deciding which level to address in casework. Science & Justice. 1998; 38(4): 231–239. Publisher Full Text\n\nAlberts B, Bray D, Lewis J, et al.: Molecular Biology of the Cell, 2nd, Garland Publishing, New York, NY. 1994. Reference Source"
}
|
[
{
"id": "2871",
"date": "09 Jan 2014",
"name": "Gustavo J. Gutierrez Gonzalez",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHanson & Ballantyne report in this article a novel use for High Resolution Melt analysis of RNA samples in order to identify single or mixed human body fluids (and tissues), namely vaginal secretions, skin, blood, menstrual blood, saliva, or semen using a series of molecular biomarkers: IL19, IL1F7 or CCL27, ALAS2, MMP10, HTN3, and TGM4, respectively. Identification of these body fluids/tissues can be of crucial importance in forensic sciences and criminal cases. The methodology appears to be quite specific and versatile, and according to the authors, less expensive and time-consuming compared to similar mRNA-based platforms such as quantitative real-time polymerase chain reaction or capillary electrophoresis. Remarkably, the technique can allow the identification of up to 6 body fluids in one single reaction (i.e., hexaplex assay). The paper is very well written, scientifically sound and their results are very clear and adequately interpreted and discussed. I support its approval in the journal F1000 Research. I also have several comments to be eventually addressed: Would there be a way to differentiate between blood and menstrual blood samples? Would that be something important to achieve? Have the authors used (or planned to use) this technique with samples such as sweat, urine, or hair? Additionally, could the technique differentiate human from animal samples (using the same biomarkers)? Can the authors give a little bit more information about the size of the amplicons generated by PCR for the different biomarkers employed and also about the design of the primers employed? How reproducible is the assay for one sample coming from an individual? We noticed that when you compare tables 2 and 4, there is a significant improvement of the specificity using the IL19 marker in the hexaplex assay. As mentioned by the authors, they have used a different reverse primer for IL19 in the hexaplex assay. We wonder whether this new reverse primer can also improve the specificity for IL19 to differentiate vaginal secretions from menstrual blood also in single, duplex, or triplex analyses. Regarding the possible amplification of MMP3 by MMP10 primers in the menstrual blood samples under certain experimental conditions. I think it could be important to test that this is indeed the case; maybe by designing MMP3 specific primers that confirm this particular peak or eventually sequencing the products that were generated. Finally, I got the impression from the experiments in which the authors mixed blood with decreasing amounts of semen that the technique does not necessarily reflect quantitative trends. Is that indeed the case? Could quantitation be ultimately important in a forensic case?",
"responses": [
{
"c_id": "682",
"date": "29 Jan 2014",
"name": "Jack Ballantyne",
"role": "Author Response",
"response": "Would there be a way to differentiate between blood and menstrual blood samples? Would that be something important to achieve?The described hexaplex assay has a biomarker for both blood and menstrual blood. Therefore they will be distinguished using this assay. However, menstrual blood will also contain varying amounts of peripheral blood and therefore both the blood (ALAS2) and menstrual blood (MMP10) biomarkers may be present in menstrual blood samples. If the sample is strictly peripheral blood, only the ALAS2 (blood) biomarker would be present and the menstrual blood (MMP10) marker would be absent. This can be important in some cases where intimate contact is implied or suggested based on the circumstances of the case and a suspect argues that it is blood from the nose of the victim, for example, during an assault but not a sexual assault. The identification of menstrual blood as the source would indicate more intimate contact. Additionally during sexual assault investigations, peripheral blood could indicate that trauma had taken place to the female reproductive system, and therefore a determination that the blood present is not simply menstrual blood from normal menstruation could be probative. Have the authors used (or planned to use) this technique with samples such as sweat, urine, or hair? Additionally, could the technique differentiate human from animal samples (using the same biomarkers)?We have not yet used this technique with other types of samples such as sweat, urine or hair. The HRM approach described here could certainly be utilized for the identification of other forensically relevant materials if suitable biomarkers were identified. This could also include internal organs and tissues tissues such as brain, heart, etc. This work was focused on those body fluids and tissues commonly found at the crime scene, but the approach could certainly be used for other fluids and tissues in the future. Can the authors give a little bit more information about the size of the amplicons generated by PCR for the different biomarkers employed and also about the design of the primers employed?All of the primers were designed using Primer 3 software. Primers were designed to either span exon-exon boundaries (no DNA amplification) or designed to be in separate exons (so that the DNA product size would be different than the RNA product size due to the inclusion of an intron). Tables 1 and 3 have been updated to include amplicon size and also the exon location of the primers. How reproducible is the assay for one sample coming from an individual?The assay is highly reproducible within an individual. Typically in a single experiment the Tm curves from the same individual are exactly superimposable upon one another The donors used in this study were also used repeatedly throughout testing, with both separate RNA extractions and multiple RT-PCR’s from the same individual extract being employed. No significant differences in Tm were observed. We noticed that when you compare tables 2 and 4, there is a significant improvement of the specificity using the IL19 marker in the hexaplex assay. As mentioned by the authors, they have used a different reverse primer for IL19 in the hexaplex assay. We wonder whether this new reverse primer can also improve the specificity for IL19 to differentiate vaginal secretions from menstrual blood also in single, duplex, or triplex analyses. Menstrual blood samples will, by nature of the source of the material, contain varying amounts of vaginal secretions. Therefore IL19 in a singleplex assay still will never provide sufficient resolution to distinguish between a vaginal and menstrual sample, as both could contain vaginal secretions. While every menstrual blood sample may not contain sufficient levels of vaginal material to result in IL19 detection, vaginal secretions will still be present in such samples to a varying extent. Therefore a menstrual blood-specific biomarker is typically needed to definitively identify the presence of menstrual blood. A duplex reaction with IL19 and MMP10 for example would permit a determination of vaginal secretions and menstrual blood. Vaginal secretions only samples should have only IL19, whereas menstrual blood samples could contain only MMP10 or a mixture of MMP10 and IL19.Regarding the possible amplification of MMP3 by MMP10 primers in the menstrual blood samples under certain experimental conditions. I think it could be important to test that this is indeed the case; maybe by designing MMP3 specific primers that confirm this particular peak or eventually sequencing the products that were generated.It is certainly of interest to determine whether the additional product is in fact MMP3 and to determine if specific MMP10 and MMP3 primer sets could be distinguished. This will be the subject of future work, although it is important to note that this uncertainty does not interfere with the accurate interpretation of the body fluid results using the current assay. Finally, I got the impression from the experiments in which the authors mixed blood with decreasing amounts of semen that the technique does not necessarily reflect quantitative trends. Is that indeed the case? Could quantitation be ultimately important in a forensic case?In our evaluation so far of data from the HRM assays, the height of the melt peak, somewhat surprisingly, does not appear to correlate with the amount of analyte. More in depth studies need to be performed in order to determine possible reasons for this. It should be noted that the melt peak is the first derivative of fluorescence with temperature. It might be the case that other transforms of the data other than the first derivative might prove to be better correlated with analyte input.The ability to make a relative quantitative assessment of the amount of each body fluid present in an unknown sample would be useful. The RNA quantitation method we employ, namely the binding of the fluorescent dye RiboGreen, provides an overall assessment of the amount of RNA present, but cannot provide quantitative information regarding the amount of each fluid present if the sample contains more than one body fluid. While a quantitative assessment is not critical for a determination of the presence or absence of a body fluid, a quantitative assessment could be provide important probative information for admixed body fluid samples."
}
]
},
{
"id": "3637",
"date": "19 Feb 2014",
"name": "Titia Sijen",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHanson and Ballantyne describe the development of HRM-based single- and multiplex assays for the identification of forensically relevant cell types. These assays are potentially a rapid and relatively inexpensive alternative for mRNA profiling methodologies such as qRT-PCR assays and CE analysis of fluorescently labeled RT-PCR products. The research is scientifically sound and follows a well-designed stepwise approach. The study is focused at assay development and specificity testing. Known markers were chosen (some more studied such as HTN3, ALAS2, MMP10, TGM4 than others eg IL19, CCL27, IL1F7) that show good specificity with the body fluids tested.The rationale, results and interpretation of the data are well-described although some figures could be improved (if possible) by using different colors: the use of pink and red to discriminate MMP10 and ALAS2 in Fig2A is somewhat challenging to the eye, and when yellow is used (Fig1D, 2B, 3A, 4B) I found some details quite hard to discern. The authors state ‘the results demonstrate the potential use of HRM assays for the screening of biological evidence’ and ‘we describe the development and initial performance evaluation of the developed HRM assays’. This raises the question what would be needed before HRM assays would be suited for casework?In our laboratory, numerous cases have been analyzed using end-point RT-PCR multiplexes on both cell and organ typing detected by CE. These cases have taught us that most/many forensic casework samples will be 1) mixtures of various cell types and 2) of limited stain size. Thus, besides good specificity, sensitivity and the ability to handle (unequal) mixtures are important performance characteristics for RNA assays. The capacity to analyze unequal mixtures will depend on the analysis method. When using CE platforms for instance, high mixture components may result in pull-up signals and artifact peaks that can mask the presence of the minor components or complicate RNA profile interpretation.Thus, I would like to invite the authors to comment on the expected performance of HRM assays when analyzing unequal mixtures.Furthermore, as this initial study did not yet assess sensitivity, I am interested in the authors’ thoughts on the sensitivity that they perceive for HRM-based RNA assays. Overall, I compliment the authors with this study and the novelty they bring to the forensic field.",
"responses": [
{
"c_id": "717",
"date": "24 Feb 2014",
"name": "Jack Ballantyne",
"role": "Author Response",
"response": "\"The rationale, results and interpretation of the data are well-described although some figures could be improved (if possible) by using different colors: the use of pink and red to discriminate MMP10 and ALAS2 in Fig2A is somewhat challenging to the eye, and when yellow is used (Fig1D, 2B, 3A, 4B) I found some details quite hard to discern.\"The colors are used to aid visually distinguishing each body fluid. There are always variations between computers and printers, and therefore the original color selected in the instrument software is not always as clearly represented. We also labeled each biomarker so that its location would be clearly indicated on each diagram. We will keep color selection in mind for future publications. \"The authors state ‘the results demonstrate the potential use of HRM assays for the screening of biological evidence’ and ‘we describe the development and initial performance evaluation of the developed HRM assays’. This raises the question what would be needed before HRM assays would be suited for casework?\"A full developmental validation in accordance with SWGDAM guidelines would be required. This would include a more comprehensive evaluation of sensitivity, specificity, reproducibility and performance with mixtures and mock casework samples.In our laboratory, numerous cases have been analyzed using end-point RT-PCR multiplexes on both cell and organ typing detected by CE. These cases have taught us that most/many forensic casework samples will be 1) mixtures of various cell types and 2) of limited stain size. Thus, besides good specificity, sensitivity and the ability to handle (unequal) mixtures are important performance characteristics for RNA assays. The capacity to analyze unequal mixtures will depend on the analysis method. When using CE platforms for instance, high mixture components may result in pull-up signals and artifact peaks that can mask the presence of the minor components or complicate RNA profile interpretation. \"Thus, I would like to invite the authors to comment on the expected performance of HRM assays when analyzing unequal mixtures.\"A blood-semen mixture, with decreasing amounts of semen in each mixture, was evaluated using the duplex assays. Both body fluids were correctly identified in each of these mixtures. Successful detection of both components of a saliva-vaginal admixed sample was also observed for the hexaplex assay. While these were not large-scale comprehensive mixture studies, we are hopeful that the HRM assays will permit the ability to identify all components in admixed body fluid samples. Similar to the experience of the referee with CE based assays, It is likely that some mixture types will be challenging to define because of the particular mixture composition or because one or more of the components are present as a minor fraction. In our experience with CE as well, highly abundant biomarkers tend to dominate detection and can mask the presence of a minor component. It will be interesting to see with future studies whether the use of the HRM assays will permit an improved detection of minor component fluids or tissues. So while more comprehensive mixtures studies are needed (and will be included as part of a full developmental validation), we are hopeful that the successful initial results presage the efficacy of HRM assays to perform reasonably well with mixtures. [See also the response to the first referee report]. \"Furthermore, as this initial study did not yet assess sensitivity, I am interested in the authors’ thoughts on the sensitivity that they perceive for HRM-based RNA assays.\"We performed initial sensitivity studies for each of the HRM duplex assays. The sensitivity of each of the duplex assays was evaluated by testing a range of input total RNA, from 15pg to 100ng (15pg, 25pg, 50pg, 100pg, 250pg, 500pg. 1mg, 5ng, 10ng, 25ng, 50ng and 100ng). For the blood/menstrual blood duplex assay, both biomarkers (ALAS2 and MMP10) were detected using picogram quantities of input total RNA (consistently detected using 250 pg or more; variable below 250pg (positive results for both biomarkers with 15 and 50pg input, but not 25 and 100pg)). For the saliva/semen assay, both biomarkers (HTN3 and TGM4) were detected using as little as 25pg input. Using 15pg input, HTN3 was also detected but not TGM4. Therefore the sensitivities of these two duplex assays appear to be highly sensitive, requiring only picogram levels of input total RNA. Additionally, it should be noted that the total RNA input amounts listed represent the amount used in the RT reaction. Detection of product in the real time assays is performed with 1/10th of the RT input amount (2ml cDNA/20ml reaction). For the vaginal secretions/skin duplex assay, 5ng of input RNA (still only 500pg for detection) was required for IL19. Skin was successfully detected with as little as 100pg total RNA RT input. Since only one donor was tested for this initial sensitivity work, the actual sensitivity limits need to be determined with additional testing. For vaginal secretions, since no human specific RNA quantitation method is currently available, there is always a potential for the obtained RNA quantitation value to be artificially higher due to the presence of endogenous bacteria. Therefore, the total RNA input used for these samples may not be fully accurate which will then affect the sensitivity results. But overall, these assays appear to require picogram levels of input total RNA, which would make them suitable for use with forensic, samples.An initial sensitivity check for the hexaplex assay was also performed (again only one donor per fluid; 5 pg to 25 ng range of total RNA input into the RT reaction). The body fluids were detected down to varying lower input limits: blood (ALAS2) - 25 pg; menstrual blood (MMP10) - 250 pg; skin (CCL27) - 1ng; semen - 5ng; vaginal secretions – consistently with 1 ng or more input (variable detection in the 5pg to 1ng range); saliva (HTN3) – not determined at this time.While we performed this preliminary sensitivity evaluation, it was not included in the manuscript since only one donor was evaluated. More comprehensive sensitivity studies, particularly for the hexaplex system, will be included in a future publication describing a comprehensive testing and evaluation of the HRM assay."
}
]
}
] | 1
|
https://f1000research.com/articles/2-281
|
https://f1000research.com/articles/2-88/v1
|
14 Mar 13
|
{
"type": "Research Article",
"title": "Learning intrinsic excitability in medium spiny neurons",
"authors": [
"Gabriele Scheler"
],
"abstract": "We present an unsupervised, local activation-dependent learning rule for intrinsic plasticity (IP) which affects the composition of ion channel conductances for single neurons in a use-dependent way. We use a single-compartment conductance-based model for medium spiny striatal neurons in order to show the effects of parameterization of individual ion channels on the neuronal activation function. We show that parameter changes within the physiological ranges are sufficient to create an ensemble of neurons with significantly different activation functions. We emphasize that the effects of intrinsic neuronal variability on spiking behavior require a distributed mode of synaptic input and can be eliminated by strongly correlated input. We show how variability and adaptivity in ion channel conductances can be utilized to store patterns without an additional contribution by synaptic plasticity (SP). The adaptation of the spike response may result in either \"positive\" or \"negative\" pattern learning. However, read-out of stored information depends on a distributed pattern of synaptic activity to let intrinsic variability determine spike response. We briefly discuss the implications of this conditional memory on learning and addiction.",
"keywords": [
"A role for modification of activation functions",
"or intrinsic plasticity (IP)",
"for behavioral learning has been demonstrated for a number of systems1–3. For instance",
"in rabbit eyeblink conditioning",
"when ion channels related to after hyperpolarization are being suppressed by a learning event",
"they can become permanently suppressed. This has been shown for pyramidal cells of hippocampal areas CA1 and CA3",
"and for cerebellar Purkinje cells4",
"5. In some cases",
"these changes are permanent and still present after 30 days6",
"7",
"in other cases",
"intrinsic changes disappear after 3–7 days",
"while the behavioral memory remains intact",
"raising questions about the long-term component of intrinsic plasticity in these systems. There are at the present time conflicting ideas on the significance of IP compared to synaptic plasticity1",
"8",
"and the range of functions that IP may have in adaptivity9–12."
],
"content": "Introduction\n\nA role for modification of activation functions, or intrinsic plasticity (IP), for behavioral learning has been demonstrated for a number of systems1–3. For instance, in rabbit eyeblink conditioning, when ion channels related to after hyperpolarization are being suppressed by a learning event, they can become permanently suppressed. This has been shown for pyramidal cells of hippocampal areas CA1 and CA3, and for cerebellar Purkinje cells4,5. In some cases, these changes are permanent and still present after 30 days6,7, in other cases, intrinsic changes disappear after 3–7 days, while the behavioral memory remains intact, raising questions about the long-term component of intrinsic plasticity in these systems. There are at the present time conflicting ideas on the significance of IP compared to synaptic plasticity1,8, and the range of functions that IP may have in adaptivity9–12.\n\nA few computational models have been proposed that show how modification in activation functions can be achieved with ion channel based models of realistic single neurons. Marder and colleagues have developed an approach, where they sample a very large parameter space for conductances of ion channels, exploring nonlinearities in the relation between conductances and neural spiking behavior13–15. The motivation for this research are observations about neuromodulation and intrinsic plasticity in specific neurons of an invertebrate ganglion (e.g. LeMasson et al.,16). They have noted that large variations in some parameters may have little effect on neuronal behavior, while comparatively small variations in certain regions in parameter space may change response properties significantly. They also suggest that neuro modulation may provide an efficient means of targeting regions in parameter space with significant effects on response properties14.\n\nA study by Stemmler and Koch17 assumed the goal of modification of activation functions is to achieve an optimal distribution of firing rates for a population of neurons. The idea was that by tuning each neuron to a different band of the frequency spectrum, the full bandwidth of frequencies could be employed for information transfer. This goal was achieved by adjusting Na+, K+, and C2+ channels for a generically defined neuron until a desired frequency was stably reached.\n\nWe present a different approach, where the modification of activation functions reflects the history of exposure to stimuli for a specific neuron. Similarly18,19, suggested that synaptic LTP/LTD and linear regulations of intrinsic excitability could operate in a synergistic fashion. However, in our approach, different types of synaptic stimulation result in state changes for the neuronal unit, influencing its capacity for read-out of stored intrinsic properties. Thus, intrinsic plasticity is conceptualized as fundamentally different from synaptic plasticity which does not encompass such a state change. The learning rule that we derive as the basis for adjustment concerns one-dimensional upregulation or down-regulation of excitability in the “read-out” state of the neuron, and affecting only this state. This rule uses neural activation, significantly determined by intracellular calcium for the learning parameter, which can be shown to be biologically well-motivated (cf. also e.g. LeMasson et al.,16).\n\n\nMaterials and methods\n\nThe membrane voltage Vm is modeled as\n\n\n\nThe individual currents are modeled by conductances, state variables and the reversal potential:\n\n\n\nThe dynamics are defined using state variables for activation (m) and inactivation (h). The types of equations used for the dynamics are:\n\n\n\n\n\n\n\nThe state variables can be defined indirectly using\n\n\n\nand\n\n\n\nand one of the (Equation 1–Equation 3) with different values for λ (λα, λβ), Vi (Viα, Viβ) and Vc (Vcα, Vcβ). We use this method for the ion channels in Table 1.\n\nThe types of equations (Eqα, Eqβ) are 1 for exponential, 2 for logistic, and 3 for linexp (see text for the equations). The reversal potential Erev is given in mV.\n\nThe state variables can also be directly defined (cf. Goldman et al.,14):\n\n\n\nThe parameters used are m∞ = m0, h∞ = h0, τm and τh as in Table 2. Again, we use one of the (Equation 1–Equation 3) with the λ parameters (λm0 and λh0) set to 1. These representations are mathematically equivalent and related by\n\n\n\nExponents (p, q), conductance g¯ (in mS), parameters m0 and h0 for a logistic function (equation type 2), and time constants τm, τh are shown. The reversal potential Erev is given in mV.\n\nStandard parameter values for the modeling of ion channels (“naive state”) were compared with several publications. Parameter values for IK, INa and Ileak were adapted from20, for L-type calcium channels (ICaL) from21 and22, see Table 1.\n\nParameters for slow A-type K channels (IAs) were adapted from23,24, for fast A-type K channels (IAf) from25, for inward rectifying K channels (IKir) from26, and the resulting parameter tables were compared with27 and28, see Table 2.\n\nThe maximum conductance of different ion channels can be expressed by a scaling factor in the membrane potential equations as in Eq. 2 (for synaptic currents) or Eq. 3 (for synaptic conductances), cf. Gruber et al.,28.\n\n\n\n\n\nBoth neuromodulator (NM)-independent and NM-dependent modifications may coexist in a neuron, as expressed in Eq. 4 ([NM] stands for the level of synaptic availability of a neuromodulator NM).\n\n\n\nIn this paper, for simplicity, we shall refer to (Eq. 2) as the generic format for intrinsic adaptation, with the understanding that µ is replaceable by [NM]k.\n\nPhysiological ranges for µ can be estimated by various means. There are measurements for variability in electrophysiologically defined membrane behavior (current threshold, spike response to current pulses etc.29,30) that are typically expressed as standard errors (e.g., 16–20% for current threshold29). There are also attempts at classifying MSN (Medium Spiny Neuron) cells into different ‘types’ based on their electrophysiological profile31,32. Modeling shows that variability of ion channel conductances with a range of ±40% matches measures of electrophysiological variability and reproduces the ranges for MSN types (data not shown). Interestingly, direct measurements for dopamine D1 receptor-mediated changes on ion channel conductances are approximately in the same ranges (±30 – 40%28). Our discussion is thus based on an estimate of µ ranging from 0.6 – 1.4 for each channel.\n\nSynaptic input is defined by overlays of the excitatory postsynaptic potentials (EPSPs) generated by N individual Poisson-distributed spike trains with mean interspike interval τsyn. Each EPSP is modeled as a spike with peak amplitude I0 = 1.2µA/cm2 and exponential decay with τ = 2.5ms similar to22,33. IPSPs are modeled in a similar way with I0 = –0.4µA/cm2. This corresponds to 0.5nA (–0.2nA) as peak current (with 1nA = 2.3µA/cm2). Synaptic conductances are calculated by gsyn = Isyn/(Vm – V0) with V0 set to 0mV. We have tuned the model to gsyn= 0.0035mS/cm2 for a first spike for the naive or standard neuron (all µ = 1). At –40mV, this is 0.0035mS/cm2 * (–40mV) = – 1.4µA/cm2 or 0.6nA, which corresponds to the experimentally measured average value for the rheobase in29. We may increase the correlation in the input by using a percentage W of neurons which fire at the same time. Higher values for W increase the amplitude of the fluctuations of the input (cf. Benucci et al.,34).\n\nThe simulator has been implemented in Matlab and is available at https://github.com/gscheler/CNeuroSim.git. The entire code is interpreted and no specific code optimizations have been applied. For numerical integration, the solver ode45 was used.\n\n\nResults\n\nWe explore the impact of small variations in ion channel conductances on the shape of the activation function. As an example, we show the current and conductance changes for a slowly inactivating A-type K+ channel (Kv1.2, IAs), L-type calcium channel (ICaL) and inward rectifying K+ channel (IKir) at different membrane potentials modulated by a scaling factor µ = {0.6, 0.8, 1.0, 1.2, 1.4} (Figure 1, Figure 2). Regulation of the voltage-dependence35 and even of the inactivation dynamics of an ion channel36 has also been shown, but these effects are not further discussed here.\n\nVariable factors (µ = {0.6 ... 1.4}) for the slowly inactivating K+-channel (Kv1.2, IAs), the L-type calcium channel (ICaL), and the inward rectifying K+ channel (IKir) are shown at different membrane voltages Vm (A) in an I-V plot, (B) as variability in conductance.\n\nVariable factors (µ = {0.6 ... 1.4}) for IAs, ICaL, and IKir as components of the activation function (gs vs. Vm). The activation function is defined as the membrane voltage response for different injected (synaptic) conductances (gs), and computed by solving Eq 3 for the membrane voltage Vm.\n\nWe can see that there are critical voltage ranges (around –50mV, around –80mV and starting at –40mV), where the conductance and the current are highest, and where scaling has a significant effect, while scaling has small or no effect in other voltage ranges. (The Na+ current has been disabled for this example to prevent the neuron from firing).\n\nIn Figure 3, we show the current over time–to graphically display the slow dynamics of the IAs and ICaL channel. Since we do not change the activation-inactivation dynamics of any channel in our model, we show currents only for µAs, µCaL = 1.\n\n(A) dynamics for the slow A channel IAs (B) the L-type Ca channel ICaL, and (C) for the set of ion channels used in the standard MSN model. We see a rise-time due to IAs and overlapping inactivation dynamics in the -55 to -40 mV range.\n\nWe can see that IAs activates moderately fast (20ms), while it inactivates with a half-time of about 300ms, depending on the voltage. For ICaL, activation is almost instantaneous, but inactivation is > 500ms.\n\nThe activation function for the MSN model shows a time-dependence only in the high-voltage range (at or above –55mV), whereas the components in the lower voltage ranges are not time-dependent.\n\nMathematically, we can consider the individual channels as a set of basis functions that allow function approximation for the activation function. Each particular adjustment of an activation function can be considered learning of a filter which is suited to a specific processing task. The activation-inactivation dynamics would provide a similar set of basis functions for the temporal domain. Of course, it is interesting to note which particular basis functions exist, and also how the temporal dimension is tied in with specific voltage-dependences. For instance, the slowly inactivating potassium channel IAs provides a skewed mirror image of the function of calcium-gated Sk/BK channels, which are responsible for after hyperpolarization, making different variants of frequency filters possible. On this basis, a mapping of ion channel components and their density or distributions in different types of neurons could provide an interesting perspective on direct interactions for neurons from different tissue types or brain areas, as well as e.g., between cholinergic interneurons and MSNs within striatum.\n\nTo further explore the influence of variability of the activation function, we apply realistic synaptic input with different amounts of correlation to individual MSNs (see Figure 4).\n\nResponse to inputs generated from N = 80 neurons with independent Poisson processes using different correlations parameters W = 0.2, 0.9 (A, B). Three slightly different neurons with µAs = 1.1,1.3,1.5 are shown under BOTH conditions. (A) Response variability and different firing rates for each neuron (here: 20, 26, 40Hz) occur with distributed (low correlation) input. (B) Highly correlated input produces reliable spiking and by implication a single firing rate (20Hz). The upper panel shows the membrane voltage, the middle panel shows the membrane conductances, and the lower panel shows the synaptic input as conductance.\n\nThis shows us that small adjustments in the contribution of a specific ion channel can result in significantly different spiking behavior even for identical synaptic input. This occurs when the input is distributed, i.e., has low correlation. In this case, the neurons spike independently of each other and with different frequencies. We can eliminate this effect by increasing the correlation of the input. Because of the slow activation/inactivation dynamics of the IAs channel, (latency of ≈ 20ms) only low correlated input activates these channels (“neuronal integrator mode”), but highly correlated inputs do not activate these channels, driving the membrane to spiking quickly (“coincidence detector mode”). Therefore correlated input can produce reliable spiking behavior for model neurons which differ in the relative contribution of the slow IAs channel. Distributed input, in contrast, activates slower ion channels, and can produce different tonic firing rates, here according to the contribution of the IAs channels, as long as strong synaptic input keeps the neuron in the relevant voltage range (“persistent activity”).\n\nSimilarly, the differential contribution of other channels (high-voltage gated L-type Ca-channels, hyperpolarization-activated GIRK channels or calcium-dependent Sk/Bk channels) will affect neuronal behavior, when the conditions for a prominent influence of these channels are met.\n\nWe are modeling a state of MSNs that exhibits regular tonic firing. In experimental studies37, showed that MSNs, similar to cortical neurons, exhibit upstate-downstate behavior, reminiscent of slow-wave sleep, under certain forms of anesthesia (ketamine, barbiturate). However, under neuroleptanalgesia (fentanyl-haloperidol)50, showed that MSNs can show driven activity, when cortical input is highly synchronized, and exhibit a state characterized by fluctuating synaptic inputs without rhythmic activity (i.e., without upstates/downstates), when cortical input is desynchronized. The regular, tonic spiking in this state is very low, much less than in the waking animal, which may be related to the dopamine block by haloperidol. This makes a waking state of MSNs characterized by regular tonic spiking at different firing rates probable.\n\nIn the following, we show how intrinsic excitability adaptation can lead to different recalled firing rates under appropriate synaptic stimulation. The model could thus reflect learning that is recalled or read out during MSN states under desynchronized cortical input–in contrast to highly synchronized input, which would homogenize the response of the coincidence detecting neurons and favor reliable transfer of spikes.\n\nThe general idea for learning intrinsic plasticity is to use a learning parameter h for each individual update of the conductance scaling factor µ. The direction of learning (h > 0 or h < 0) is determined from the neural activation (An) for each individual neuron. Neural activation is largely determined by intracellular calcium but here we estimate the neural activation An from the spike rate of the neuron, measured over 1s of simulated behavior (see Discussion).\n\nWe define a bidirectional learning rule dependent on an initial firing rate θ: excitability is increased by a step function h (with stepsize σ) when An is greater than θ, excitability is decreased when An is lower than θ (“positive learning”). This means, when the actual neural activation is higher than the initial firing rate, membrane adaptations aim to move the neuron to a higher excitability in order to create a positive memory trace of a period of high activation (which can then be replicated under distributed synaptic stimulation). The same mechanism applies to lower the excitability of a neuron.\n\n\n\nThis rule can also be implemented by individual increases in excitability after each action potential, and decreases of excitability for periods of time without action potentials. Initial experiments38,50 indeed show such adaptation of intrinsic excitability after individual spikes.\n\nThe function h can be applied to a single ion channel, such as IAs, but also to a number of ion channels in parallel: e.g., to mimic dopamine D1 receptor activation, h may be applied to µAs (upregulated with high An), µCaL (downregulated with high An), and µCaL (downregulated with high An).\n\nWe can show the effect of this learning rule on pattern learning. We generate synaptic inputs from a grid of 200 input neurons for a single layer of 10 MSNs. On this grid we project two stripes of width 4 as a simple input pattern Plearn by adjusting the mean interspike interval (ISI) for the corresponding input neurons to a higher value (ISI = 350ms for on vs. 750ms for off neurons, see Figure 5).\n\n200 input neurons (arranged as 20 × 10), 10 learning neurons, and definitions for 3 patterns. Only 37 of 80 data points for Pnoise are shown.\n\nWe apply the learning rule to each of the currents INa, ICaL and IAs. This mimics changes in dopamine D1 receptor sensitivity, which targets these ion channels. Adaptation can be weaker or stronger, depending on learning time (e.g., σ = 0.01, t = 20s (20 steps) (weak), t = 40s (40 steps) (strong)). After a number of steps, we achieve a distribution of µ-values that reflects the strength of the input (Table 3A).\n\nCaL and Na channels are adapted in the opposite direction to K channels.\n\nIn Figure 6, we obtain spike frequency histograms from the set of MSNs under different conditions. Figure 6A shows the naive response to the input pattern Plearn- high activation in two medial areas. After adaptation, this response is increased (Figure 6B). When we apply a test input of a random noise pattern Pnoise, we see that the learned pattern is still reflected in the spike histogram (Figure 6C). For positive learning, this process is theoretically unbounded, and only limited by the stepsize and the adaptation time. A saturation state could be defined to prevent unbounded learning, which would also allow to perform capacity calculations.\n\nspike frequency histograms for 10 adaptive neurons (θ=11.5Hz) (A) response of naive neurons to Plearn (B) response of Plearn-adapted neurons to Plearn (C) Plearn-adapted neurons tested with Pnoise. Average synaptic input (nA/cm2) for each neuron is shown on top. Responses in (A) and (B) to the same input Plearn are different, a pattern similar to Plearn emerges in response to uniform (noise) pattern input in (C).\n\nWe should note that applying just one pattern continuously results in a very simple learning trajectory: each update results in a step change in the relevant ion channel currents. However, we also show that the effects of stepwise adaptation of individual ion channels do not necessarily lead to a completely parallel adaptation of firing rate. In Figure 6 we see that adaptation is much stronger for high input rather than low input neurons. In this case, θ at 11.5Hz is a fairly low value for neurons to continue to lower their firing rate with stepwise adaptation of the chosen ion channels. This shows the importance of using appropriate tuning (“harnessing”) mechanisms to make highly nonlinear channels work in a purely linear learning context.\n\nClearly one of the results of learning is an altered spiking behavior of individual neurons dependent on their history. It is important to realize that this rule is based on neural activation, not synaptic input as a learning parameter – since synaptic input is constant during learning.\n\nWe show that this mechanism can be employed not only for positive trace learning, when excitability adaptation corresponds to frequency response, but also for negative trace learning, when excitability adaptation counteracts frequency response and approximates a target firing rate θ. This target rate could be set as a result of global inhibitory mechanisms corresponding to the expected mean Ai values under physiological stimulation. Accordingly, the neuron responds with decreases of excitability to high input ranges and increases of excitability to low input ranges (Figure 7).\n\nLearning results in different activation functions for high (4, 8), medium (3, 5, 7, 9) and low (1, 2, 6, 10) input.\n\n\n\nThis emphasizes that “homeostatic” responses – adjusting excitability in the opposite direction to the level of input can implement trace learning (pattern learning and feature extraction) as well.\n\nNegative learning rule results in a mirror image of parameter values compared to positive learning, as shown in Table 3B. The naive response is the same as before (Figure 6A). But here, after adaptation, the neurons have habituated to the input, and do not produce a strong response anymore (Figure 8A). When neurons are tested with Pnoise, an inverse version of the original pattern appears (Figure 8B). Similarly, when we apply a different pattern Ptest, we obtain a spike histogram, where the learned pattern is overlayed with the new input, resulting in a dampening of the frequency response for Ptest (Figure 8C).\n\nspike frequency histograms for 10 adaptive neurons (θ=11.5Hz) (A) habituation for neurons adapted to Plearn, (B) an inverse pattern for Plearn-adapted neurons tested with Pnoise and (C) interference (dampening of response) for a new pattern Ptest (naive: line-drawn bars, adaptive: filled bars). Average synaptic input (nA/cm2) for each neuron is shown on top. (A) shows a uniform response to patterend synaptic input and (B) a patterned response to uniform (noise) input. (C) shows a difference of response for naive vs. Plearn-adapted neurons to a new pattern Ptest.\n\nFor both positive and negative traces, learning is pattern-specific, i.e., training with homogeneous, fluctuating (high-low) noise, such as Pnoise, results in no adjustments (or computes an average). However, any prolonged sequence of neuron-selective stimulation results in neuron-selective patterns. This requires the population to be protected from prolonged stimulation with random patterns in a biological setting. We may assume most patterns to be meaningful and highly repetitive, while the neuron exists in a plastic state, while patterns may be random, when the neuron is not plastic (because it is stimulated with highly correlated or very low frequency input, saturated in its parameters or undergoes ion channel block by selected neuromodulators).\n\nThe whole approach to pattern storage and responses elicited to stimulation is summarized again in Figure 6 and Figure 8. We can see that pattern storage by changes in intrinsic excitability is useful for a short-term buffer system for complete patterns. Patterns are imprinted upon a set of neurons and remain available as long as they are not obliterated or overwritten by an opposite pattern. Presumably the pattern degrades over time. Training with a new pattern – during the period of active maintenance of the pattern–would result in cross-activation, i.e., the generation of a mixed pattern. This may well be a useful feature of a short-term pattern storage system. It allows for pattern integration, or pattern completion from different sources. Adapting intrinsic excitability has inherent limitations of storage capacity. We do not fully understand where patterns go after they have passed through the intrinsic buffer system, but we assume that synaptic growth, intracellular changes and membrane adaptations in a variety of trafficking proteins (receptors and channels) all play a role. In the simplest case, the intrinsic buffer system serves only to integrate and maintain a pattern of neural excitation until all the necessary synaptic adjustments that the memory system requires for permanent storage have been made. However, it is not clear, and actually highly doubtful at this time that the difference between short-term and long-term storage is clear-cut between intrinsic (neuronal) and synaptic (esp. glutamatergic synaptic) storage systems.\n\n\nDiscussion\n\nA number of experimental results show that intrinsic plasticity in MSNs may be prominently induced and regulated by intracellular calcium: It has been shown that e.g., the regulation of delayed rectifier K+-channels (Kv2.1 channels) is effectively performed by Ca2+ influx and calcineurin activation in cultured hippocampal neurons, which can be achieved by glutamate stimulation35. The regulation concerns marked dephosphorylation (reduction of conductance) plus a shift in voltage-dependence. It has also been shown that 20s of NMDA stimulation, or alternatively, increase of intracellular calcium, increases functional dopamine D1 receptor density at the membrane, which corresponds to an alteration in k for D1 parameters, targeting a number of ion channels simultaneously39. For deep cerebellar neurons, there has recently been some direct evidence on the conditions required that induce intrinsic plasticity. Here, alterations in intrinsic excitability can be induced by bursts of EPSPs and IPSPs, accompanied by dendritic calcium transients38. In striatal MSNs, it has been determined that synaptic stimulation at 1Hz does not cause significant calcium signals, but 10Hz stimulation causes moderate increases, and higher stimulation (up to 100Hz) significantly raises calcium levels40.\n\nIn the simulations, neural activation (An) is estimated from the number of spikes generated, measured over the simulated behavior. In the model case, the membrane potential is not used as a separate parameter, because membrane potential and spiking behavior are closely linked. However, when a neuron exhibits prominent upstates (periods of high membrane voltages with a variable number of actual spikes), membrane potential may need to be treated as an additional, independent component of An, since a great part of the intracellular calcium signal in striatal MSNs is being generated from high voltage activated NMDA and L-type calcium channels41. The number of spikes produced nonetheless seems important because of the phenomenon of backpropagating spikes. Backpropagating spikes enhance the calcium signal, thus providing a basis for a prominent role for spiking behavior, or firing rate, for defining intracellular calcium. The presence of backpropagation of spikes has recently been confirmed for MSNs41.\n\nIn general, the induction of intrinsic plasticity may be linked not only to intracellular calcium. There exists an intricate intracellular system of interactions between diffusible substances like calcium and cAMP, as well as a number of crucial proteins (RGS, calcineurin, PKA, PKC, other kinases and phosphatases) for regulating receptor sensitivity and ion channel properties, which are furthermore influenced by NM receptor activation. Thus the learning parameter h may be analyzed as being dependent not only on An, but also on [NM], and possibly even a third variable for a–slowly changing–intracellular state.\n\nLearning by intrinsic excitability seems particularly suitable for striatal MSNs, since they have few lateral connections, which provide only a small part of their total input42. When we have strong recurrent interaction, as in cortex, intrinsic excitability learning needs to adjust activation functions relative to each other, e.g., to ensure optimal distribution of activation functions. This probably happens in the cortical maps, such as frequency maps in auditory cortex43.\n\nIn hippocampus, synaptic and intrinsic modulation may potentiate each other (E-S potentiation1), but in other systems (e.g., striatum) antagonistic regulation may also exist (such as LTD combined with positive learning), with effects on the balance of synaptic vs. whole-cell localization for the storage of information.\n\nWhen ion channels are regulated by neuromodulation, we can use a factor [NM]k, where [NM] is the extracellular concentration of the ligand and k the receptor sensitivity (see Eq. 4). k stands for the influence that a NM signal of a certain strength has on a particular ion channel, i.e., the degree of coupling between NM receptor ligand binding and ion channel modification44. Typically, a signal [NM] will regulate several ion channels in parallel, but there may be different Ki for each ion channel.\n\nIf activation function adaptation proceeds by NM-activated k parameters, rather than unconditioned µ parameters, response to stimuli will consist of an early, non-modulated component, where the input pattern is reflected directly in the spiking frequency, and a later, modulated component, where habituation occurs for a learned pattern, or the stored pattern is reflected by overlaying a new stimulus and the stored pattern.\n\nNM signals orchestrate both adjustments in activation function and synaptic input, with NM activation often depressing synapses, but increasing the variability in the activation function through selected conductance changes (activating k-parameters). As a result, the input component of the signal is reduced in comparison to the stored intrinsic component after NM activation. Presumably, this has a dynamic component, such that for a short time after a strong signal there is an input-dominant phase which is then followed by an intrinsic-dominant phase.\n\nThere are different ideas at the present time what intrinsic plasticity can achieve within a network model of neuronal interaction. Reviews of intrinsic plasticity1,10,18 are undecided, whether IP acts mainly to maintain homeostasis, adapting to changes in synaptic strength by keeping neurons within certain ranges but without significant informational capacity, as in the model of17, or whether they are themselves capable of being modified in response to particular patterns of activity in ways that facilitate learning and development45. However, as we have shown, homeostatic adaptation does not exclude information storage under conditions of conditional readout. The synergy between synaptic and intrinsic plasticity may take different forms, beyond E-S potentiation. Based on experimental evidence in different systems1,9,19 have listed many possible functions and roles of intrinsic adaptive plasticity.\n\nWe have greatly simplified the exposition here by concentrating on spike frequency as a major indicator of neural behavior. Certainly the type of firing (e.g., burst firing) is also under control of neuromodulators, and may be influenced by the distribution and density of ion channels. Single neuron computation is more complex than what can be shown with a single compartment model. In dendritic computation, the coupling of different compartments may be prominently affected by intrinsic plasticity. For instance35, showed a loss of clustering for K+ channels on the membrane, induced by high glutamate stimulation, indicating a possible input-dependent regulation of dendritic integration.\n\nStudies of concurrent simulation of synaptic coupling parameters and intrinsic ion channel conductances has concluded that intrinsic and synaptic plasticity can achieve similar effects for network operation15. We have suggested that synaptic and intrinsic plasticity can substitute for each other, and furthermore that this essential functional parallelism could be an indication for information flow over time from one modality to the other. The direction of this information flow may be from intrinsic to synaptic for the induction of permanent, morphological changes (such as dendritic spine morphology)–however in some systems (e.g., cerebellum) intrinsic plasticity may also have a permanent component (Purkinje cells)5,46. The detailed interaction between synaptic and intrinsic plasticity is still an open question. Here we have shown a simple, local learning mechanism for intrinsic plasticity that allows to store pattern information without synaptic plasticity. This is different from theoretical approaches, where activation functions are only being modulated to optimize global measures of information transmission between neurons while the information is exclusively stored in synaptic weights. Further work will be needed to investigate the smooth integration of synaptic and intrinsic plasticity and their respective functions in different systems.\n\n\nConclusions\n\nWe wanted to show quantitatively that IP can have significant effects on spike frequency, dependent on the statistical structure of the input. In particular, low correlated input, or input during sensitive (high-voltage membrane) states induces the strongest variability of spike responses for different activation functions, while highly correlated input acts as drivers for neurons, eliminating subtle differences in activation function. We suggested that starting from a very general, natural format for a learning rule, which can be biologically motivated, we arrive at simple pattern learning, the basis for feature extraction, and realistic types of neural behavior: population-wide increases/decreases of neural firing rates to novel input stimuli, habituation to known stimuli and history-dependent distortions of individual stimuli. A significant application of this theoretical model exists in the observation of pervasive whole-cell adaptations in selected ion channels (INa, ICaL) after cocaine sensitization47–49, with implications of the type of learning that underlies addiction. This would reduce the dynamic range of intrinsic plasticity. Potentially, then, learning in striatum is mediated in part by intrinsic plasticity50, and a reduction in inducible intrinsic plasticity or dynamic range of intrinsic plasticity after cocaine sensitization may contribute to the pathology of addiction.",
"appendix": "Author contributions\n\nN/A\n\n\nCompeting interests\n\nNo relevant competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nZhang W, Linden DJ: The other side of the engram: experience-driven changes in neuronal intrinsic excitability. Nat Rev Neurosci. 2003; 4: 885–900.\n\nMahon S, Charpier S: Bidirectional Plasticity of Intrinsic Excitability Controls Sensory Inputs efficiency in Layer 5 Barrel Cortex Neurons in Vivo. J Neurosci. 2012; 32(33): 11377–11389.\n\nRosenkranz JA: Neuronal Activity Causes Rapid Changes of Lateral Amygdala Neuronal Membrane Properties and Reduction of Synaptic Integration and Synaptic Plasticity In Vivo. J Neurosci. 2011; 31(16): 6108–6120.\n\nSchreurs BG, Gusev PA, Tomsic D, et al: Intracellular correlates of acquisition and long-term memory of classical conditioning in Purkinje cell dendrites in slices of rabbit cerebellar lobule HVI. 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Science. 1993; 259: 1915–7.\n\nStemmler M, Koch C: How voltage-dependent conductances canadapt to maximize the information encoded by neuronal firing rate. Nat Neurosci. 1999; 2: 521–7.\n\nDaoudal G, Debanne D: Long-Term Plasticity of Intrinsic Excitability:Learning Rules and Mechanisms. Learn Mem. 2003; 10: 456–465.\n\nDisterhoft JF, Oh MM: Learning, aging and intrinsic neuronal plasticity. Trends Neurosci. 2006; 29(10): 587–99.\n\nWang XJ, Buzsaki G: Gamma oscillation by synaptic inhibition in a hippocampal interneuronal network model. J Neurosci. 1996; 16: 6402–13.\n\nBargas J, Howe A, Eberwine J, et al: Cellular and molecular characterization of Ca+ currents in acutely isolated, adultrat neostriatal neurons. J Neurosci. 1994; 14(11): 6667–6686.\n\nTsubo Y, Kaneko T, Shinomoto S: Predicting spike timings of current-injected neurons. Neural Netw. 2004; 17: 165–73.\n\nGabel LA, Nisenbaum ES: Biophysical characterization and functional consequences of a slowly inactivating potassium current in neostriatalneurons. J Neurophysiol. 1998; 79: 1989–2002.\n\nNisenbaum ES, Mermelstein PG, Wilson CJ, et al: Selective blockade of a slowly inactivating potassium current in striatal neurons by (+/-) 6-chloro-APB hydrobromide (SKF82958). Synapse. 1998; 29: 213–24.\n\nSurmeier DJ, Bargas J, Kitai ST: Two types of A-current differing in voltage-dependence are expressed by neurons of the rat neostriatum. Neurosci Lett. 1989; 103: 331–7.\n\nNisenbaum ES, Wilson CJ: Potassium currents responsible for inward and outward rectifcation in rat neostriatal spiny projection neurons. J Neurosci. 1995; 15: 4449–63.\n\nMahon S, Deniau JM, Charpier S, et al: Role of a striatal slowly inactivating potassium current in short-term facilitation of corticostriatal inputs: a computer simulation study. Learn Mem. 2000; 7: 357–62.\n\nGruber AJ, Solla SA, Surmeier DJ, et al: Modulation of striatal single units by expected reward: a spiny neuron model displaying dopamine-induced bistability. J Neurophysiol. 2003; 90: 1095–114.\n\nOnn SP, Fienberg AA, Grace AA: Dopamine modulation of membrane excitability in striatal spiny neurons is altered in DARPP-32 knockout mice. J Pharmacol Exp Ther. 2003; 306: 870–9.\n\nMahon S, Delord B, Deniau JM, et al: Intrinsic properties of rat striatal output neurones and time-dependent facilitation of corticalinputs in vivo. J Physiol. 2000; 527: 345–54.\n\nWickens JR, Wilson CJ: Regulation of action-potential firing inspiny neurons of the rat neostriatum in vivo. J Neurophysiol. 1998; 79: 2358–64.\n\nOnn SP, West AR, Grace AA: Dopamine-mediated regulation ofstriatal neuronal and network interactions. Trends Neurosci. 2000; 23: S48–56.\n\nSachdev RN, Ebner FF, Wilson CJ: Effect of subthreshold up and down states on the whisker-evoked response in somatosensory cortex. J Neurophysiol. 2004; 92: 3511–21.\n\nBenucci A, Verschure PF, Konig P: Two-state membrane potential fluctuations driven by weak pairwise correlations. Neural Comput. 2004; 16: 2351–78.\n\nMisonou H, Mohapatra DP, Park EW, et al: Regulation of ion channel localization and phosphorylation by neuronal activity. Nat Neurosci. 2004; 7: 711–8.\n\nHayashida Y, Ishida AT: Dopamine receptor activation can reduce voltage-gated Na+ current by modulating both entry into and recovery from inactivation. J Neurophysiol. 2004; 92: 3134–41.\n\nWilson CJ, Kawaguchi Y: The origins of two-state spontaneous membrane potential fluctuations of neostriatal spiny neurons. J Neurosci. 1996; 16: 2397–410.\n\nZhang W, Shin JH, Linden DJ: Persistent changes in the intrinsic excitability of rat deep cerebellar nuclear neurones induced by EPSP or IPSP bursts. J Physiol. 2004; 561: 703–19.\n\nScott L, Kruse MS, Forssberg H, et al: Selective up-regulation of dopamine D1 receptors in dendritic spines by NMDA receptor activation. Proc Natl Acad Sci U S A. 2002; 99: 1661–4.\n\nBonsi P, Pisani A, Bernardi G, et al: Stimulus frequency, calcium levels and striatal synaptic plasticity. Neuroreport. 2003; 14: 419–22.\n\nCarter AG, Sabatini BL: State-dependent calcium signaling indendritic spines of striatal medium spiny neurons. Neuron. 2004; 44: 483–93.\n\nTepper JM, Koos T, Wilson CJ: GABAergic microcircuits in the neostriatum. Trends Neurosci. 2004; 27: 662–9.\n\nBao S, Chan VT, Merzenich MM: Cortical remodelling induced by activity of ventral tegmental dopamine neurons. Nature. 2001; 412: 79–83.\n\nScheler G: Regulation of neuromodulator receptor efficacy-implications for whole-neuron and synaptic plasticity. Prog Neurobiol. 2004; 72: 399–415.\n\nO'Leary T, Wyllie DJ: Neuronal homeostasis: time for a change? J Physiol. 2011; 589(20): 4811–4826.\n\nNelson AB, Krispel CM, Sekirnjak C, et al: Long-lasting increases in intrinsic excitability triggered by inhibition. Neuron. 2003; 40: 609–20.\n\nHu XT, Basu S, White FJ: Repeated cocaine administration suppresses HVA-Ca2+ potentials and enhances activity of K+ channels in rat nucleus accumbens neurons. J Neurophysiol. 2004; 92: 1597–607.\n\nZhang XF, Cooper DC, White FJ: Repeated cocaine treatment decreases whole-cell calcium current in rat nucleus accumbens neurons. J Pharmacol Exp Ther. 2002; 301: 1119–25.\n\nZhang XF, Hu XT, White FJ: Whole-cell plasticity in cocaine withdrawal: reduced sodium currents in nucleus accumbens neurons. J Neurosci. 1998; 18: 488–98.\n\nMahon S, Casassus G, Mulle C, et al: Spike-dependent intrinsic plasticity increases firing probability in rat striatal neurons invivo. J Physiol. 2003; 550: 947–59."
}
|
[
{
"id": "936",
"date": "09 May 2013",
"name": "Fidel Santamaria",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting paper that aims to elucidate the contribution of intrinsic excitability plasticity on neuronal learning using computer models. The paper would benefit from presenting a statistical analysis of the learning process. What is the probability of distinguishing a learnt pattern as opposed of detecting a false positive?The fact that Pnoise also evokes a close firing rate response as Plearn should be closely addressed. Is the output for Pnoise and Plearn significantly different? There should be controls for having the same number of input neurons to any pattern. Otherwise, the results in Figure 6 could be due to the overlap of Pnoise and Plearn. Does the author propose a metric by which the neurons distinguish the learnt from the noisy pattern?Figure 7 and 8 are mixed up in the text and with their captions. Please re-arrange. In the histogram of what is now Fig. 7 the author again shows that Pnoise is capable of eliciting a response that seems to be different from background. Is this significantly different (statistically speaking)? I think there’s a typo in Eq. 3.Although the premise of the paper is quite interesting I think the results and discussions would be much better supported with a thorough statistical analysis of the spiking response of the model.",
"responses": [
{
"c_id": "818",
"date": "13 May 2014",
"name": "Gabriele Scheler",
"role": "Author Response",
"response": "“Figure 7 and 8 are mixed up in the text and with their captions. Please re-arrange.”This was a mixup during publication, and has now been corrected. “I think there’s a typo in Eq. 3”This has been corrected, so it reads: [μ1g1(Vm-V0) + .... + gs(Vm-V0)]"
}
]
},
{
"id": "3068",
"date": "27 Jan 2014",
"name": "Arvind Kumar",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the first part of the manuscript the author describes the voltage dependent sensitivity of the VI curves of the different ion channels as a function of neuromodulation. This result follows directly from the fact that different ion channels are active at different voltages. Next, the author shows that a plasticity rule can be realised by changing the conductance of different ion channels. The author provides a nice simple demonstration of this interesting idea. But the manuscript needs a major revision to clarify several issues. Below are my comments, and I have tried not to repeat the first reviewer. I do not think it is correct to refer to the VI curve as the activation function of a neuron. In general it is not correct to use the word ‘variability’ to refer to the modulation of the VI curve due to μ. I think ‘sensitivity’ better captures the observation. In Figure 1 the author shows that modulation of the VI curve is maximal around specific voltages. But it seems that this modulation in the VI curve depends on the value of current at that voltage. Obviously, e.g. when I_{Kir} is zero for μ=1 , it does not matter how much you change μ, there will not be any change in the VI curve at that voltage. To make a claim about the sensitivity of the VI curves in different voltage ranges, it should scaled for the VI curve for μ=1. The claim that the VI curves can act as basis functions is made in a loose sense and should be strengthened, else remove it from the text. Figure 4: Is there a range of μ values for which correlations fail to induce reliable spiking response. In general 0.9 is a very high correlation. How do the neurons behave for more acceptable correlations values e.g. 0.1-0.2?Induction and maintenance of plasticity: The author wrote “Neural activation is largely determined by intracellular calcium but here we estimate…”. What is the meaning of ‘Neural activation’? Why is it determined by calcium? If it is spike output, then it is mainly determined by the kind of its input.The learning rule is formulated in such a way that for every input to these neurons, there will be increased response (positive learning) and other way around in the negative learning. So this learning will increase the noise level in the networks. The learning rule is unstable, because one neuronal activity is above the threshold θ its firing can only increase. Authors argue that adaption could be a way to stabilise the firing rates, however is not clear whether the model will converge, because if the firing rate drops below θ, then neuron response can only decrease.Finally, there is another implementation related problem with the learning rule. An external agent is assumed which checks the firing rate of the neuron above or below a baseline firing rate and then updates the ion channel parameters. If we compare it to synaptic plasticity do not require such an agent, because every spike can causes a weight update.An important question is how the learning rule could switch between negative and positive learning regimes. In general, the methods are poorly described. See minor comments for more details. Minor points:Page 1: left col. last para, II line: The sentence does not link with the previous and next sentences. Moreover, it needs to be rephrased; the reference nos. are in superscript, that's why the sentence does not read correctly. I do not understand what the author means by “Thus, intrinsic plasticity is conceptualized as fundamentally different from synaptic plasticity which does not encompass such a state change”. It is obvious that synaptic plasticity can cause state changes. The difference in Eq. 2 and Eq. 3 is not clear with regard to the implementation of the ion channel conductances. What does g_s refer to in Eq. 3? Equation 4: In the expression [NM]K, it is not clear if ‘K’ is a multiplier to [NM] or subscript. The notations in the Eq. and in the corresponding text do not match. Page 4: EPSP’s should be expressed in the units of voltage and not in terms of the current. As far as I understand this is a point neurone implementation of the MSN, so why should the synaptic strengths be expressed in units of area? The method to calibrate the synapses is not clear. What is the meaning of “ …0.6nA, which corresponds to the experimentally measured average value for the rheobase in\". At -40mV MSN is already close to its spiking threshold? And rehobase is measured from resting membrane potential? How were the correlations in input generated? Figure 1: Please state which variable is plotted on the x- and y-axis. Providing just units is confusing. Also it is not clear which line refers to which μ value? And therefore it is not possible to know in which direct μ affects the v-i curves. I assume that all of the three VI curves are modulated in the same direction. None of the axis measures ‘variability’ in this figure. What is plotted is the change in the VI curve for different values of μ. So you want to refer to this effect as sensitivity of the VI curve. Figure 2: Once again I am not sure which axis shows the variability? And axis on the insets are missing. Figure 3: Render the different lines in colours which reflect the voltage. In its current form it's not easy to read these figures. Figure 4: the o/p is shown for only one value of i/p firing rate (couldn't find any mention of what value it is). Hence, in addition to the present figure 4, a figure showing i/p firing rate Vs o/p firing rate for more than one data point of i/p firing rate could be included. This can be plotted for all the three values of μ. This on the other hand could be drawn for few values of i/p correlations. Figure 8: It is not clear if the traces are drawn for +ve or -ve learning. The labels of neuron numbers (No. 4,8) are right if it's for negative learning, but should be in reverse order if it's for positive learning. Page 9, first paragraph: Update the reference to figures after the changing the figure captions of Fig. 7,8.",
"responses": [
{
"c_id": "817",
"date": "13 May 2014",
"name": "Gabriele Scheler",
"role": "Author Response",
"response": "“I do not think it is correct to refer to the VI curve as the activation function of a neuron. In general it is not correct to use the word ‘variability’ to refer to the modulation of the VI curve due to μ. I think ‘sensitivity’ better captures the observation. In Figure 1 the author shows that modulation of the VI curve is maximal around specific voltages. But it seems that this modulation in the VI curve depends on the value of current at that voltage. Obviously, e.g. when I_{Kir} is zero for μ=1 , it does not matter how much you change μ, there will not be any change in the VI curve at that voltage. To make a claim about the sensitivity of the VI curves in different voltage ranges, it should scaled for the VI curve for μ=1.”Have added the variable names to the axes, as well as the μ values.“Variability” has been replaced with “modulation” throughout the text.Activation function has been replaced by I-V curve. “The claim that the VI curves can act as basis functions is made in a loose sense and should be strengthened, else remove it from the text.”This has been removed. “Figure 4: Is there a range of μ values for which correlations fail to induce reliable spiking response. In general 0.9 is a very high correlation. How do the neurons behave for more acceptable correlations values e.g. 0.1-0.2?”These extreme values have been used for demonstration in this figure. “The learning rule is formulated in such a way that for every input to these neurons, there will be increased response (positive learning) and other way around in the negative learning. So this learning will increase the noise level in the networks. The learning rule is unstable, because one neuronal activity is above the threshold θ its firing can only increase. Authors argue that adaption could be a way to stabilise the firing rates, however is not clear whether the model will converge, because if the firing rate drops below θ, then neuron response can only decrease.”This is not guaranteed to be stable; additional mechanisms (additional research), for example synaptic plasticity in the other direction, may be needed. Finally, there is another implementation related problem with the learning rule. An external agent is assumed which checks the firing rate of the neuron above or below a baseline firing rate and then updates the ion channel parameters. If we compare it to synaptic plasticity do not require such an agent, because every spike can causes a weight update.”Have added a clarifying sentence. “An important question is how the learning rule could switch between negative and positive learning regimes.”The negative/positive learning rule can depend on each neuron. We did not investigate this for networks of neurons.Minor Comments:“I do not understand what the author means by “Thus, intrinsic plasticity is conceptualized as fundamentally different from synaptic plasticity which does not encompass such a state change”. It is obvious that synaptic plasticity can cause state changes.”Have added a clarifying sentence. “The difference in Eq. 2 and Eq. 3 is not clear with regard to the implementation of the ion channel conductances. What does g_s refer to in Eq. 3?”There was a typo in Eq. 3, which has now been fixed (please see response to Fidel Santamaria). “Equation 4: In the expression [NM]K, it is not clear if ‘K’ is a multiplier to [NM] or subscript. The notations in the Eq. and in the corresponding text do not match.”This was an error introduced during typesetting; it has been corrected to κ (lowercase kappa). “Page 4: EPSP’s should be expressed in the units of voltage and not in terms of the current. As far as I understand this is a point neurone implementation of the MSN, so why should the synaptic strengths be expressed in units of area?”All units in the simulation are expressed in area units; VEPSP = 1/gs * IEPSP “How were the correlations in input generated?”Referred to code. “Figure 1: Please state which variable is plotted on the x- and y-axis. Providing just units is confusing. Also it is not clear which line refers to which μ value? And therefore it is not possible to know in which direct μ affects the v-i curves. I assume that all of the three VI curves are modulated in the same direction. None of the axis measures ‘variability’ in this figure. What is plotted is the change in the VI curve for different values of μ. So you want to refer to this effect as sensitivity of the VI curve.”Added plotted variables at the axes, and added μ values. “Figure 2: Once again I am not sure which axis shows the variability? And axis on the insets are missing.”Added plotted variables at the axes, and added μ values. “Figure 3: Render the different lines in colours which reflect the voltage. In its current form it's not easy to read these figures.”This has been done. “Figure 4: the o/p is shown for only one value of i/p firing rate (couldn't find any mention of what value it is). Hence, in addition to the present figure 4, a figure showing i/p firing rate Vs o/p firing rate for more than one data point of i/p firing rate could be included. This can be plotted for all the three values of μ. This on the other hand could be drawn for few values of i/p correlations.”One could have a figure with correlation on the x axis, and firing rate on the y. For low correlations all three neurons are at 20, 26, 40. At a certain, higher, correlation rate it switches from these frequencies to reliable firing modes correlated with the input – there might be different correlation values for different neurons. “Figure 8: It is not clear if the traces are drawn for +ve or -ve learning. The labels of neuron numbers (No. 4,8) are right if it's for negative learning, but should be in reverse order if it's for positive learning.”It is negative learning, consistent with the original caption. “Page 9, first paragraph: Update the reference to figures after the changing the figure captions of Fig. 7,8.”This has been done."
}
]
}
] | 1
|
https://f1000research.com/articles/2-88
|
https://f1000research.com/articles/3-36/v1
|
04 Feb 14
|
{
"type": "Data Article",
"title": "Matched molecular pair-based data sets for computer-aided medicinal chemistry",
"authors": [
"Ye Hu",
"Antonio de la Vega de León",
"Bijun Zhang",
"Jürgen Bajorath",
"Ye Hu",
"Antonio de la Vega de León",
"Bijun Zhang"
],
"abstract": "Matched molecular pairs (MMPs) are widely used in medicinal chemistry to study changes in compound properties including biological activity, which are associated with well-defined structural modifications. Herein we describe up-to-date versions of three MMP-based data sets that have originated from in-house research projects. These data sets include activity cliffs, structure-activity relationship (SAR) transfer series, and second generation MMPs based upon retrosynthetic rules. The data sets have in common that they have been derived from compounds included in the latest release of the ChEMBL database for which high-confidence activity data are available. Thus, the activity data associated with MMP-based activity cliffs, SAR transfer series, and retrosynthetic MMPs cover the entire spectrum of current pharmaceutical targets. Our data sets are made freely available to the scientific community.",
"keywords": [
"The matched molecular pair (MMP) concept is widely applied in medicinal chemistry1–4. An MMP is defined as a pair of compounds that are only distinguished by a structural modification at a single site1",
"i.e.",
"the exchange of a substructure",
"termed a chemical transformation5. MMPs are attractive tools for computational analysis because they can be algorithmically generated and they make it possible to associate defined structural modifications at the level of compound pairs with chemical property changes",
"including biological activity2–4. MMPs are usually chemically intuitive and easily accessible",
"which helps to bridge the gap between computational analysis and the practice of medicinal chemistry."
],
"content": "Introduction\n\nThe matched molecular pair (MMP) concept is widely applied in medicinal chemistry1–4. An MMP is defined as a pair of compounds that are only distinguished by a structural modification at a single site1, i.e., the exchange of a substructure, termed a chemical transformation5. MMPs are attractive tools for computational analysis because they can be algorithmically generated and they make it possible to associate defined structural modifications at the level of compound pairs with chemical property changes, including biological activity2–4. MMPs are usually chemically intuitive and easily accessible, which helps to bridge the gap between computational analysis and the practice of medicinal chemistry.\n\nIn the context of different studies, we have systematically generated MMPs through the mining of publicly available compound activity data. All possible MMPs have been derived from compounds active against currently available pharmaceutical targets. Then, MMPs have been used to explore structure-activity relationships (SARs) on a large-scale and from different viewpoints.\n\nIn a previous data article, we have reported and made publicly available a number of different data sets and computational tools developed in our laboratory6. Here we describe three recently developed MMP-based data structures, which should be of interest for SAR analysis and compound design, and we also provide up-to-date versions of the corresponding data sets. It is anticipated that these data sets will be helpful as a resource for computer-aided medicinal chemistry applications. The data sets include MMP-based activity cliffs (i.e., MMP-cliffs), SAR transfer series, and MMPs derived on the basis of retrosynthetic fragmentation rules and were derived from all bioactive compounds currently available in the ChEMBL database7,8. Only high-confidence activity data (as specified below) were considered. MMP-cliffs, SAR transfer series, and retrosynthetic MMPs provide comprehensive sources of SAR information. In addition, retrosynthetic MMPs are thought to increase the utility of computational MMP analysis for practical chemistry efforts because these second generation MMPs can be associated with specific chemical reactions.\n\n\nMaterials and methods\n\n(1) Activity cliffs are generally defined as pairs or groups of compounds that are structurally similar and have large differences in potency9–11. Accordingly, activity cliffs usually have high SAR information content (because small chemical changes in similar or analogous compounds lead to large potency effects). The assessment of activity cliffs requires clearly defined similarity and potency difference criteria9–11. The formation of an MMP can be considered as a similarity criterion, which is similarity metric-free and often chemically more intuitive than the use of calculated molecular similarity11,12. MMP formation as a similarity criterion has led to the introduction of MMP-cliffs12. For MMP-cliffs, a difference in potency of at least two orders of magnitude between cliff-forming compounds was set as a potency difference criterion12. Figure 1 shows exemplary MMP-cliffs.\n\nSix representative MMP-cliffs for three targets belonging to different target families are shown; (a) muscarinic acetylcholine receptor M3, (b) serine/threonine-protein kinase c-TAK1, (c) matrix metalloproteinase-2. The pKi value of each compound is provided and the structural differences between cliff-forming compounds are highlighted in red.\n\n(2) SAR transfer can be rationalized in different ways. For example, a compound series might display similar potency progression against two different targets13. Alternatively, two different compound series with corresponding analogs, i.e., series having different core structures and containing compounds with pairwise corresponding substitutions, might display similar potency progression against a given target14. Such SAR transfer series displaying similar target-specific SAR behavior are often sought after in medicinal chemistry as alternative compounds for optimization. Here we focus on these target-based SAR transfer series. Figure 2 shows an example.\n\nAn exemplary target-based SAR transfer series is shown. Compound pairs are arranged in the order of increasing potency (from the bottom to the top). Potency progression is monitored by corresponding pairs of color-coded dots using a continuous color spectrum from green (lowest potency value (pKi = 5.7) in the compound data set), over yellow to red (highest potency value; pKi = 9.0). The pKi value of each compound is provided. The core structures are drawn in black and the substituents in red. The compounds are active against serine/threonine-protein kinase D2.\n\n(3) Computational generation of MMPs typically involves molecular fragmentation through the systematic deletion of exocyclic single bonds5. Hence, the resulting fragments representing a molecular core and substituent are not derived considering chemical reactions. Accordingly, a transformation relating MMP-forming compounds to each other might not necessarily be interpretable from a synthetic perspective. Hence synthetic accessibility of MMPs might be further improved by considering the reaction information during molecular fragmentation. This has been accomplished by applying the well-known retrosynthetic combinatorial analysis procedure (RECAP) rules15, leading to the introduction of RECAP-MMPs16. Representative examples are shown in Figure 3.\n\nIn (a)–(d), four exemplary RECAP-MMPs representing different retrosynthetic rules are shown. For each RECAP-MMP, the chemical transformation is highlighted in red.\n\nFor the generation of MMP-cliffs, SAR transfer series, and RECAP-MMPs, transformation size restrictions that limit transformations to meaningful chemical substitutions were introduced12. Specifically, the common core structure had to be at least twice the size of each exchanged substructure. Furthermore, the difference in size of the exchanged fragments was limited to at most eight non-hydrogen atoms and the maximal size of an exchanged fragment was set to 13 non-hydrogen atoms12. Therefore, the largest permitted transformations included, for example, the addition of a substituted ring to a compound or the replacement of a five- or six-membered ring with a substituted condensed two-ring system (with a maximum of 13 atoms). All possible transformation size-restricted MMPs and RECAP-MMPs were calculated using an in-house implementation of the algorithm by Hussain and Rea5.\n\nCompound data were taken from the latest version of ChEMBL (release 17)7,8. Only compounds with direct interactions (i.e., target relationship type “D”) against human targets at the highest confidence level (target confidence score 9) were selected. Two types of potency measurements were separately considered, i.e., Ki (equilibrium constant) and IC50 (half-maximal inhibition concentration) values. In order to ensure high data confidence, inconclusive compounds and compounds with approximate measurements such as “>”, “<”, or “∼” were not considered. For compounds with multiple measurements against the same target, the geometric mean was calculated as the final potency annotation, provided that all values fell within one order of magnitude; otherwise, the compound was discarded. All qualifying compounds were further organized into target sets. A total of 661 and 1203 target sets (consisting of compounds with reported specific activity against a given target) were collected for the Ki- and IC50-based subsets, respectively, as reported in Table 1. The target sets contained a total of 45,353 and 95,685 compounds and 77,421 and 135,291 potency measurements for the Ki and IC50 subsets, respectively. These target sets provided the basis for the generation of all MMPs.\n\nFor the Ki and IC50 subsets from the latest version of ChEMBL (release 17), the total number of targets, compounds, and corresponding potency measurements are reported.\n\n\nResults\n\nAs a follow-up on the original publications in which MMP-cliffs12, SAR transfer series14, and RECAP-MMPs16 were introduced, all corresponding data sets have been re-generated on the basis of ChEMBL release 17, hence providing up-to-date versions for release. Separate data subsets have been generated for different types of well-defined potency measurements (i.e., assay-dependent IC50 vs. assay-independent Ki values) to avoid inconsistencies due to simultaneous consideration of different potency measurements that cannot be directly compared.\n\nFigure 1 illustrates small chemical changes in compound pairs leading to large potency differences that are captured by MMP-cliffs. For ease of structural interpretation, we currently prefer MMP-based activity cliff representations compared to alternative representations that rely on calculated similarity values11. Table 2 provides the MMP-cliff statistics for the current data set. On the basis of Ki and IC50 measurements, more than 20,000 and 25,000 MMP-cliffs were obtained, respectively, requiring an at least 100-fold difference in potency between cliff-forming compounds. The MMP-cliffs corresponded to ~5% of all MMPs that were generated from ChEMBL compounds with high-confidence activity data. They covered 293 and 500 different targets on the basis of Ki and IC50 measurements, respectively.\n\nFor the Ki- and IC50-based compound subsets, the number of MMPs, the number of targets for which MMPs were obtained, and the number (and ratio) of compounds that formed MMPs are reported. In addition, the number and proportion of MMP-cliffs derived from all MMPs are provided as well as the number of targets for which MMP-cliffs were obtained and the number (and ratio) of cliff-forming compounds.\n\nSAR transfer series are best rationalized as pairs of compound series active against the same target that have a distinct core structure, and consist of corresponding pairs of analogs, as illustrated in Figure 2 for a small series with three pairs. Different from the original analysis of target-based SAR transfer14 that was based upon MMPs without transformation size restrictions, the current analysis has been carried out on the basis of size-restricted MMPs. This modification further supports SAR exploration (because only small chemical changes are considered) and explains a reduction in series numbers compared to the original publication. In Table 3, the numbers of different series available for the current data set are reported. A total of 1270 and 2109 matching series were obtained from the Ki and IC50 subsets, respectively. Matching series met the structural requirement of consisting of at least three pairs of corresponding analogs. In addition, the potency values of compounds associated with individual series had to span at least two orders of magnitude. From these pre-selected matching series, 157 (Ki) and 513 (IC50) SAR transfer series with at least approximate potency progression and activity against 42 and 54 targets, respectively, were obtained. A subset of 60 (Ki) and 322 (IC50) SAR transfer series displayed strictly corresponding (regular) potency progression (often over different potency ranges)14. These series were active against 23 (Ki) and 27 (IC50) different targets. The size of SAR transfer series with approximate and regular potency progression ranged from three to 12 corresponding pairs of analogs. On average, the SAR transfer series consisted of three to four pairs.\n\nFor the Ki and IC50 subsets, the number of qualifying matching compound series is reported. In addition, the number of target-based SAR transfer series with at least approximate potency progression (T_SAR-TS), the subset of SAR transfer series with regular potency progression (T_SAR-TS-RP), and the corresponding numbers of targets are given.\n\nThe replacement of systematic fragmentation of exocyclic single bonds with a set of 13 retrosynthetic rules for MMP generation reduced the number of MMPs that were obtained by more than half. RECAP-MMP numbers are reported in Table 4. However, (perhaps surprisingly) large numbers of RECAP-MMPs remained for further consideration and assessment of synthetic feasibility. From the Ki and IC50 subsets, nearly 170,000 and more than 240,000 RECAP-MMPs were obtained with activity against 371 and 778 targets, respectively. Examples are shown in Figure 3.\n\nFor the Ki and IC50 subsets, the number of RECAP-MMPs, the number of targets for which RECAP-MMPs were obtained, and the number (and ratio) of compounds that formed RECAP-MMPs are reported.\n\n\nData availability\n\nAll MMP-cliffs, SAR transfer series, and RECAP-MMPs are provided in canonical SMILES representation17 on a per-target basis separately for the Ki and IC50 subsets.\n\nZENODO: Data sets of MMP-cliffs, SAR transfer series and RECAP-MMPs, doi: 10.5281/zenodo.775018\n\n\nSummary\n\nWe have described new and up-to-date MMP-based data sets comprising activity cliffs, SAR transfer series, and second generation retrosynthetic MMPs that have been systematically generated from currently available public domain compounds with high-confidence activity data. Hence, these data sets are comprehensive and have broad target coverage. They are made available without restrictions to the scientific community to aid in SAR analysis, compound design, and other medicinal chemistry applications. It is hoped that these data sets might be of interest and useful to many investigators in this field and catalyze further research efforts.",
"appendix": "Author contributions\n\n\n\nJB designed the study, YH, AVL, and BZ collected and organized the data, JB and YH wrote the manuscript, all authors examined the manuscript and agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nKenny PW, Sadowski J: Structure modification in chemical databases. In Chemoinformatics in Drug Discovery; Oprea, T. I., Ed.; Wiley-VCH: Weinheim, Germany, 2005; 271–285. Publisher Full Text\n\nGriffen E, Leach AG, Robb GR, et al.: Matched molecular pairs as a medicinal chemistry tool. J Med Chem. 2011; 54(22): 7739–7750. PubMed Abstract | Publisher Full Text\n\nWassermann AM, Dimova D, Iyer P, et al.: Advances in computational medicinal chemistry: matched molecular pair analysis. Drug Dev Res. 2012; 73(8): 518–527. Publisher Full Text\n\nDossetter AG, Griffen EJ, Leach AG: Matched molecular pair analysis in drug discovery. Drug Discov Today. 2013; 18(15–16): 724–731. PubMed Abstract | Publisher Full Text\n\nHussain J, Rea C: Computationally efficient algorithm to identify matched molecular pairs (MMPs) in large data sets. J Chem Inf Model. 2010; 50(3): 339–348. PubMed Abstract | Publisher Full Text\n\nHu Y, Bajorath J: Freely available compound data sets and software tools for chemoinformatics and computational medicinal chemistry applications [v1; ref status: indexed, http://f1000r.es/Mu9krs]. F1000Res. 2012; 1: 11. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGaulton A, Bellis LJ, Bento AP, et al.: ChEMBL: a large-scale bioactivity database for drug discovery. Nucleic Acids Res. 2012; 40(Database issue): D1100–D1107. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBento AP, Gaulton A, Hersey A, et al.: The ChEMBL bioactivity database: an update. Nucleic Acids Res. 2014; 42(1): D1083–D1090. PubMed Abstract | Publisher Full Text\n\nStumpfe D, Bajorath J: Exploring activity cliffs in medicinal chemistry. J Med Chem. 2012; 55(7): 2932–2942. PubMed Abstract | Publisher Full Text\n\nStumpfe D, Hu Y, Dimova D, et al.: Recent progress in understanding activity cliffs and their utility in medicinal chemistry. J Med Chem. 2014; 57(1): 18–28. PubMed Abstract | Publisher Full Text\n\nHu Y, Stumpfe D, Bajorath J: Advancing the activity cliff concept [v1; ref status: indexed, http://f1000r.es/1wf]. F1000Res. 2013; 2: 199. Publisher Full Text | Free Full Text\n\nHu X, Hu Y, Vogt M, et al.: MMP-Cliffs: systematic identification of activity cliffs on the basis of matched molecular pairs. J Chem Inf Model. 2012; 52(5): 1138–1145. PubMed Abstract | Publisher Full Text\n\nZhang B, Hu Y, Bajorath J: SAR transfer across different targets. J Chem Inf Model. 2013; 53(7): 1589–1594. PubMed Abstract | Publisher Full Text\n\nZhang B, Wassermann AM, Vogt M, et al.: Systematic assessment of compound series with SAR transfer potential. J Chem Inf Model. 2012; 52(12): 3138–3143. PubMed Abstract | Publisher Full Text\n\nLewell XQ, Judd DB, Watson SP, et al.: RECAP--retrosynthetic combinatorial analysis procedure: a powerful new technique for identifying privileged molecular fragments with useful applications in combinatorial chemistry. J Chem Inf Comput Sci. 1998; 38(3): 511–522. PubMed Abstract | Publisher Full Text\n\nde la Vega de León A, Bajorath J: Matched molecular pairs derived by retrosynthetic fragmentation. Med Chem Commun. 2014; 5(1): 64–67. Publisher Full Text\n\nWeininger D: SMILES, a chemical language and information system. 1. Introduction to methodology and encoding rules. J Chem Inf Comput Sci. 1988; 28(1): 31–36. Publisher Full Text\n\nHu Y, de la Vega de León A, Zhang B, et al.: Data sets of MMP-cliffs, SAR transfer series and RECAP-MMPs. 2014. Data Source"
}
|
[
{
"id": "3509",
"date": "10 Feb 2014",
"name": "Ajay Jain",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe data set described by Hu et al. is a large set of carefully curated small molecule matched-molecular pairs (MMPs) with high-quality activity data derived from ChEMBL. The set includes examples of structure-activity cliffs, as well as matched SAR-transfer series, both of which are important in the development and validation of activity prediction algorithms. The availability of the MMP data set will be very valuable to researchers that are focused on methods development. The data should also be of interest to those interested in fundamental questions about molecular activity (e.g. questions about the independence and additivity of activity changes that are linked with substituent changes).",
"responses": []
},
{
"id": "3707",
"date": "17 Feb 2014",
"name": "Peter Ertl",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe matched molecule pairs approach provides a “chemistry friendly” and intuitive way of expressing relationships among molecules and therefore this manuscript is of importance to all cheminformatics scientists interested in the study of activity cliffs, SAR analysis and in the design of bioactive molecules in general.The authors extracted several datasets of matched molecule pairs from public databases that could be used as benchmarks in further analysis of this phenomenon. Care has been taken to assure a high quality of data.The manuscript is well written and the procedure and all results are sufficiently documented and, in addition, all datasets are available for download; therefore I am suggesting only a few minor modifications to the text:Introduction: replace \"the latest release of the ChEMBL\" with the version number. Provide a bit more technical information about the in-house implementation of a molecule fragmentation procedure used to generated matched pairs. Was the procedure implemented entirely in-house, or is it based on a publicly available cheminformatics toolkit? (If this is the case, please cite the respective toolkit). The authors mention that structures in the download file are available as canonical SMILES. The form of canonical SMILES however will depend on the particular program used to generate it. Please specify whether the original ChEMBL SMILES is included or the canonical SMILES was created by another toolkit",
"responses": []
},
{
"id": "3508",
"date": "18 Feb 2014",
"name": "Patrick Walters",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper provides a review of the Bajorath group's recent work on matched molecular pairs (MMP), a technique for exploring structure activity relationships, and identifying chemical transformations that can readily modulate biological activity. The authors focus on recent applications of MMP to large datasets from the publicly accessible ChEMBL database. The paper provides an excellent introduction to those unfamiliar with the MMP technique and with concepts such as activity cliffs. In addition to providing an overview of the recent literature, the authors also provide links to publicly available software and datasets that will provide tutorial materials for those interested in learning more about these powerful techniques. Datasets and software like those described in this paper are valuable resources. A logical next step from this work would be to create interactive tutorials using tools like the iPython Notebook Viewer or knitr.The presentation is clear, but a few changes may help readers unfamiliar with some of the concepts.On p2 the authors refer to \"second generation MMPs\". It would be useful to add a sentence explaining the differences between first and second generation MMPs. MMP Cliffs which differ in activity by 10-fold may also be interesting. It would be informative to see the number of examples available with a 1 log vs 2 log difference. In the section (3) on page 3, it would be interesting to provide a specific example of how the results of RECAP generated MMPS differ from those generated using a more \"traditional\" approach.This paper provides an excellent gateway to a topic that is becoming increasingly more important in drug discovery. The paper should be of interest to computational and medicinal chemists as well as biologists.",
"responses": []
},
{
"id": "3733",
"date": "20 Feb 2014",
"name": "Shana Posy",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHu et al have compiled a useful set of matched pair datasets based on the CHEMBL database of biological activity. They describe in a straightforward manner the derivation of the datasets and basic concepts relevant for matched pairs. The following suggested modifications to the data provided would enhance their usefulness and completeness:As a result of switching the method for generating MMPs from cleavage of single bonds to a RECAP-based method, the pool of MMPs now includes substitutions of internal fragments (e.g. in Figure 3c) as well as substitution of a terminal R-group (as in Figure 3 examples a, b, and d). Although both types of MMPs involve replacement of a single structural fragment, it may be desirable for many applications to distinguish between core scaffold replacement and R-group variation. It would therefore be helpful to annotate the datasets to easily separate these two classes of MMPs. Since the authors filter out IC50s/Kis of indeterminate values, it is unclear how compounds that were clearly inactive were processed. Were compounds with IC50s/Kis that could not be quantified due to a flat dose response curve included in the datasets? The authors present a filtered dataset where a number of factors have contributed to rejection of potential MMPs, namely: the difference in size of the exchanged fragments was limited to 8 heavy atoms; the ratio of the common core fragment to the size of each exchanged fragment had to be>2; and the exchanged fragment could have maximum 13 heavy atoms. While these are reasonable filters to obtain MMPs that truly represent small structural changes, the cutoffs selected are arbitrary and for some targets may exclude MMPs that another user might consider relevant. Rather than providing the final filtered dataset, it would be helpful if the authors would provide the full original datasets with the values of the features used for filtering annotated as extra columns. This would allow maximal flexibility in designing custom MMP sets. In the files that list the RECAP MMPs, key fields are missing that would require the user to retrieve the relevant data from CHEMBL in order to perform any analysis: (a) the Target name (only the target CHEMBL ID is provided); and (b) more importantly, the compound activities are not included. In the files that list the transfer series, for each matched pair the authors provide the two series cores and full compound smiles, but not the substituted fragments.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-36
|
https://f1000research.com/articles/2-217/v1
|
15 Oct 13
|
{
"type": "Method Article",
"title": "MethylExtract: High-Quality methylation maps and SNV calling from whole genome bisulfite sequencing data",
"authors": [
"Guillermo Barturen",
"Antonio Rueda",
"José L. Oliver",
"Michael Hackenberg",
"Guillermo Barturen",
"Antonio Rueda"
],
"abstract": "Whole genome methylation profiling at a single cytosine resolution is now feasible due to the advent of high-throughput sequencing techniques together with bisulfite treatment of the DNA. To obtain the methylation value of each individual cytosine, the bisulfite-treated sequence reads are first aligned to a reference genome, and then the profiling of the methylation levels is done from the alignments. A huge effort has been made to quickly and correctly align the reads and many different algorithms and programs to do this have been created. However, the second step is just as crucial and non-trivial, but much less attention has been paid to the final inference of the methylation states. Important error sources do exist, such as sequencing errors, bisulfite failure, clonal reads, and single nucleotide variants.We developed MethylExtract, a user friendly tool to: i) generate high quality, whole genome methylation maps and ii) detect sequence variation within the same sample preparation. The program is implemented into a single script and takes into account all major error sources. MethylExtract detects variation (SNVs – Single Nucleotide Variants) in a similar way to VarScan, a very sensitive method extensively used in SNV and genotype calling based on non-bisulfite-treated reads. The usefulness of MethylExtract is shown by means of extensive benchmarking based on artificial bisulfite-treated reads and a comparison to a recently published method, called Bis-SNP.MethylExtract is able to detect SNVs within High-Throughput Sequencing experiments of bisulfite treated DNA at the same time as it generates high quality methylation maps. This simultaneous detection of DNA methylation and sequence variation is crucial for many downstream analyses, for example when deciphering the impact of SNVs on differential methylation. An exclusive feature of MethylExtract, in comparison with existing software, is the possibility to assess the bisulfite failure in a statistical way. The source code, tutorial and artificial bisulfite datasets are available at http://bioinfo2.ugr.es/MethylExtract/ and http://sourceforge.net/projects/methylextract/, and also permanently accessible from 10.5281/zenodo.7144.",
"keywords": [
"DNA methylation at the cytosine carbon 5 position (5meC) is an important epigenetic mark in eukaryotic cells that is predominantly found in CpG or CpHpG (H = A",
"C",
"T) sequence contexts1. Epigenetic modifications at the DNA level play important roles in embryonic development2",
"3",
"transcription4",
"chromosome stability5",
"genomic imprinting6 and in the silencing of transposons in plants7. Furthermore",
"aberrant methylation is involved in the appearance of several disorders as cancer",
"immunodeficiency or centromere instability8. The methylation pattern along the genome sequence carries biologically relevant information. For example: methylated promoter regions are generally associated with silenced transcription and DNA methylation in the gene body of transcribed genes is often increased8. Given these findings",
"the generation of high quality whole genome methylation maps at a single cytosine resolution is an important step towards the understanding of how DNA methylation is involved in the regulation of gene expression or the generation of a pathologic phenotype. In addition",
"methylation maps may provide new insights into how the methylation patterns themselves are established."
],
"content": "Introduction\n\nDNA methylation at the cytosine carbon 5 position (5meC) is an important epigenetic mark in eukaryotic cells that is predominantly found in CpG or CpHpG (H = A,C,T) sequence contexts1. Epigenetic modifications at the DNA level play important roles in embryonic development2,3, transcription4, chromosome stability5, genomic imprinting6 and in the silencing of transposons in plants7. Furthermore, aberrant methylation is involved in the appearance of several disorders as cancer, immunodeficiency or centromere instability8. The methylation pattern along the genome sequence carries biologically relevant information. For example: methylated promoter regions are generally associated with silenced transcription and DNA methylation in the gene body of transcribed genes is often increased8. Given these findings, the generation of high quality whole genome methylation maps at a single cytosine resolution is an important step towards the understanding of how DNA methylation is involved in the regulation of gene expression or the generation of a pathologic phenotype. In addition, methylation maps may provide new insights into how the methylation patterns themselves are established.\n\nSeveral high-throughput techniques have been developed able to generate whole genome methylation maps. In general, the techniques consist of a methylation-sensitive pre-treatment and a read-out step. The pre-treatments generally consist of digestion by methyl-sensitive endonucleases, methyl-sensitive immunoprecipitation or bisulfite conversion, while the read-out of the methylation information is done by hybridization, amplification or sequencing9. Recently, several promising techniques have been developed that link the bisulfite conversion with High-Throughput Sequencing (MethylC-Seq10, BS-Seq11 or RRBS12). Briefly, the bisulfite treatment converts un-methylated cytosines into uracil (converted to thymine after PCR amplification) while leaving methylcytosines unconverted. After sequencing the bisulfite-treated genomic DNA, the methylation state can be recovered from the sequence alignments. Therefore, the methylation profiling from High-Throughput Bisulfite Sequencing data can be divided into two steps: the alignment of the reads, and the read-out of the methylation levels from the alignment. The alignment of bisulfite-treated reads is highly non-trivial due to the reduced sequence complexity given that all cytosines except methylcytosines are converted to thymines. This challenge has been extensively addressed over the last years and several algorithms are available that either align the reads in a 3-letter space or adapt the alignment scoring matrix in order to account for the C/T conversions. Among these algorithms are BSMAP13, Bismark14, MethylCoder15, NGSmethPipe16, BS Seeker17, Last18 and BRAT-BW19. Note that some of these tools are not just alignment programs but can, in addition, perform the profiling of the methylation levels such as Bismark and MethylCoder. After alignment, the methylation states can be recovered: C/T mismatches indicate un-methylated cytosines while C/C matches reveal methylcytosines. However, several error sources−like sequencing errors, clonal reads, sequence variation, bisulfite failure and mis-alignments−can lead to a wrong inference of the methylation levels16,18,20. For example, C→T or T→C (on converted cytosines) sequencing errors would be incorrectly interpreted as un-methylated or methylated respectively biasing the results towards lower or higher methylation levels. On the other side, bisulfite failures bias the methylation levels only to higher levels; un-methylated cytosines are not converted and therefore detected as methylcytosines. The existence of sequence variation is another important error source that was traditionally disregarded in the data analysis of whole genome bisulfite sequencing (WGBS) experiments. A C/T SNV would be interpreted as un-methylated cytosine. Given that over two thirds of all Single Nucleotide Polymorphisms (SNPs) occur in a CpG context, having two alleles: C/T or G/A21, sequence variation needs to be addressed as an important error source. A C/T SNV manifests on the complementary DNA strand as an adenine, while bisulfite deamination does not affect the guanine on the complementary strand (see Figure 1). This fact allows in principle to distinguish between sequence variation and bisulfite conversion and therefore to i) avoid wrong inference of the methylation state due to sequence variation and ii) detect sequence variation in the same sample preparation as the methylation levels. Profiling the methylation levels and the genotype of the sample from one experiment will be a very important step towards \"putting the DNA back into methylation”22, as the impact and importance of certain DNA sequences on the methylation levels have been recently demonstrated23. To our knowledge, the first program that performed a threshold-based detection of sequence variation in bisulfite sequencing experiments was NGSmethPipe16. This program detects sequence variation mainly to avoid wrong inference of the methylation level reporting those genome positions in the output. Only recently, the first state-of-the-art SNP calling algorithm based on the Genome Analysis Toolkit (GATK)24 was implemented to detect both methylation levels and sequence variation at high precision in a single experiment (Bis-SNP).\n\nSequence variation can be detected for a cytosine position analyzing the nucleotide frequency at the same position but on the complementary strand. Bisulfite conversion does not affect the guanine on the complementary strand, therefore the presence of any other base (H=A,C,T) might indicate the existence of an SNV. The figure illustrates three different situations: (a) a methylated cytosine in a CpG context without sequence variation (all reads that map to the position independently of the strand carry a cytosine in the corresponding position), (b) a heterozygous SNV (genotype C/T, SNV detected on the ‘+’ strand) and (c) a homozygous SNV (genotype T/T, SNV detected on the ‘-’ strand). The example in b) shows a heterozygous SNV; the 6 reads with A/G mismatch from a total of 11 reads mapping the position indicate a heterozygous variation. Furthermore, we can conclude that the cytosine allele is methylated (7 reads with C/C matches to the ‘-’ strand). The case illustrated in part c), shows 12 reads that show C/T mismatch (‘+’ strand in blue in the upper part). Without looking at the complementary strand, the inference would be a completely un-methylated cytosine. However, the 11 reads that map to the complementary strand show an A/G mismatch at the corresponding position (we would expect guanines in the case of bisulfite conversion). Note that on bisulfite treated datasets only G/A mapped on the ‘+’ strand and C/T on the ‘-’ strand (refereed to the ‘+’ strand) can be used for SNV calling purposes. The figure was generated using the UCSC Genome Browser41.\n\nHere we present MethylExtract, a multi-threaded tool for methylation profiling and sequence variation detection from alignments in standard BAM/SAM format25. The tool is able to generate high quality methylation maps taking into account SNVs, putative bisulfite failures, reducing also the contribution of sequencing errors by means of the base quality PHRED score26,27. In addition, it detects sequence variation based on VarScan methodology28 reporting all detected SNVs in VCF format29. Therefore, from a single sequencing experiment, MethylExtract obtains both the methylation levels and the sequence variation, which will increase the reliability of downstream analyses23. We confirm its usefulness using extensive artificial BS data and a comparison to Bis-SNP. We show that while its SNV-calling performance is slightly less specific but more sensitive compared to Bis-SNP, MethylExtract performs better in methylation profiling, is easier to use and over twice as fast on a typical whole genome experiment.\n\n\nImplementation\n\nMethylExtract is implemented in Perl and consists of one main script and two auxiliary scripts that are exclusively dedicated to the statistical assessment of the bisulfite error. In general, the program takes standard BAM/SAM file format as input (previously aligned reads) and performs methylation profiling and SNV calling taking into account several error sources like sequencing errors, clonal reads and bisulfite failures. MethylExtract writes two output files. First, the methylation information for each cytosine including the coordinates, sequence context (CG, CHG, CHH), number of methylcytosines, read coverage and mean base quality (PHRED) score. The second output file reports the sequence variation in standard VCF format29.\n\nFrequently, whole genome bisulfite experiments include the estimation of the bisulfite conversion rate through a completely un-methylated genome (lambda phage for example). If the bisulfite conversion rate is known, statistical tests can be applied to infer whether an observed methylation level might be only due to failures of bisulfite conversion. The two auxiliary scripts allow i) estimating the bisulfite conversion rate by mapping the bisulfite-treated reads from the un-methylated genome only and ii) to apply a binomial statistics based test to infer the probability that the “real” methylation value lies within a given interval of the observed value.\n\nThe PCR step can lead to duplicated (clonal) reads, thus causing a bias in the read coverage. This bias might lead to incorrect inference at positions with allele-specific methylation (genetic imprinting), sequence variation, hemi-methylation, sequencing errors, bisulfite failure or those that are heterogeneous over the cell population. Frequently, the start coordinates of the alignments are used to eliminate duplicates like in SAMtools25, adding a criterion to keep the best read among the duplicates. However, those approaches do not take into account that at a heterozygous locus two reads with the same start coordinate could represent two different alleles, thus not being clonal reads. The same applies for loci with genetic imprinting or hemi-methylation. To avoid the elimination of meaningful biological information, MethylExtract groups all reads that start at the same position in the genome and that have the same seed nucleotides with Q ≥ ‘minQ’; and selects the read that has the highest number of bases with Q ≥ ‘minQ’ (by default ‘minQ’ = 20) and the longest read in case of equal number of high quality positions. Furthermore, if there are multiple reads with the same selection values, only one will be selected in a random way. Two non-identical reads that align to exactly the same position in the chromosome can represent either the existence of sequence variation or putative clonal reads with a sequencing error in at least one read (disregarding mis-alignments). To restrict the impact of sequencing errors we used only the seed region of the read, i.e. the region with the highest quality. The seed is defined as those nucleotides at the 5´ end of the read (first 26 nt by default) that have a higher PHRED score than ‘minQ’.\n\nNote that the two types of methods, the ones that use only the coordinates and our method using the coordinates and the sequence, have advantages and disadvantages. If the sequence differences are considered, biological meaningful information like sequence variation, genetic imprinting or hemi-methylation is maintained; however, our approach will be vulnerable to sequencing errors and bisulfite errors. The default option is to not perform the detection of duplicated reads, and thus any of the publically available tools can be used optionally to remove clonal reads prior to run MethylExtract.\n\nThe first nucleotides can be removed from the 5´ end of the read (3 bp for the MspI restriction sites of reduced representation bisulfite sequencing (RRBS) protocol), as also implemented by Bismark14.\n\nThe bisulfite conversion error probability of un-methylated cytosines is usually below 1% in modern protocols. However, even for such low values, some positions could be incorrectly profiled, i.e. some methylated cytosines are actually un-methylated. MethylExtract implements a method proposed by Lister et al.30 to detect those reads with a high number of un-converted cytosines. By default, it eliminates reads with at least 90% of (presumably) unconverted cytosines in non-CpG contexts (Lister et al. used ≥ 3 methylated non-CpG cytosines). The default threshold is very conservative and only a rather small fraction of reads will be eliminated. Caution is needed if the user knows that the analyzed species (plants) or tissues (e.g. embryonic stem cells) contain an elevated number of DNA methylation in non-CpG contexts. In those cases, this step should be better skipped as otherwise a bias will be introduced into the analysis.\n\nSequencing errors are another important cause of incorrect methylation profiling (and SNV calling). The contribution of the individual bases can be controlled by means of the assigned PHRED score (i.e. an upper limit of sequencing error contribution to the wrongly inferred methylation states). For example, when setting PHRED score ≥ 20, thus accepting bases with a probability < 0.01 to be incorrectly called, the contribution of sequencing errors to the overall error would be less than 1%. By default, MethylExtract sets the minimum PHRED score to 20 (‘minQ’ parameter) which is then used for both methylation profiling and SNV calling (see below on the determination of the default values).\n\nSNVs are the most disregarded error source in the analysis of whole genome bisulfite sequencing data. Most tools would interpret a C to T substitution as an un-methylated cytosine, although a certain number of them are actually SNVs, and therefore this inference would be wrong. A C/T SNV manifests on the complementary DNA strand as an adenine, while bisulfite deamination does not affect the guanine on the complementary strand31 (Figure 1). The SNVs detection algorithm implemented in MethylExtract is an adaptation of the widely used varScan algorithm28. The main difference compared to SNV calling from non-bisulfite-treated DNA is the reduced amount of sequence information that can be used to detect sequence variation. The bisulfite treatment converts the un-methylated cytosines into thymines, and therefore, at cytosine positions nucleotides that might result from the bisulfite conversion cannot be used to detect sequence variation. For adenine and thymine, both strands can be used like in re-sequencing experiments. The algorithm works as follows: i) filter out positions that are covered by fewer reads than the minimum read depth (‘minDepthSNV’) – by default ‘minDepthSNV’ is set to 1, thus analyzing all positions that are covered by at least one read; ii) calculate the nucleotide frequencies including all base calls that pass the minimum PHRED score threshold (‘minQ’); iii) discard nucleotides with frequencies below a given threshold (‘varFraction’); iv) calculate a p-value for the variant positions (more than two nucleotides above ‘varFraction’) by means of Fisher’s exact test, v) only those positions with a p-value below a given threshold are considered as SNVs (‘maxPval’), and vi) the two nucleotides with the highest frequencies are determined as the putative genotype of the sample at this position. Detected sequence variation is reported in VCF output format, which can be used as input for SNP-annotation programs32 or VCFtools29.\n\nBisulfite conversion failure has been addressed using binomial statistics for the two possible outcomes; methylated and un-methylated33. However, intermediate biologically meaningful states exist like allele specific methylation (with expected methylation levels of 0.5, if both homologous chromosomes have the same sequencing depth), or the reported partial methylation levels30. Therefore, we developed a statistical test for the methylation levels and not for the methylation state previously proposed30,34. To apply this test, the user needs to know the bisulfite conversion rate obtained in the experiment. This rate needs to be established using an un-methylated genome (lambda phage, chloroplast, etc). We supply two additional scripts to i) estimate the bisulfite conversion rate using the appropriate experimental data, and ii) associate a p-value, based on binomial statistics, to each of the extracted methylation levels, as well as a procedure to control the false discovery rate35.\n\nIn order to calculate a p-value for a given methylation level, we first need to select an interval as we want to calculate the probability that the real methylation level lies within an interval of the observed methylation level. Once the interval is fixed, we can calculate the number of false methylcytosines that would not change the methylation level, e.g. the methylation level would stay within the error interval.\n\nOnce we have detected the maximum number of false methylcytosines that would maintain the methylation level within the error interval, we can calculate the p-value by means of the binomial distribution:\n\n\n\nbeing: p the bisulfite error rate, mc the number of observed methylcytosines at a given position and fmc the maximum number of allowed false methylcytosines. The p-value corresponds then to the probability to find more than fmc false methylcytosines at this position, e.g. the probability that the real methylation level lies outside the defined error interval.\n\nTo illustrate the method, let’s assume that we have a position that is covered by 21 reads with 17 methylcytosines. In this situation, we would have a methylation level of 0.81. If we fix the error interval at 0.1, we could accept up to 2 false methylcytosines. For two false methylcytosines, the methylation level would be (17–2)/21 = 0.714 which lies within the error interval of 0.81–0.1 < 0.714 while 3 false methylcytosines would lead to a methylation level of 0.67 which lies outside the tolerated error interval. Note that the coverage depth of the position (number of reads) does not appear in the equation, but it does to calculate the maximum number of false methylcytosines. In this way, a higher coverage will lead to a higher number of allowed false methylcytosines and therefore to smaller p-values. Finally, we implemented the Benjamini–Hochberg step-up procedure35 to control for the false discovery rate in multiple testing. This step can be optionally activated by the user.\n\n\nResults\n\nMethylExtract is currently one of the programs with most implemented features related to quality control. Together with Bis-SNP it is the only program that detects sequence variation, both to avoid incorrect methylation profiling and to assess the genotype of the used sample. Table 1 shows a comparison of the main features of all programs that allow methylation profiling from aligned reads. Apart from the used method to call the sequence variation, another important difference between MethylExtract and Bis-SNP is the number of scripts involved to run a full analysis. Bis-SNP requires the execution of: i) 3 scripts to sort, add read group tags (required by GATK tools) and mark duplicates, ii) 4 scripts to realign the reads and recalibrate the base quality score, iii) 2 scripts to obtain and sort the SNVs and number of methylcytosines and iv) an additional script to calculate the methylation levels on a standard format. In summary, Bis-SNP needs 10 different scripts to process reads from bisulfite-treated experiments. On the other hand, MethylExtract unifies all analysis steps into a single program which makes it especially suitable for users without a bioinformatics background. Another feature that is currently unique to MethylExtract is the possibility to assess the bisulfite failure in a statistical way. In order to achieve this, MethylExtract provides an auxiliary script to estimate the bisulfite conversion rate, and a second script that calculates the probability that the observed methylation level lies outside the selected interval of the real methylation level due to bisulfite conversion failures.\n\n# Input formats: input formats used by each software. 5´ trim: allows the trimming of the 5´ end of the reads. Bisulfite failure: implementation of a step to discard reads where the bisulfite might have failed converting the un-methylated cytosines. Minimum depth: allows the user to discard positions with low coverage. Base call errors: discards positions that do not exceed a given minimum PHRED score value. SNVs calling: detects variation that can lead to wrong methylation levels or context estimation. Methylation output formats: available formats for the methylation results. Variation output formats: output formats for the sequence variation results. The asterisk (*) represents a non-standard input or output format, or the impossibility of extracting the methylation ratios from other alignment tools. The dash (-) represents the inexistence of SNV output format, because the software does not allow to detect them.\n\nAs mentioned above, sequence variants can lead to incorrect inference of methylation values. Figure 2 illustrates the impact of C/T variation on the methylation values (C/(C+T) ratio) within CpGs contexts. Around 470,000 SNVs within CpG contexts (affecting to 2.08% of the CpG contexts on the genome) covered by at least 10 reads have been detected by MethylExtract in Lister’s H1 dataset30. Figure 2 shows the methylation levels for non-variant positions (both alleles coincide with the reference) and for the variant sites, both in homozygosis and heterozygosis. The observed distribution of the methylation levels without variation has two maxima close to 0 and 1, which is similar to previous studies30. However, for heterozygous positions detected by MethylExtract, the methylation levels present a local maximum at approximately 0.5 (the T allele on one of the parental chromosomes biases the methylation levels to intermediate values, if the C allele is methylated) and a peak at 0 (if the C allele is un-methylated). Finally, for the homozygous positions where both chromosomes present the T allele, most of the methylation values are exactly 0. However, we know that no cytosine exist at those locus in the analyzed sample and therefore these values are incorrect and should be eliminated from the analysis. The incorrectly inferred methylation values for variant positions, both in homozygosis and heterozygosis, stress the need to detect and remove them from the analysis. For example, a CpG position with C→T SNV on both homologous chromosomes is eliminated by MethylExtract, as actually at this position no CpG exists in the sample. Furthermore, MethylExtract outputs the detected genotype of all profiled positions and therefore heterozygotic loci can be detected easily by the user and treated apart if wished.\n\nC/(C+T) values for cytosines at non-variant and variant (homo- and heterozygotic) positions were shown. The minimum read coverage was set to 10 reads.\n\nMethylExtract implements several quality controls and is among the programs with most implemented features. Main features of MethylExtract are compared in Table 1 to a number of other, widely used programs. The implementation was validated in several ways. MethylExtract takes aligned reads as input and therefore we first compared the methylation profiling quality achieved on artificial bisulfite data when using two different tools for aligning bisulfite-treated reads; NGSmethPipe16 and Bismark36. Next, we quantify the correctly profiled methylation levels and SNVs as a function of the main quality parameters using NGSmethPipe as aligner. Finally the predictive power of MethylExtract to detect methylation levels and sequence variation was compared to Bis-SNP24, both in terms of sensitivity and positive predictive value as it was proposed for datasets for which the number of true negatives tend to be much higher than false positives37.\n\nGeneration of artificial BS data. For all further comparisons we will use artificial bisulfite data. The usage of this kind of data for benchmarking has the advantage that the true methylation levels and genotypes are known for each position, which is not true when using other experimental methods like microarrays as a golden standard. Artificial sequencing data has been used before in other studies assessing the SNV prediction quality of different algorithms38. To generate the artificial bisulfite data we used DNemulator18. We obtained two datasets from the human contig GL000022.1 (11.2Mb), one with all CpGs completely methylated, and the other one with all CpGs completely un-methylated. DNemulator allows also simulating the genotypes of a diploid genome by introducing the sequence variation from a set of confirmed SNPs (dbSNP135)39. Finally, we simulate a bisulfite conversion rate of 99%. The read quality scores are taken from real experimental data (Lister’s H1 dataset30). All together, we generated artificial bisulfite sequencing datasets at two different coverages; 15× and 20× which corresponds to the coverage usually achieved in whole genome bisulfite sequencing experiments.\n\nMethylExtract with NGSmethPipe and Bismark input. NGSmethPipe16 is a tool to align bisulfite-treated reads which was developed by our group. It is based on the Bowtie aligner and uses a 3-letter alphabet to map the bisulfite-treated reads. The program implements a pre-processing to improve the mapping accuracy18 and an alignment seed extension in order to increase the number of mapped reads.\n\nWe launched both, NGSmethPipe and the well-established Bismark tool with default options to obtain the SAM/BAM input. Next we used MethylExtract on both input files to obtain the number of covered CpGs and the number of correctly recovered methylation values. Note that we know the correct methylation value for each position due to the use of artificial bisulfite data. A position is considered as correctly profiled, only if the obtained methylation value is identical to the real value. Figure 3 shows the result of this comparison. It shows that the obtained CpG coverage and number of correctly profiled positions is nearly identical both as a function of read coverage (15× and 20×) and for the methylated and un-methylated input data. The only remarkable difference is that NGSmethPipe leads to a slightly higher CpG coverage at 20× for both data sets. Nevertheless, the main conclusion is that MethylExtract yields nearly identical results for input sets obtained from NGSmethPipe and Bismark.\n\nThe results obtained from MethylExtract (correctly profiled methylation values and CpG coverage) using two bisulfite short read aligners, NGSmethPipe and Bismark are compared. The results are nearly independent of the used alignment algorithm.\n\nAnalysis of the MethylExtract quality parameters. Next, we aimed to assess the impact of certain quality parameters implemented in MethylExtract on the methylation profiling and SNV calling capacity. To detect sequence variation, MethylExtract relies on two main parameters, i) the relative nucleotide frequencies (‘varFraction’) and ii) the corresponding p-value. The ‘varFraction' parameter determines if a position shows putatively variation: the position is analyzed only if at least one nucleotide that differs from the reference sequence has relative frequencies higher than ‘varFraction’. Only for these positions the corresponding p-value is calculated by means of a Fisher exact test. Figure 4 shows the impact of these parameters on the prediction sensitivity (Sn) and positive predictive value (PPV). Sequence variation is best detected by setting the ‘varFraction’ threshold close to 0.1 (yielding around 91% Sn and only 2% of false positives at a statistical significance of 0.05). If the ‘varFraction’ threshold is increased further, the probability to eliminate heterozygous loci increases steadily for positions with high bias in the read coverage between the two homologous chromosomes. If the p-value threshold is set to 0.01, a small increase in positive predictive value (PPV) is observed, but it causes a strong decrease in sensitivity. Therefore, we determined a ‘varFraction’ of 0.1 and a p-value threshold of 0.05 as the best (default) parameters to detect sequence variations.\n\nThe figures show the sensitivity (Sn) and the positive predictive value (PPV) for SNV detection using two different p-value thresholds. The graphs are based on the methylated (top) and un-methylated (bottom) artificial bisulfite datasets at a mean 20× read coverage.\n\nThe minimum base quality (‘minQ’) and the coverage depth (‘minDepthMeth’ for the methylation profiling) thresholds might be also important parameters to control de quality of methylation profiling and SNV calling. To analyze the impact of the minimum PHRED score parameter (‘minQ’) we fix the minimum read coverage (‘minDepthMeth’) in 3, as suggested by Laurent et al.40, ‘varFraction’ = 0.1 and ‘maxPval’ = 0.05 (default values derived above). Figure 5 shows the fraction of correctly profiled methylation values and the PPV for SNVs. It can be seen that the correctly profiled positions increase approximately 31% (from 68% to 99%) and the SNVs around 71% (27% – 98%), when the minimum PHRED score is increased from 0 (all base calls are accepted) to 30 (0.001 error probability). The major difference between the methylated and un-methylated datasets is observed for the profiling of the methylation level for which the percentage increases only from approximately 52% to 86%. The simulated bisulfite conversion failures will affect mainly un-methylated positions which can explain the observed differences. These results confirm that the ‘minQ’ threshold is critical to obtain high quality methylation profiling and genotyping results. The default value was set to 20 as higher values will lead to a coverage reduction compromising the SNV calling sensitivity.\n\nBoth graphs show the positive predictive value (PPV) for SNV calling and the fraction of correctly profiled CpG methylation values (methylation profiling) as a function of the minimum base quality (PHRED score parameter ‘minQ’). The graphs are based on the methylated (top) and un-methylated (bottom) artificial bisulfite datasets at a mean 20× read coverage.\n\nThe comparison between MethylExtract and Bis-SNP needs to be based on identical alignment input files in BAM/SAM format. We obtained these files in a two-step process: First, we trim the input reads as it was done by Lister et al.30 and second, we align the bisulfite treated reads to the reference genome using Bismark36 with default parameters. Note that we based this comparison on Bismark, as the realignment and recalibration steps implemented in Bis-SNP require the read mapping quality, which is currently not available in NGSmethPipe.\n\nBoth methods were used with default parameters. We first compared the detection of sequence variation (SNVs) in terms of Sn and PPV. Figure 6, shows that in general Bis-SNP is more specific (between 1.9% and 3.9% higher PPV), being MethylExtract more sensitive (between 1% and 3.1% higher Sn). This trend can be seen for both artificial bisulfite datasets as well as for both read coverages. However, when comparing the fraction of correctly recovered methylation values, drastic differences can be seen (Figure 7). While Bis-SNP yields a slightly higher number of covered positions (Fraction of covered CpGs), MethylExtract is strikingly more specific. In all four comparisons, MethylExtract yields over 20% more correctly profiled positions compared to Bis-SNP. One explanation for this difference might be the PHRED score quality threshold implemented in MethylExtract.\n\nThe top graph shows the sensitivity (Sn) and the bottom graph the specificity (PPV) obtained for the methylated and un-methylated artificial bisulfite datasets at two different mean coverages (15× and 20×).\n\nBoth methods are compared in terms of fraction of correctly profiled CpG methylation values (top) and the fraction of recovered CpG positions (bottom).\n\nAs mentioned before, only Bis-SNP and MethylExtract perform the detection of SNVs which constitutes an additional CPU demanding task. Therefore, we only compared these two programs in terms of CPU time using Lister’s H1 dataset30 on a 24 core Intel(R) Xeon(R) CPU X5650 2.67GHz machine. Available memory is crucial for both methods. In order to not bias the comparison, we limited the available memory to 15GB for both programs allowing up to 15 threads. We used a gnu-zipped SAM input file for MethylExtract (122GB) and a BAM input file for Bis-SNP (151GB). Note that only the input file format is different but the data was exactly the same. After aligning with Bismark, we carried out the entire process for both tools (from the aligned reads to the methylation and SNV profiling). MethylExtract needed 51 hours 7 minutes to process the entire dataset including the sorting by coordinates and the removal of duplicated reads. Bis-SNP spend 40 hours 37 minutes sorting the file and removing putative clonal reads, 46 hours and 51 minutes realigning and recalibrating the reads, and 36 hours 33 minutes genotyping and retrieving the methylation levels. Therefore, it seems that MethylExtract is notably faster than Bis-SNP (2.49 times on this whole genome data set), with the additional advantage that MethylExtract uses a more compressed data-input file.\n\n\nConclusions\n\nWe present a user-friendly tool for methylation profiling and SNV calling in whole genome bisulfite sequencing experiments. MethylExtract takes standardized input formats (BAM/SAM) and writes out likewise broadly used file formats like WIG, BED and VCF. To show its usefulness, we compared it to Bis-SNP, a recently published method that is very similar in scope. Although Bis-SNP is more specific (less false positive predictions) in the detection of SNVs, MethylExtract is more sensitive (higher number of recovered SNVs). However, the main advantages of MethylExtract when compared to Bis-SNP seem to rely in the higher percentage of correctly profiled methylation values, as it reaches values over 20% higher compared to Bis-SNP. Other aspects that favor MethylExtract are its user-friendliness (everything is implemented into one script) and the run-time in comparison to Bis-SNP (over 2 times faster in a whole genome bisulfite sequencing experiment).\n\n\nAvailability and requirements\n\nMethylExtract is freely available. The source code, the tutorial and artificial bisulfite datasets can be downloaded from the page http://bioinfo2.ugr.es/MethylExtract/ and are also permanently accessable from 10.5281/zenodo.7144.\n\n\nList of abbreviations used\n\n5meC: DNA methylation at cytosine carbon 5 position; SNV; Single Nucleotide Variation; WGBS: whole genome bisulfite sequencing; SNP: Single Nucleotide Polymorphism; PPV: positive predictive value; PHRED score: the quality score to each base call assigned by the program PHRED; SAM format: Sequence Alignment/Map format used for storing large nucleotide sequence alignments; BAM format: the compressed binary version of the SAM format.",
"appendix": "Author contributions\n\n\n\nGB wrote the code and carried out the experiments, AR helped with the benchmark experiments, and MH, JLO and GB designed the software and wrote the manuscript. All the authors critically read and approved the final version.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the Spanish Government [BIO2008-01353 to JLO and BIO2010-20219 to MH], and Basque country 'AE' grant (GB).\n\n\nReferences\n\nOliveira DC, Tomasz A, de Lencastre H: The evolution of pandemic clones of methicillin-resistant Staphylococcus aureus: identification of two ancestral genetic backgrounds and the associated mec elements. Microb Drug Resist. 2001; 7(4): 349–61. PubMed Abstract | Publisher Full Text\n\nGu F, Doderer MS, Huang YW, et al.: CMS: a web-based system for visualization and analysis of genome-wide methylation data of human cancers. PLoS One. 2013; 8(4): e60980. 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}
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[
{
"id": "2103",
"date": "25 Oct 2013",
"name": "Michael B. Stadler",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present the tool MethylExtract that combines extraction of methylation states and SNV calling, given alignments of bisulfite-converted reads to a reference in SAM/BAM format. Methylated CpG are mutation hotspots - dealing with SNVs is therefore an important part of any analysis of Bis-seq data.The main functions of MethylExtract are implemented in a single Perl script, which should make it easy to use - unfortunately I could not verify this because it failed to run in my environment (see below).The performance of MethylExtract is evaluated using simulated sequence data (completely methylated or completely unmethylated containing sequencing errors and SNVs) and compared to Bis-SNP, a conceptually similar tool that is based on the GATK variant calling package. The simulations cover most important aspects of the tool; however, the paper would benefit from extended simulations and a test on a real dataset. For example, the current evaluation does not cover Bis-seq datasets with very low or very high coverage or intermediate methylation levels (see minor issues below).The paper is clearly written, and the conclusions are supported by the presented results.Major issues:The MethylExtract perl script (version 1.3) did not run in my environment (RHEL 6, with Kernel 2.6.32-220.7.1.el6.x86_64, perl 5.10.1 built for x86_64-linux-thread-multi). Trying to run the main script resulted in a compilation error: >perl MethylExtract_1.3.pl Type of arg 1 to keys must be hash (not hash element) at MethylExtract_1.3.pl line 318, near \"}) \" Execution of MethylExtract_1.3.pl aborted due to compilation errors.It is possible that the problem lies in the combination of the script and the test environment. However, the test environment fulfills the stated requirements and dependencies. Figure 6 and corresponding text: A single point comparison of Sn/PPV between MethylExtract and Bis-SNP, both with default parameters, is not very informative. Typically there is a trade-off between sensitivity and specificity which can be influenced by the choice of parameter values such as the score or P value cutoffs. It is possible that with slightly altered parameter values, the improved Sn/reduced PPV of MethylExtract compared to Bis-SNP turn into the opposite. The two tools should therefore be compared using varying parameter settings or cutoffs (altering the trade-off between Sn and PPV) and then relating the resulting specificity and sensitivity in an ROC analysis. The same applies in principle to the results presented in Figure 3.Minor issues:Simulations - readout: The current evaluation of \"correct methylation\" requires exact identity of simulated and estimated methylation states. This criterium is very stringent yet may not be able to uncover systematic problems. In practice, a very small deviation from the true methylation level may be tolerable. For illustration: A tool that produces many incorrect values that are off only by a small amount may be preferable to a tool that produces fewer incorrect values that are several-fold off. I would suggest using a continuous measure of performance (e.g. the differences between true and estimated methylation levels) or to allow for a minimum deviation. Simulations - methylation levels: In the introduction, the authors point out the value of methylation levels as opposed to methylation states. Also, intermediate methylation is present in virtually all real world Bis-seq data sets. The simulations should take this into account and also contain C's with intermediate methylation levels (e.g. around 50% methylation). Simulations - coverage: The current simulations are performed at 15- and 20 fold coverage and the two yield very similar results. More informative differences in performance may be observed when simulating data at even lower (~5-fold) or higher (>30-fold) coverage, which are commonly found in published Bis-seq datasets. Runtime comparison: It's surprising that even though MethylExtract supports BAM input, SAM.gz was used for runtime measurements, while Bis-SNP was reading from BAM input. Unpacking of alignments from BAM files is CPU-intensive, and I wonder if MethylExtract would take more time if it was run on the same input as Bis-SNP. Table 1: MethylExtract is listed to support both SAM and BAM inputs. However, it does not directly read BAM files, but converts them to SAM using SAMtools. Using such a conversion, BSMAP and Bismark also support BAM input. I would suggest not to discriminate between SAM and BAM inputs in the table to avoid confusion based on this subtle difference. Impact of parameter choice when analyzing real world data: For some parameters (e.g. \"duplicated reads filter\" and \"elimination of bisulfite conversion failure\"), it is unclear how they would impact results in a real world analysis. A comparison of the results obtained on an experimental dataset with different parameter values could identify sensitive parameters and guide users when choosing parameters for their own analysis. Figure 5: The labels of the two y-axes are missing. In addition, CpG coverage (blue line) was probably scaled to be plotted on the same axis; if that is the case, it should be described in the legend and/or indicated in the plot.",
"responses": []
},
{
"id": "2607",
"date": "13 Jan 2014",
"name": "Jörn Walter",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe mapping and calling of cytosine methylation in whole genome bisulfite sequencing is a challenging task. Following sequence alignment, the localisation and scoring of reliable and quantitative positional methylation information requires a number of control functions including the detection and high quality scoring of SNVs. So far BiSNP has been used as the major tool for these tasks. The MethylExtract tool now offers a (slightly) improveed software suite compiling state of the art (BiSNP-like) features with additional QCs. One advantage of MethylExtract is that the package can be executed in a single PERL script. Compared to BiSNP, MethylExtract reduces the error rate of false SNV calling by including an optimized PHRED score and controlling for bisulfite conversion error rates. MethylExtract accepts SAM and BAM alignment files and cretaes an independent SNV output file (VCF). With SAM as an input MethylExtract runs about 2x faster compared to BiSNP. A performance test on 15x and 20x artificial test alignment sets shows a better performance of MethylExtract in comparison to BiSNP but only with respect to specificity, while BiSNP has the better sensitivity. Unfortunately the authors did not run a direct comparison on real datasets. Overall MethylExtract is a nice compilation of surely useful tools for a comprehensive and quality controlled WGBS data analysis. The key features of the package are nicely documented. I only have my doubts that conversion rate errors calculated on spiked in control DNA really generates a meaningful background correction.",
"responses": []
},
{
"id": "3164",
"date": "27 Jan 2014",
"name": "Felix Krueger",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral comments MethylExtract is a new tool that implements several QC related steps on already aligned Bisulfite-Seq data. This includes the deduplication of clonal reads, filtering based on base call quality, identification of potential bisulfite conversion errors and most notably the detection of single nucleotide variants (SNVs) that would affect methylation calls, the latter of which is until now only being performed by Bis-SNP.After reading the documentation it was straight forward to set MethylExtract off, and it ran to completion in an acceptable time frame (~1d 7h for one lane of HiSeq data aligned against the human genome using Bismark). I liked that the deduplication tool that would potentially allow reads aligning to the same position in the genome to pass if they contain SNVs and thus originate from two distinct alleles (even though it is probably questionable whether one would expect to see this sort of reads a lot for large eukaryotic genomes at moderate read depth such as 15x).Even though I've got a few questions about how certain things are handled or documented in the current implementation of MethylExtract, I was quite impressed at how easy it was to get hold of SNV information using just one command. I am sure MethylExtract will prove a useful tool in the genuine analysis of bisulfite data. Handling paired-end alignmentsThe only time I found mention of paired-end reads was in the explanation of the deduplication option 'delDup' in the MethylExtract Manual. One can further see that the options 'tagW' and 'tagC' for FLAG values of reads aligning to the Watson and Crick strands default to 0 and 16, respectively, which are standard FLAG values for forward or reverse mapping single-end reads. If MethylExtract currently only handles single-end experiments this is fair enough, even though it would be a serious limitation seeing that most data we generate is in fact paired-end data. Paired-end reads will have at least four different FLAG values, so it is unclear if and how one would specify these? Would the SNV detection would still work with paired-end reads? Are there options to remove overlapping parts in the reads? In any case, I think paired-end reads should be documented better, both in the manuscript and the manual. Handling SNV positionsIf I understood it correctly, CpG positions with a homozygous C to T SNV are eliminated by MethylExtract before reaching the final methylation output, but does this also happen for heterozygous loci? The manual mentions: \"(v) SNV can be detected and removed\" - can they or are they?It seems that MethylExtract reports only a single position (presumably the most 5' one?) for cytosines in CG or CHG context (this could also be mentioned in the manual). Are symmetrical cytosine positions completely eliminated from the output if SNVs are detected on at least one strand? Similarly, is the context of a cytosine determined purely by the genomic sequence or would a homozygous SNV effectively also change a C's context (e.g. would an A to G SNV from 'CAG' to 'CGG' change the context from CHG to CG?). I would find it useful to read some more information on how these cases are dealt with, maybe in the User Guide? Finally, to make it a bit more user-friendly I would welcome an option to include or exclude positions from the output specifically that were detected as homozygous or heterozygous SNVs. M-biasMethylExtract addresses several important aspects affecting the accuracy of BS-Seq experiments, however it doesn't mention the issue of methylation bias (M-bias) in the reads (described in Hansen et al., 2012). M-bias may have several causes, such as 5' bisulfite conversion failure (described in BSeQC - Lin et al., 2013), fill-in bias during library construction which is especially pronounced for paired-end reads (as an example see read 2 in this report: http://www.bioinformatics.babraham.ac.uk/projects/bismark/PE_report.html) or other technical reasons (e.g. PBAT libraries, an example report is available here: http://www.bioinformatics.babraham.ac.uk/projects/bismark/SE_report.html). Are there any plans to take M-bias into consideration in future versions of MethylExtract? 5' end trimmingBoth the manuscript and the MethylExtract manual mention that there is an option to ignore bases at the 5' end of reads, e.g. \"3 bp for the MspI restriction sites of reduced representation bisulfite sequencing (RRBS) protocol\". While the option is certainly useful, e.g. for the removal of M-bias in the reads, it should be noted that for standard (= directional) RRBS libraries the first 3bp should reflect the true genomic methylation state of the MspI site and do not have to be ignored. The situation is somewhat different for non-directional reads or when reading through filled-in positions at the 3' end of reads. We have tried to illustrate this in a bit more detail in this brief RRBS guide (http://www.bioinformatics.babraham.ac.uk/projects/bismark/RRBS_Guide.pdf). Very minorThe version of MethylExtract hosted on zenodo.org which I downloaded first was outdated (v1.3) and failed to run at some point. The latest version from http://bioinfo2.ugr.es/MethylExtract/ (v1.4) worked fine.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-217
|
https://f1000research.com/articles/2-248/v1
|
15 Nov 13
|
{
"type": "Web Tool",
"title": "StatsDB: platform-agnostic storage and understanding of next generation sequencing run metrics",
"authors": [
"Ricardo H. Ramirez-Gonzalez",
"Richard M. Leggett",
"Darren Waite",
"Anil Thanki",
"Nizar Drou",
"Mario Caccamo",
"Robert Davey",
"Ricardo H. Ramirez-Gonzalez",
"Darren Waite",
"Anil Thanki",
"Nizar Drou",
"Mario Caccamo"
],
"abstract": "Modern sequencing platforms generate enormous quantities of data in ever-decreasing amounts of time. Additionally, techniques such as multiplex sequencing allow one run to contain hundreds of different samples. With such data comes a significant challenge to understand its quality and to understand how the quality and yield are changing across instruments and over time. As well as the desire to understand historical data, sequencing centres often have a duty to provide clear summaries of individual run performance to collaborators or customers. We present StatsDB, an open-source software package for storage and analysis of next generation sequencing run metrics. The system has been designed for incorporation into a primary analysis pipeline, either at the programmatic level or via integration into existing user interfaces. Statistics are stored in an SQL database and APIs provide the ability to store and access the data while abstracting the underlying database design. This abstraction allows simpler, wider querying across multiple fields than is possible by the manual steps and calculation required to dissect individual reports, e.g. ”provide metrics about nucleotide bias in libraries using adaptor barcode X, across all runs on sequencer A, within the last month”. The software is supplied with modules for storage of statistics from FastQC, a commonly used tool for analysis of sequence reads, but the open nature of the database schema means it can be easily adapted to other tools. Currently at The Genome Analysis Centre (TGAC), reports are accessed through our LIMS system or through a standalone GUI tool, but the API and supplied examples make it easy to develop custom reports and to interface with other packages.",
"keywords": [
"Next generation short-read sequencers are now capable of generating hundreds of gigabases of sequence data per run. This increase in throughput is complemented by technologies such as long-read single molecule platforms",
"multiplex sequencing and RADSeq1",
"2",
"that lead to differing analytical techniques and enable hundreds of samples to be combined into a single sequencing experiment",
"respectively. With this data heterogeneity and abundance comes a significant challenge to keep track of samples",
"to understand data quality and to understand how quality control (QC) and yield are changing across instruments and over time. As well as the desire to understand historical data",
"centres often have a duty to provide clear summaries of individual run performance to collaborators or customers."
],
"content": "Introduction\n\nNext generation short-read sequencers are now capable of generating hundreds of gigabases of sequence data per run. This increase in throughput is complemented by technologies such as long-read single molecule platforms, multiplex sequencing and RADSeq1,2, that lead to differing analytical techniques and enable hundreds of samples to be combined into a single sequencing experiment, respectively. With this data heterogeneity and abundance comes a significant challenge to keep track of samples, to understand data quality and to understand how quality control (QC) and yield are changing across instruments and over time. As well as the desire to understand historical data, centres often have a duty to provide clear summaries of individual run performance to collaborators or customers.\n\nA user’s initial attempts to understand the QC profile of a run usually involve using manufacturer-supplied software, for example Illumina’s Sequence Analysis Viewer or PacBio’s SMRTAnalysis. As well as these, a number of community-developed tools have emerged for assessing run quality and they are often included in the primary analysis pipelines of sequencing centres. FastQC3 is a popular tool for analysing FASTQ files and is able to report a wide range of information related to the quality profile of the reads, as well as analysing GC content and over-represented sequence, such as PCR duplicates or over-abundance of adaptors. HTQC, another toolkit for FASTQ data analysis, is composed of a set of six tools for analysis and trimming of reads4. PRINSEQ also analyses and trims reads, with an emphasis on metagenomic datasets5. Other tools include NGSQC6, QRQC7 and SAMStat8. The latter tool, as the name implies, works with SAM files rather than FASTQ files.\n\nThe new generation of single molecule sequencing technologies, such as the RS platform from Pacific Biosciences with longer reads and different error profiles, have brought their own QC challenges. While it is still possible to get useful information from next generation tools such as FastQC, new tools are emerging which are tailored to the platform. Examples include stsPlots, which provides graphical summaries of data included in the sts.csv files output by the instrument9 and PacBioEDA which operates on the .bas.h5 files produced by the PacBio primary analysis10.\n\nWhile there are a range of useful tools available to generate QC statistics for individual runs, we are not aware of any currently available solution for facilitating the easy storage and access of this valuable information. For this reason, we have created StatsDB, a platform-independent, tool-independent run QC and metadata database with APIs in Perl and Java. StatsDB features a generic database schema which enables the storage of data from any QC tool designed for any sequencing platform. StatsDB is designed to automate the storage of run QC metrics, enabling more granular queries over the data held within. Installation is simple and use of the software and API requires no knowledge of SQL.\n\n\nMethods and implementation\n\nFigure 1 illustrates the overall structure of the StatsDB system. At the core of StatsDB is a MySQL database which stores run metrics on a per-base, per-partition or per-run basis. On top of the database sits an API - currently implemented in both Perl and Java - which abstracts the database design from the tools that use the data and provides a simple interface for adding or querying data.\n\nParsers are simple scripts to write the output of QC tools to the database, while Consumers present the database contents to users.\n\nWe envisage two types of programs utilising the API parsers and consumers. Parsers process external data output from QC tools and use the StatsDB API to write the data. Conversely, consumers query the data stored in StatsDB and present it to the user, typically in the form of text and graphs. The StatsDB package includes parser implementations and examples of consumers, and the API enables others to be developed quickly and easily. Additionally, integrating these consumers into third-party applications is promoted - the open source LIMS system currently in development at TGAC, MISO11, provides support for accessing data in StatsDB out-of-the-box.\n\nThe StatsDB framework supplies two SQL files that are used to set up the database for use, comprising the schema and the stored procedures, respectively, and detailed in the following sections.\n\nThe first step is to create a new MySQL database called ‘statsdb’ and grant a user read-write access to it, e.g. a new ’statsdb’ user with a suitable password, e.g. 'statsdb’:\n\n\n\nThe two SQL files are then imported into the database as follows:\n\n\n\nThis will populate the ‘statsdb’ database with the tables and procedures required.\n\nTesting a successfully installed database can be undertaken by running the FastQC parser on the supplied example data, as follows:\n\n\n\nThis will result in data being inserted into the database. In order to revert back to an empty state, reimport the schema SQL as detatiled previously.\n\nThe StatsDB database is designed to be flexible and to hold virtually any type of QC analysis. Figure 2 illustrates the database schema. The database is normalised to the third normal form (3NF) and has stored procedures and views to facilitate consistent access to the stored information. The tables in the core of the database are as follows:\n\nanalysis holds the ID and timestamp of when the analysis was recorded.\n\nanalysis_property holds general information about the analysis and the run from which it originated. The values contained within this table are populated directly from a user-specified tab-delimited table of property headings and respective values, i.e. the RunTable object (see ’Parsers’ below). The following properties are used to define common denominators across platforms and analyses:\n\ntool - the name of the tool that was used to undertake the analysis (for example, FastQC, PRINSEQ).\n\nencoding - the encoding of the input, for example ‘Illumina 1.5’, ‘fasta+qual’.\n\nchemistry - a short name for the chemistry or type of experiment run.\n\ninstrument - the ID of the instrument.\n\nsoftware - the basecaller software or software version of the instrument.\n\ntype - type of experiment, for example ‘RNA-Seq’, ‘WGS’.\n\npair - if the experiment is paired end or mate pair, then 1 if the first read or 2 if the second read.\n\nsample_name - the name of the sample as assigned during the library construction.\n\nlane - the lane or flowcell of the instrument.\n\nrun - the name of the run, as given by the instrument.\n\nbarcode - if the sample is multiplexed, the adaptor barcode of the sample, for example ‘ACCTTG’.\n\nanalysis_value is used to store the properties that can be represented as a value for the whole run, e.g. overrepresented sequences, counts of multiplexed tags.\n\nper_position_value stores values related to a absolute position that is never grouped, such as counts of how many times a certain quality is represented.\n\nper_partition_value is similar to per_position_value, but allows grouping by some range or factor.\n\nvalue_type is an auxiliary table that holds descriptions for the values and the scope of the value.\n\ntype_scope is an auxiliary table defining the scope relevant to the analysis to group the values consistently. Currently, the following are the scopes supported by StatsDB.\n\nanalysis - global values for the analysis, for example ‘Total GC content’.\n\nbase_partition - values of summaries per base, allowing grouping of ranges, for example quality mean per position.\n\nsequence_cumulative - for cumulative counts, such as quality scores or read counts.\n\nIn addition to the tables, StatsDB has three auxiliary views, used to facilitate complex queries:\n\nproperty transposes the analysis_property table, transforming the row values to columns.\n\nrun merges the corresponding values from property, according to the run.\n\nlatest_run selects the latest run from the run view, so if an analysis has been run more than once, only the latest results are stored.\n\nMost of the functionality of StatsDB is embedded directly in the database as views and stored procedures. This enables it to have lightweight APIs which behave consistently across languages and means that the API can easily be ported to other languages should the implementation require it.\n\nThe following stored procedures are used by the APIs to query the database:\n\nlist_runs will list all available runs.\n\nlist_selectable_properties will list the properties that can be used as queries.\n\nlist_selectable_values_from_property will list the values for a given property.\n\nlist_summary_per_scope will return the descriptions present for a given scope. This is useful to know if a value_type is already present or not, before trying to insert it again.\n\nThe following stored procedures are used to generate reports. To make queries, the common arguments to specify the run or runs to group are: instrument_in, run_in, lane_in, pair_in and barcode. The arguments are optional and if more than one analysis meets the query criteria, a summary is produced. This can be used, for example, to query the quality of a given lane and use this information to find systematic issues.\n\ngeneral_summaries_for_run returns a summary of all the values global to the analysis.\n\nsummary_per_position_for_run returns all the values queried from the per_partition_value table.\n\nsummary_value_with_comment returns the summary values with a descriptive text, if it was present as a note for a value. For example, this can be used to retrieve further description of the over-represented sequences in a FastQC report.\n\nThe StatsDB framework provides two APIs, one in the Perl language and the other in Java, both of which offer the same functionality. They call the stored procedures and provide a sufficient layer of abstraction such that parsers and consumers do not need to access the stored procedures directly. Therefore, the typical method of accessing the data held within StatsDB is through the following APIs.\n\nBefore using the Perl API, a connection to the database has to be created as defined by the Perl DBI API12. A template for the configuration file is provided in Perl/examples/template_db.txt, and the values need to match those used in the database installation instructions above:\n\n\n\nThe Perl API comprises modules to import analysis information to the StatsDB database, as well as functions to query the database. The abstraction of the database is contained in the QCAnalysis module, which is used to add an analysis to the database. It automatically fills the missing types of value in the database, so it is not necessary to have a comprehensive list of types of analysis that are going to be stored a priori. However, a parser needs to define valid types for the QCAnalysis object, i.e. value_type, and to which scope they should be assigned, i.e. the type_scope, as described in the Database design section above, and in the following code:\n\n\n\nAfter defining the valid values and scopes, the properties of the analysis should be added. The following example adds a 'tool’ property with a 'FastQC’ value, to represent a FastQC analysis property type:\n\n\n\nGlobal values are supported in StatsDB to represent generalised properties that are permissible across analyses. To add global values to the analysis, the add_general_value function is used. General values can have an optional description. When the description is present, and if the value is new to the database, the description is added. If the value is already present in the database, the description is ignored. This is by design and allows for consistency across analyses:\n\n\n\nTo add values with a discrete count the add_position_value function is called. The following example specifies that 15,000 reads in the analysis had a quality score of 30:\n\n\n\nFinally, to add values that can be grouped in ranges the function add_partition_value is called. A range is an array specifying the first and last position (inclusive). The following example inserts a quality mean from position 10 to position 14 (5 values) of the run:\n\n\n\nAn auxiliary function, parse_range, is provided to convert a string representation of a range into an array type. If a single string value is provided, it returns an array with the value repeated, representing a partition of size 1. If a range string is passed, it returns an array with the positions as needed by add_partition_value:\n\n\n\nOnce the QCAnalysis object is constructed with all the required values, it can be inserted to the database with db- > insert_analysis$(analysis).\n\nTo query the database from the Perl API, the Reports.pm module is used. To allow flexibility in the querying and to be able to get summaries at different granularities (barcode, lane, pair, etc) the convention is that all the queries accept as an argument a properties hash comprising the required key-value pairs to build the query. A constant, declared in the Reports.pm module, represents the set of controlled platform-agnostic keys and is defined as follows:\n\n\n\nAn example script to query the different types of tables is provided with the StatsDB framework, i.e. examples/example_consumer.pl.\n\nThe Java API is supplied as a Maven13 project to ease building and testing. For convenience, a pre-built JAR file is available to use in existing Java projects by simply downloading the JAR file from the TGAC Maven repository14, or including the following repository and dependency Maven declarations in your pom.xml build descriptor to download the artifact:\n\n\n\nThe API is built using the standard Maven command:\n\n\n\nThis will compile the source code and provide a library JAR comprising the API, but does not attempt the StatsDB database-level tests. These unit tests make sure the database is accessible, that the schema is correct, and that the API calls available work correctly. To turn these tests on, supply the relevant database connection properties in Java/statsdb – api/src/test/resources/test.statsdb.properties:\n\n\n\nThen use the following profile activation when building the library:\n\n\n\nAn option to build an executable JAR file is available which includes a dedicated command-line application that allows API access for loading and querying a StatsDB database. To enable this option, use the following build command:\n\n\n\nThe resulting JAR can then be executed by the user. This helper application requires either an input file representing the analysis report to be parsed, e.g. a f astqc_data.txt file, or a StatsDB metadata table file (see Table 1) comprising multiple analysis reports. The helper application will then process the analysis file(s) and load the data into StatsDB. Supplying the –t option allows testing of a given parser without writing any information into the database.\n\n\n\nBefore using the Java API library, a connection to the database has to be created in a similar way to the Perl API. This file needs to be called statsdb.properties, needs to reside on the classpath (in the case of the executable JAR, this would simply be in the same directory, for example), and contains the same fields as the test properties example above. Finally, as with the Perl API, the values need to match those used in the database installation instructions above.\n\nIn terms of building and initiating queries within custom applications, the Java API mirrors the Perl API whereby the abstraction of the database is contained in the QCAnalysis class. Similarly, the Reports class is used to query the database from the Java API, and the query key granularity is represented as a Map < RunProperty, String > comprising the required key-value pairs to build the query. For convenience, the API tolerates missing values and, in such a case, the average is returned by default. When explicitly specifying these values, in the same way as the Perl API constants, the RunProperty enumeration represents the set of controlled platform-agnostic keys.\n\nThe following is a minimal example to query each of the different types of tables. Note that the first step is to construct a map of the values to query. In this particular example, the barcode is explicitly specified, i.e. \"ACCGTT\". If this was to be omitted, a general summary for lane 1 would be returned instead of a specific sample, allowing for an overview assessment of the run:\n\n\n\nAll javadoc for the StatsDB Java API can be found at https://repos.tgac.ac.uk/statsdb/javadoc/latest/.\n\nStatsDB parsers are small programs, usually scripts, which take the output of a QC tool and use one of the APIs to store the data. Parsers for StatsDB should have the same structure, e.g. adhering to the contractual Java interfaces, and only ever need implement the specific parsing code for the analysis to be added.\n\nBoth parsing APIs require analyses to be defined in an analysis metadata table (see Table 1). This is represented as a simple tab-delimited flat file describing the list of analysis fields and values. This file should supply one analysis per line, for example a path to a FastQC data file and related properties.\n\nEvery new parser should conform to the contract shown in Figure 3. To write a parser it is not necessary to know the schema of the database or to modify the contents directly. StatsDB conveniently provides the database access objects within its APIs which then connect to the database. The Wrapper object represents a managerial entity that calls a SpecificParser to actually undertake the parsing, but also manages connections to the underlying StatsDB DB object. The Wrapper should open a connection to the database, then create a RunTable object to contain a list of runs, its properties and a path to the analysis to be stored, as described by the metadata table. The path is then used on the call to the SpecificParser, an object that creates an Analysis object with the properties from the run and the values in the file with the analysis. The Analysis object holds the values in the categories described above. Once the parsing of the file is complete, the parser forwards the Analysis object to the DB object, which inserts the properties and values of the analysis. Finally, the Wrapper object should close the connection in the DB object.\n\nTo add new parsers, only the SpecificParser has to be written. The rest of the classes abstract the interaction with the database and ensure the consistency of the data. The Java API utilises different class and method names but employs the same generalised interactions.\n\nAn example Wrapper application would be implemented in Perl as follows:\n\n\n\nA concrete Wrapper and SpecificParser implementation that interrogates FastQC output can be found in Perl/parse_ fastqc.pl and Perl/QCAnalysis/FastQC.pm in the Github repository. Similarly, the Java version of a SpecificParser can be found in FastQCReportParser.java. Examples of wrapper construction of the parser and analysis objects can be seen in the StatsDBApp.java and TestFastQCParser.java classes.\n\nConsumers are programs that process StatsDB data through the API. StatsDB provides a comprehensive API to query for summaries or results of an analysis related to a specific run. The client needs to know if the value is global to the run, per position, or per partition in order to select the relevant method to call. To provide a consistent interface, all the queries are summaries and the following properties can be used as selecting criteria: encoding, chemistry, instrument, softwareOnInstrument, typeOf Experiment, pair, sampleName, lane, barcode, run. If all the properties are specified, only the latest corresponding analysis for the run is returned. This approach allows consumers to make complex comparative analyses.\n\nThe Perl API examples/example_consumer.pl script provides example calls to the Reports.pm reporting module, which forms the basis for any consumer implementation. Similarly, to facilitate these complex queries in the Java API, we provide a ReportsDecorator class which contains specific methods to interact with each report, but also encapsulates related reports. One example is getPerPositionBaseContent() which produces a matrix of the base content of each base per position, rather than producing individual queries for each base.\n\nFigure 4 shows the general approach to query the database. The consumers call the corresponding summary or the report decorator. The queried properties are then used to call the stored procedures. StatsDB reads the result set to produce an internal representation of the table, which can then be used directly by the consumer, or can be formatted as CSV or JSON. For further details and the available methods, the inline perldoc or javadoc documentation provides a comprehensive description of each method.\n\nThe client only needs to be aware of the Java/Perl API and StatsDB will format the result in CSV or JSON, so that the client can display the summary.\n\nReporting tools such as FastQC produce informative plots to visualise run metrics. StatsDB supplies example plots based on those produced by FastQC, written using the d3.js Javascript library15, to demonstrate downstream processing and representation of consumer-generated StatsDB JSON. In this way, developers can easily integrate StatsDB plots on their own web pages. A dedicated helper class to generate a set of required JSON fragments is supplied, i.e. D3PlotConsumer.java, and downstream Javascript and HTML that renders the JSON fragments can also be found in the GitHub repository in the Web directory. Examples of interactive plots generated from FastQC per-base quality and per-base GC content can be seen in Figure 6.\n\n\nUse case\n\nAt TGAC, we use StatsDB as part of our Primary Analysis Pipeline. Each Illumina run sequenced at TGAC, both with HiSeq and MiSeq instruments, passes through this pipeline. Two important steps in the process are QC analysis with FastQC and contamination analysis using an in-house kmer-based screening tool. The output of both of these tools is parsed using two separate Perl scripts provided as part of the StatsDB package, and loaded into StatsDB. As well as these QC output, we load details of the instrument, chemistry version, RTA version and Casava version into StatsDB.\n\nOur PacBio primary analysis pipeline is still under development, but this currently includes using FastQC to analyse FASTQ files output as part of the process. We are also working on parsers for the sts.csv files that are produced by the instrument. With StatsDB, it is perfectly possible to mix data from different platforms and different tools into one database. When querying the data, the consumer application can make the decision about what data comparisons are meaningful.\n\nWe currently access data stored in StatsDB using two consumers: TGAC’s open-source LIMS, MISO, and the prototype StatsDB Reporter tool. Using the MISO web-based interface, it is possible to access StatsDB information and produce graph plots of FastQC data, which are based on the d3.js consumer examples supplied with the framework (Figure 6). StatsDB Reporter allows selection of runs by instrument, lane, run, sample or barcode and provides comparison of data across runs (Figure 5).\n\n\nConclusions\n\nMany software tools exist for the generation of run quality statistics and FastQC is possibly the most notable example. However, until now, there has been no easy solution to the problem of storage and analysis of historical run metadata and statistics. StatsDB has been designed to address this problem and offers a flexible, easy to use, platform-agnostic and tool-independent framework for consolidated access to run metrics.\n\nInstallation of StatsDB and integration with existing analysis pipelines is achieved with minimal effort. To perform data entry into StatsDB, a parser is included for the popular FastQC tool, and parsers for other tools can be written in less than a day by a competent programmer or scripter. Similarly, command line tools are provided in both Perl and Java to load parsed data into StatsDB.\n\nTo perform downstream analysis and visualisation of data held within StatsDB, reporting helper entities are provided. Furthermore, an example consumer tool is supplied in the form of the StatsDB Reporter application which currently exists in prototype form but a mature version will be available in due course from the GitHub repository for StatsDB, as well as parsers for other tools (for example, PacBio sts files).\n\n\nSoftware details\n\nHomepage: http://www.tgac.ac.uk/tools-resources/Source code: https://github.com/TGAC/statsdb and 10.5281/zenodo.7534 Licence: GPL v3.",
"appendix": "Author contributions\n\n\n\nMC initiated the project, which RD now leads. RHRG designed the database, implemented the Perl API and, with RD, implemented the Java API. RML and DW wrote parsers and consumers to use the API. AT developed the d3.js plots. RML, RHRG, RD, DW and AT all contributed to the manuscript.\n\n\nCompeting interests\n\n\n\nThe authors declare that there are no competing interests.\n\n\nGrant information\n\nThe development of StatsDB has been funded by a Biotechnology and Biological Sciences Research Council (BBSRC) National Capability Grant at TGAC.\n\n\nAcknowledgements\n\nWe are grateful to the Library Preparation and Sequencing Operations teams at TGAC for generating the data that has been used to test StatsDB.\n\n\nReferences\n\nMiller MR, Dunham JP, Amores A, et al.: Rapid and cost-effective polymorphism identi.cation and genotyping using restriction site associated DNA (RAD) markers. Genome Res. 2007; 17(2): 240–248. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBaird NA, Etter PD, Atwood TS, et al.: Rapid SNP discovery and genetic mapping using sequenced RAD markers. PLoS One. 2008; 3(10): e3376. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAndrews S: FastQC: A quality control tool for high throughput sequence data. Reference Source\n\nYang X, Liu D, Liu F, et al.: Htqc: a fast quality control toolkit for Illumina sequencing data. BMC Bioinformatics. 2013; 14: 33. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchmieder R, Edwards R: Quality control and preprocessing of metagenomic datasets. Bioinformatics. 2011; 27(6): 863–864. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDai M, Thompson RC, Maher C, et al.: Ngsqc: cross-platform quality analysis pipeline for deep sequencing data. BMC Genomics. 2010; 11(Suppl 4): S7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBuffalo V: QRQC - quick read quality control. Reference Source\n\nLassmann T, Hayashizaki Y, Daub CO: Samstat: monitoring biases in next generation sequencing data. Bioinformatics. 2011; 27(1): 130–131. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAshby M, Lee L: stsPlots. Reference Source\n\nSkelly T: PacBio Exploratory Data Analysis. Reference Source\n\nDavey RP, Bian X, Thanki A, et al.: MISO: An open-source LIMS for small-to-large scale sequencing centres. Reference Source\n\nPerl DBI. Perl DBI. Reference Source\n\nThe Apache Software Foundation. Maven. Reference Source\n\nThe Genome Analysis Centre. TGAC Maven Repository. Reference Source\n\nD3.js - Data-Driven Documents. 2012. Reference Source"
}
|
[
{
"id": "2467",
"date": "10 Dec 2013",
"name": "Mick Watson",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is well written and describes a tool which will be very useful to the community.I only have a few minor issues:Throughout I felt that the terms used were confusing: \"analysis\" and \"run\" for example. These can take on multiple meanings in sequencing (for example, what the SRA calls a run is not what I call a run). I feel these terms need a better definition when they are first introduced, with examples where appropriate. Is it valid to still use the term \"short-read\" sequencers? There is a lot of technical information about the design, database and APIs, but only a single example (using Illumina data) at the end; examples using PacBio and Ion Torrent data would show the true flexibility of the tool.",
"responses": [
{
"c_id": "679",
"date": "24 Jan 2014",
"name": "Robert Davey",
"role": "Author Response",
"response": "We would like to thank Mick for taking the time to read and comment on our manuscript.Admittedly, there is much interplay when considering terminology such as \"run\" and \"analysis\", but throughout the paper we use the term \"run\" to represent a sequencing run, and similarly, the term \"analysis\" to represent a QC process. We feel this is adequate given the focus of the paper, but we have clarified one potential misuse (changed \"run\" to \"carried out\", where appropriate).Yes, the field refers to these terms regularly, given that \"long read\" sequencers are available and distinct from their \"short-read\" counterparts, i.e. we consider the new 2x300bp and upcoming 2x400bp Illumina techniques to still be \"short-read\".As long as the data produced from non-Illumina machines is in the FASTQ format (as we state in the existing Use Case text), then there are no differences from the method outlined in the paper, e.g. FASTQC output parsed and stored in StatsDB. Where differences may exist, e.g. STS files from PacBio, different parsers can be written to accommodate this, and we are working on producing such a parser. By no means is StatsDB inherently tied to the FASTQ format, but we feel this reflects the most common QC methods available currently."
}
]
},
{
"id": "2565",
"date": "16 Dec 2013",
"name": "Cyriac Kandoth",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have developed a tool that is well documented, open sourced, and version controlled. The first two thirds of the article read like a user's manual, which is appropriate in a technical article, but not in its entirety. The neatly organized code snippets and commentary will help a programmer who is considering using the API. However, to encourage them to use it, the Use case section must be expanded with figures and performance metrics. I have added more details in the comments below.Major comments:Title - \"platform-agnostic storage and understanding...\" - The word \"understanding\" seems either ambiguous or inappropriate since StatsDB is just the facilitator for interpreting/visualizing the run metrics. Consider a title like \"StatsDB: platform-agnostic storage schema and API for interpreting next-generation sequencing run metrics\" Page 4, Database design - The relationship between the analysis table and the auxiliary views is unclear. For example, the analysis table stores a unique ID and timestamp of when an analysis run was performed. But the latest_run view mentions that an analysis can be run more than once. Please clarify. Page 10, Use case - Add a figure with a flowchart showing the TGAC pipeline that is explained in the text. It is critical to show how StatsDB plugs into a standard analysis pipeline. For the source code, an LGPL license might be more appropriate than GPL because StatsDB is an API, rather than a standalone tool in a workflow. LGPL would also allow parsers/consumers for manufacturer-supplied metrics.Minor comments:Page 3, Database installation - The code snippets show file names with accidental whitespace. Specifically: statsdb_schema.sql, stored_procedures.sql, and parse_fastqc.pl Page 3, Introduction - \"With this data heterogeneity and abundance, comes a...\" - missing comma. Page 5, Database design - How would you store metrics that depend on genomic positions or ranges? e.g targeted sequencing coverage. If tables per_position_value and per_partition_value stored values based on genomic positions or ranges, it would also depend on which reference sequence build (e.g. NCBI36, GRCh37) was in use for that analysis. Would we define that build as a value_type? And in the stored procedures used by the APIs, how can we handle a mix of different reference builds used across runs? Page 7, Java API - accidental whitespace in filename fastqc_data.txt",
"responses": [
{
"c_id": "678",
"date": "24 Jan 2014",
"name": "Robert Davey",
"role": "Author Response",
"response": "We would like to thank Cyriac for taking the time to read and comment on our manuscript.In terms of the Use Case expansion, we believe the usefulness is inherent in the ability to store, retrieve and therefore compare historical run metrics, based on a variety of user- or analysis-specific attributes. We envisage tools like StatsDB Reporter will be the simplest and more widely-used interface with StatsDB for the average lab technician or bioinformatician. Similarly, we believe performance metrics at this level would be unhelpful rather than beneficial, as these would be very dependent on infrastructure, hardware and DMBS used. We aim to publish incremental updates to the API and surrounding tools, e.g. a full StatsDB Reporter release, and a Python API.The StatsDB schema was designed to allow the same analysis to be stored multiple times, with potentially differing or identical parameters. We foresee that if analysis uniqueness is required, this would be down to an API implementation to check for previous analyses with the same tool and parameters supplied, rather than at the database level. We have added a description to the Database Design section (analysis and latest_run table outlines) of the manuscript.The pipeline that utilises StatsDB has been covered in detail in our recent open-access FrontiersIn publication, so we have added a reference to this paper in the Use Case main text.The GPLv3 aims to give free software developers an advantage over proprietary developers. A parser that supports a proprietary format does not fit within the scope of our vision - we aim to continue the trend in bioinformatics software whereby fully open-source licences are preferred to maximise the reusability of a given tool or library.Yes, you are correct in using value_type for this kind of attribute. There are API calls that let you pull out analyses by a value_type value, so this should be supported out of the box.The accidental whitespace has been introduced in the HTML view, from which the PDF download is generated. We shall contact the editorial office to ensure these are corrected. Thank you for spotting those!"
}
]
},
{
"id": "2794",
"date": "27 Dec 2013",
"name": "Anuj Kumar",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript by Ramirez-Gonzalez et al. presents a software package to store and analyze metrics from next-generation sequencing runs. The open-source software package, StatsDB, stores statistics from sequencing reactions in a MySQL database, and APIs provide means to access and analyze the data. The StatsDB package facilitates easy querying, particularly across multiple fields from respective tables in the database. StatsDB provides modules that can be used in conjunction with FastQC as well as with other tools suited for the analysis of sequence data. From the perspective of a wet-lab genomicist, the StatsDB package seems to be a useful tool, and I can certainly see the utility in storing data from the sequencing reactions, particularly for a genome analysis center. I only have a few minor comments. Since FastQC is commonly used for the analysis of sequencing runs, and since the StatsDB package works with FastQC, it would be useful to provide a little more explicit information describing the output/types of data provided by FastQC. Some of these data are presented in the StatsDB schema, but it would be nice to add a bit more detail in this regard (possibly a very simple figure) into the Introduction. I appreciate the use case provided, but I think it would be helpful to provide an additional small example of how the StatsDB package could be useful to an individual lab that uses a moderate level of next-generation sequencing in their research (as opposed to a genome center). Possibly, a summary of the StatsDB Reporter application would suffice. It would be helpful to add a few sentences to the Introduction to indicate how other sequencing centers, etc. store output from FastQC or other similar tools. This does not have to be an extensive overview.",
"responses": [
{
"c_id": "676",
"date": "20 Jan 2014",
"name": "Robert Davey",
"role": "Author Response",
"response": "We would like to thank Anuj for taking the time to read and comment on our manuscript.We have added a sentence in to the Introduction, outlining briefly the data formats outputted by FASTQC, i.e. a set of HTML files, and a single plain-text flat file from which we parse the data to be loaded into StatsDB.We envisage no differences in scaling terms between a small-scale lab with one or two sequencers and a large multi-platform centre. StatsDB has applications in the smaller centre where quick but potentially more sporadic access to historical run data would be investigated through the StatsDB Reporter tool rather than the more \"heavyweight\" integration with a LIMS or via a web server. As such, we have added a sentence describing its relevance in this context. We will be publishing the StatsDB Reporter application separately, or as a software update to this publication, in due course.We have added a short overview of how the output of QC tools might be kept in different centres, highlighting the usefulness of StatsDB."
}
]
}
] | 1
|
https://f1000research.com/articles/2-248
|
https://f1000research.com/articles/3-61/v1
|
19 Feb 14
|
{
"type": "Research Article",
"title": "Characterization of a SAM-dependent fluorinase from a latent biosynthetic pathway for fluoroacetate and 4-fluorothreonine formation in Nocardia brasiliensis",
"authors": [
"Yaya Wang",
"Zixin Deng",
"Xudong Qu",
"Yaya Wang",
"Zixin Deng"
],
"abstract": "Fluorination has been widely used in chemical synthesis, but is rare in nature. The only known biological fluorination scope is represented by the fl pathway from Streptomyces cattleya that produces fluoroacetate (FAc) and 4-fluorothreonine (4-FT). Here we report the identification of a novel pathway for FAc and 4-FT biosynthesis from the actinomycetoma-causing pathogen Nocardia brasiliensis ATCC 700358. The new pathway shares overall conservation with the fl pathway in S. cattleya. Biochemical characterization of the conserved domains revealed a novel fluorinase NobA that can biosynthesize 5’-fluoro-5’-deoxyadenosine (5’-FDA) from inorganic fluoride and S-adenosyl-l-methionine (SAM). The NobA shows similar halide specificity and characteristics to the fluorination enzyme FlA of the fl pathway. Kinetic parameters for fluoride (Km 4153 μM, kcat 0.073 min-1) and SAM (Km 416 μM, kcat 0.139 min-1) have been determined, revealing that NobA is slightly (2.3 fold) slower than FlA. Upon sequence comparison, we finally identified a distinct loop region in the fluorinases that probably accounts for the disparity of fluorination activity.",
"keywords": [
"The introduction of fluorine into organic molecules can often improve their molecular stability and pharmacological properties1",
"2. Organo-fluorines have been widely used in pharmaceuticals",
"diagnostics",
"agrochemicals",
"and materials3",
"4",
"and it is estimated that 20–30% of commercial drugs",
"including many top-sellers",
"contain fluorine5. The increasing prevalence and success of organo-fluorines have instigated enormous efforts over the past decades in developing methodologies for efficiently introducing fluorine into organic molecules5",
"6",
"however the unique properties of fluorine make these chemical incorporations challenging",
"usually needing harsh synthetic conditions and giving rise to moderate chemo- or stereo- selectivity5",
"6."
],
"content": "Introduction\n\nThe introduction of fluorine into organic molecules can often improve their molecular stability and pharmacological properties1,2. Organo-fluorines have been widely used in pharmaceuticals, diagnostics, agrochemicals, and materials3,4, and it is estimated that 20–30% of commercial drugs, including many top-sellers, contain fluorine5. The increasing prevalence and success of organo-fluorines have instigated enormous efforts over the past decades in developing methodologies for efficiently introducing fluorine into organic molecules5,6, however the unique properties of fluorine make these chemical incorporations challenging, usually needing harsh synthetic conditions and giving rise to moderate chemo- or stereo- selectivity5,6.\n\nIn contrast to chemical synthesis, biocatalytic synthesis or biosynthesis provide better efficacy and selectivity under mild conditions, and can be easily scaled up for industrial production7. Although natural fluorination is rare, efforts in identifying new bio-fluorination machineries are continuing. Pioneered by O’Hagan and co-workers, the natural bio-fluorination pathway to produce fluoroacetate (FAc) and 4-fluorothreonine (4-FT) was biochemically characterized in the actinomycete Streptomyces cattleya8. Enzymatic fluorination in S. cattleya occurs via a fluorine-fixation step that is catalyzed by the unique fluorinase enzyme FlA through the nucleophilic attack of fluoride to SAM9–11. The resulting fluoro-intermediate 5′-fluoro-5′-deoxyadenosine (5′-FDA) is further converted by five enzymes to give rise to the FAc and 4-FT (Figure 1A and B)8. This powerful bio-fluorination machinery has been proved very successful in incorporating both 18F and 19F into nucleotides12–16 and in producing the anticancer drug candidate fluorosalinosporamide17. Very recently, its enormous potential for producing complex fluorinated molecules was demonstrated by Chang and co-workers18. Using an engaging biosynthetic strategy they were able to achieve site-specific incorporation of FAc into the polyketide backbone of 2-desmethyltriketide lactone. Merging this strategy with the FAC producing pathway, such as the fl pathway in vivo, could present opportunities to use living cells for the production of acetate-derived fluorinated natural products that include polyketides, fatty acids, terpenoids and steroids.\n\n(A) Organization of relevant genes in the fl and nob pathway, encoding the conversion of fluoride to FAc and 4-FT (crosshatched), and auxiliary function (blackened). Iso: isomerase; Deh: dehydrogenase and Tran: transaldolase. Identical genes are indicated by dotted lines. (B) Proposed biosynthetic pathways to the FAc and 4-FT in the S. cattleya and N. brasiliensis.\n\nSynthetic biology approaches need pools of “genetic elements” to mix and match to build and optimize synthetic bio-pathways19. However, the only natural bio-fluorination tool available so far is the fl pathway. The limited natural bio-fluorination machinery forms the bottleneck for development of efficient synthetic bio-pathway to produce fluorinated natural products, thus an expansion of the genetic resources of biological fluorination is highly desired. Here, we report the identification of a latent fluorination pathway for FAc and 4-FT biosynthesis from the pathogen Nocardia brasiliensis ATCC 700358 and the biochemical characterization of a novel SAM-dependent fluorinase enzyme.\n\n\nMaterials and methods\n\nDNA isolation and manipulation in Escherichia coli were performed according to standard methods20,21. PCR amplifications were carried out on an authorized thermal cycler (Veriti 96 Well, ABI) using PrimeSTAR Max DNA polymerase according to the manufacturer protocol (TaKaRa, Japan). Primer syntheses and DNA sequencing were performed at Genewiz BiotechCo., Ltd. (China). The E. coli DH5α cells were purchased from Invitrogen (Carlsbad, CA), and E. coli BL21 (DE3) cells were purchased from Novagen (Madison). N. brasiliensis HUJEG-1 (ATCC 700358) was purchased from the American Type Culture Collection (ATCC), and S. cattleya DSM 46488 was provided by Prof. Hong-Yu Ou. Restriction enzymes were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China). All other chemicals and reagents were purchased from Santa Cruz Biotechnology, Inc (USA) or Shanghai Sangon Biotech (China) Co., Ltd., unless noted otherwise. Analytical HPLC was carried out on an SHIMADZU LC-20A Prominence HPLC system. LC-MS analysis was conducted on a Thermo Instruments HPLC system connected to a LCQ Fleet electrospray ionization (ESI) mass spectrometer (ThermoFisher Scientific Inc.). NMR data were collected using a Bruker 400 MHz spectrometer.\n\nSequence analysis. The genetic open reading frame sequences (ORFs) in N. brasiliensis were identified using the FramePlot 4.0 beta program (http://nocardia.nih.go.jp/fp4/). The corresponding proteins were compared with other known proteins in the NCBI databases by using available BLAST methods (http://www.ncbi.nlm.nih.gov/blast/). Amino acid sequence alignments were performed by using the Strap program (http://www.bioinformatics.org/strap/).\n\nCloning, overexpression and purification of NobA. The synthetic gene codon-optimized nobA for expression in E. coli using the OptimumGeneTM algorithm was sourced from GenScript (Nanjing, China). The optimized sequence exhibited 82% identity to the original sequence and is shown below (codons optimized are underlined, restriction sites are in italics): GAATTCCATATGACGACGACGAATGGCCGCCGCCCGATTATTGCGTTTATGAGCGATCTGGGTATCACCGACGACTCCGTGGCACAGTGTAAGGGTCTGATGCTGAGCGTGTGCCCGGATGTGACGATTGTTGACATCTGTCATACCATGCAGCCGTGGGATGTGGAAGAAGGTGCGCGTTATATTGTTGACCTGCCGCGCCTGTTTCCGGAGGGTACGGTTTTCGCAACCACGACCTACCCGGCAACCGGTACGACCGCACGTAGCGTCGCTCTGCGTATCGCACATGCCTCTAAAGGCGGTGCACGTGGCCAGTGGGCAGGTAGTGGTGCAGGTTTCGAACGCAAGGAAGGCTCATATATTTACATCGCGCCGAACAATGGTCTGCTGACGACCGTTATTAAAGAACACGGCTATCTGGAAGCCTACGAAGTTAGCTCTCCGGAAGTCATCCCGGAACAACCGGAACCGACCTTTTATTCACGTGAAATGGTGGCACTGCCGTCGGCTCATCTGGCAGCAGGTTTCCCGCTGGAAAAAGTCGGTCGTCGCCTGGCAGATGACGAAATTGTGCGTTTTGAACGCAAGGATCCGGAACTGGTTGCCGATCACGACCTGGTCGGTTATGTGACCAACATTGATCATCCGTTTGGCAACGTTTGGACGAATATCCACCGTACCGACCTGGAAAAACTGGGTGTCGGCTACGGTACGAAGCTGCGCATTACCCTGGATGGTGTGCTGCCGTTTGAACTGCCGCTGTCCCCGACCTTCGCAGATGCTGGCGAAATCGGTGCAGCTGTGGCATATCTGAGTTCCCGTGGTTACCTGGCACTGGCACGTAATGCTGCGTCGCTGGCGTATCCGTATAATCTGAAGGCGGGTATTTCGGTCCAAGTCAAAGTGGGCTAAGCTT. The gene of FlA was PCR amplified from genomic DNA of S. cattleya using the forward primer 5′-TTCATATGGCTGCGAACAGCACACGTC-3′ and reverse primer 5′-TTAAGCTTATCAGCGGGCCTCGACCCG-3′. The purified PCR product was ligated to pMD18-T simple (TaKaRa, Japan) following the manufacturer protocol and its fidelity was confirmed by DNA sequencing (multi-color fluorescence-based DNA analysis). The NdeI-HindIII fragments of nobA and flA were ligated to pET28a to yield the plasmids pWHU2401 and pWHU2402, respectively, which were then been used to overexpress the NobA and FlA proteins as N-terminal 6×His-tagged fusion proteins. The resulting expression plasmids were transformed into E. coli BL21 (DE3) cells. The cells were grown in 0.8 L of liquid culture (1×Luria broth medium with 50 μg/mL kanamycin) at 37°C to an OD600 of 0.6. The cells were allowed to cool room temperature and induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 10 hours at 25°C. The cells were centrifuged (6000 rpm, 10 minutes, 4°C) resuspended in 15 mL lysis buffer (25 mM HEPES pH 7.5, 300 mM NaCl, 5 mM imidazole, 10% glycerol) and lysed by sonication. Cellular debris was removed by centrifugation (15000 rpm, 30 minutes, 4°C). Two mL Ni-NTA agarose resin was added to the supernatant and the solutions were shaken at 4°C for 1 hour. The protein resin mixtures were loaded into a gravity flow column, and proteins were sequentially eluted with 10 mL Buffer A (25 mM HEPES pH 7.5, 300 mM NaCl, 10% glycerol) supplemented with 50 mM, 100 mM and 300 mM imidazole, respectively. Purified proteins (10 mL) were buffer exchanged by dialysis in 1 L Buffer B (25 mM HEPES, pH 7.5, 50 mM NaCl, 10% glycerol) and concentrated by centrifugation using an Amicon Ultra-4 (10 KDa, GE Healthcare). Proteins purity (90–95%) was evaluated by 12% acrylamide SDS-PAGE. Protein concentrations of the NobA (5 mg mL-1), FlA (4 mg mL-1) and NobA-S158A (25 mg mL-1) were determined by the Bradford method using a BSA calibration curve. The final proteins were flash-frozen in liquid nitrogen and stored at -80°C. Gel filtration chromatography was used to determine the native molecular mass of NobA. Experiments were performed at a flow rate of 1 ml min-1 using an AKTA Purifier FPLC system (Amersham Pharmacia Biotech) and a Superdex 200 GL column (Amersham Pharmacia Biotech). The elution buffer used was 50 mM phosphate buffer (pH 7.8). The native molecular mass of the enzyme was estimated from a calibration curve plotted by using the standard proteins carbonic anhydrase (29 kDa), bovine serum albumin (66 kDa), alcohol dehydrogenase (150 kDa), β-amylase (200 kDa), apoferritin (443 kDa), and thyroglobulin (669 kDa).\n\nIn vitro enzymatic assays of NobA. Typical assays were performed in 100 μL of 20 mM sodium phosphate buffer (pH 7.5) containing 1 mM SAM, 1–5 μM enzyme and 20 mM NaF or NaCl22. For chlorination reaction, additional 1 μM L-amino acid oxidase (Sigma-Aldrich Co., Ltd.) was added in the reaction mixture. Reactions were incubated at 26°C, quenched by 10 μL 100% trichloroacetic acid (TCA) and the supernatants were subjected to HPLC or LC-HRMS analysis using an analytic Inertsil ODS-3 column (5 μm, 4.6×250 mm, GL Science Inc). HPLC analysis was normally performed with a linear gradient of 5% to 20% CH3CN (v/v) over 20 minutes, 20% to 5% CH3CN (v/v) over 1 minute, and 5% CH3CN (v/v) for further 4 minutes at a flow rate of 1 mL/min under 260 nm. HPLC-ESI-MS analysis was performed as described above. The kinetic parameters for NaF were determined with SAM maintained at a concentration of 0.4 mM and NaF at increasing concentrations from 0 to 20 mM. The kinetic parameters for SAM were determined with a concentration of NaF maintained at 5 mM and SAM at increasing concentrations from 0 to 1 mM. The formation of 5′-FDA was determined by HPLC and quantified using a standard curve of S-adenosyl-l-homocysteine (SAH). Each data point represents a minimum of three replicate, end point assays were fitted to the Michaelis-Menten equation by OriginPro 9.0 (OriginLab software, Northampton, MA) to obtain estimates for kcat and Km.\n\nDetermination pH and metal ion effect on the activity of NobA and FlA. A reaction mixture (100 µl) containing 20 mM NaF, 1 mM SAM in 20 mM sodium acetate (pH 4.5–5.5), sodium phosphate (pH 6.0–7.0), Tris-HCl (pH 7.5–9.0), or Gly–sodium hydroxide (pH 9.5–10.0) buffer was prepared. The reactions were initiated by the addition of 5 μM NobA or FlA, incubated at 26°C for 30 minutes where the velocity is in the linear range, and then subjected to HPLC analysis. To measure the metal ion effect on the activity, a group of 100 μl reaction mixtures containing 20 mM NaF, 1 mM SAM in 20 mM Tris-HCl (pH 6.5) buffer were spiked with 1 mM different divalent metal chloride salts (Mg2+, Mn2+, Fe2+, Cu2+ or Zn2+) or 1 mM ethylenediaminetetraacetic acid (EDTA) to remove the potential associated metal ions of the reaction mixture. Reactions were initiated by addition of 5 μM NobA or FlA, and then incubated at 26°C for 60 min to HPLC analysis. Neither metal ions nor EDTA was added in the negative control reaction mixture. The formation of 5′-FDA was determined by HPLC and quantified using a standard curve of S-adenosyl-l-homocysteine (SAH). Each data point represents a minimum of two replicate, end point assays were fitted to the Excel (Microsoft Corporation) to obtain scatter diagram (PH) and histogram (metal ions) for estimating relative activity.\n\nConstruction and purification of nobA mutant. A nobA mutant was constructed using a standard PCR method21 and the pWHU2401 as a template. The NobA-S158A mutant was constructed with the primer pair 5′-GACCTTTTATGCACGTGAAATGG-3′ and 5′-CCATTTCACGTGCATAAAAGGTC-3′ according to a standard protocol21. The construct of pWHU2403 was verified by DNA sequencing and was overexpressed in E. coli BL21 (DE3) as previously described. The expression and purification procedures of recombinant NobA-S158A were carried out in a manner similar to that described for NobA.\n\n\nResults and discussion\n\nA putative gene (nobA) encoding a protein with overall 79% identity to the fluorinase FlA was identified from the genome of N. brasiliensis ATCC 70035823 (Figure 1A). By extending the searching region to the up-downstream of nobA, we further identified a gene cluster of 10 genes (nobA-I, Figure 1A and Table 1). In this gene cluster, four catalytic genes (nobA, B, D and M) encode the homologues of 5′-FDA synthase, 5′-FDA phosphorylase, 5′-FDRP isomerase and 4-FT transaldolase which are responsible for the conversion of fluoride to 5-FDRibulP and fluoroacetaldehyde to 4-FT in the fl pathway)8. Flanked those are genes for encoding auxiliary functions, including regulation (nobC, F and G), transportation (nobC, H) and SAM recycling (nobE and I), among of which four (nobF, G, I and H) are homologous to the counterparts in the fl biosynthetic pathway. The overall identity to the fl pathway genes suggested that the nobA cluster might be related to the 4-FT and FAc biosynthesis.\n\nBesides the four catalytic genes described above, other two genes encoding an aldolase and a dehydrogenase to mediate the conversion of 5-FDRibulP to fluoroacetaldehyde and fluoroacetaldehyde to FAc are necessary. A homology search for the fluoroacetaldehyde dehydrogenase in the N. brasiliensis resulted in the identification of a gene with 78% identity (nobJ, YP_006807765.1; Figure 1A and Table 1) from a remote site in the genome, suggesting that N. brasiliensis has the potential to produce FAc. Unlike the others, the gene encoding aldolases in the fl pathway is still elusive. Recently, four aldolase genes were identified from S. cattleya by genome sequencing and two of them were thought to encode the conversion of 5-FDRibulP to fluoroacetaldehyde24. However, in the N. brasiliensis genome we could not find identical homologues by BLAST searching. Because this reaction can also be catalised by fuculose aldolase8 enzyme, we searched for fuculose aldolase homologues, with no success. Instead, five putative aldolase genes (YP_006810507.1, YP_006812798.1, YP_006805383.1, YP_006812725.1 and YP_006809408.1) were identified in N. brasiliensis based on the gene annotation in IMG database (https://img.jgi.doe.gov/cgi-bin/w/main.cgi). We then compared the genetic sequences of the putative aldolases to their homologues in S. cattleya. Interestingly, two of these aldolase genes (YP_004910624 and YP_004919742.1) which were previously not idenfied in S. cattleya shared sequence homology to the aldolase genes (YP_006810507.1 and YP_006812798.1 respectively) of N. brasiliensis. Their homology (both have 62% identity) is in line with the average identity (58%) between the counterpart genes of nob and fl pathway, suggesting that these genes are probably involved in the FAc biosynthetic pathway.\n\nPrevious studies revealed the thioesterase FlK confers self-immunity to the FAc in the fl pathway25,26. Inactivation of the flK gene does not affect cell growth in 2 M NaF27, however can prevent production of fluorometabolites27. Interestingly, we were not able to find the flK homologue in N. brasiliensis, although there are a few genes encoding thioesterases. To test the ability of N. brasiliensis to produce fluorometabolites, the bacteria were subjected to fermentation with a supplement of 2 mM NaF as a fluorine source28. Despite the N. brasiliensis grows normally, no new fluorine signal in the culture except fluoride could be detected by F-NMR (data not shown). The lack of fluorometabolites might be caused by improper fermentation conditions or deficiency of the FlK homologue in the genome. In vivo gene manipulations including promoter activation and heterologous complementation of the flK in N. brasiliensis were also attempted, but failed due to the inaccessibility of genetic system.\n\nSince the in vivo studies were inaccessible, we turned to in vitro characterization. The 5′-FDA synthase which catalyzes the unique fluorine-fixation reaction is one of the most interesting enzymes for the communities of enzymology and synthetic biology, thus we focused on the biochemical characterization of the NobA enzyme. The nobA gene was first codon optimized, cloned as an N-terminal 6×His tagged protein, and then expressed and purified from E. coli (Figure 2A). In our experiment, the expression yield of NobA (7.9 mg/L) was lower than the yield of FlA (9.2 mg/L), and like FlA it natively occurs as a hexamer (Figure 3)10. The fluorination activity of the recombinant protein was assessed in parallel with FlA which was utilized to produce the 5′-FDA as a control. After the reaction, the assay mixtures were quenched by 10% trichloroacetic acid (TCA) and subjected to HPLC analysis. A product strictly dependent on the addition of SAM, NaF and NobA was detected, and its identity was finally confirmed to be 5′-FDA by both HRMS and comparison to the product of FlA (Figure 2B and C). These results, taken together, indicate that NobA, like FlA, can confer fluorine fixation, and that the nob pathway might be related to FAc and 4-FT biosynthesis.\n\nA) SDS-PAGE analysis of purified recombinant proteins. The expected sizes of NobA-S158A (34.7 kDa), NobA (34.7 kDa) and FlA (34.6 kDa) are indicated. Lane M, protein molecular weight standards. B) HPLC showing NobA-catalyzed production of 5′-FDA with various control reactions. Adenine and MTA (S-methyl-5′-thioadenosine) are from the degradation of SAM, and X is an impurity associated with NobA. C) HRMS confirming the identity of 5′-FDA. The observed value (m/z [M+H]+ 270.1002) is consistent with the calculated value (m/z [M+H]+ 270.0996).\n\nStandard proteins (a–f) used are the following: thyroglobulin (669 kDa), apoferritin (443 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa) and carbonic anhydrase (29 kDa). NobA has similar molecular weight to β-amylase (200 kDa) suggesting it naturally presence as a hexamer.\n\nWith a new fluorinase in hand, we next compared the enzymatic properties of the protein. It has been previously reported that in the presence of L-amino acid oxidase (L-AAO), FlA can utilize Cl- but not Br- or I- to form the 5′-Chloro-5′-deoxyadenosine (5′-ClDA)22. Similarly, we observed that NobA was also able to perform this reaction with a similar reactivity (Figure 4). The optimal pH of NobA was determined as 6.5, slightly lower than FlA at 7.0 (Figure 5A). NobA shows no obvious effects on fluorination reactivity in the presence of metal ions, instead 1 mM Cu2+ or Zn2+ can severely inhibit its activity (Figure 5B). The activity can be slightly increased by adding EDTA, suggesting the fluorination reaction is metal independent. The optimal temperature for fluorination reactivity is 37°C, however at this temperature SAM degradation is also expedited. In contrast, as 26°C can provide acceptable activity and slow down the degradation of SAM, this temperature was chosen throughout our kinetic studies. Although previous FlA kinetic studies have been reported10,29, the data are inconsistent. Thereby, to precisely compare their reactivities, both NobA and FlA were kinetically measured in this study (Figure 5C). The Km values of NobA were 4153 μM for NaF and 416 μM for SAM, about twice the values of FlA (2167 μM for NaF and 210 μM for SAM). The turnover numbers (kcat) of NobA were 0.073 min-1 for NaF and 0.139 min-1 for SAM, which were similar to the values of FlA (0.11 min-1 for NaF and 0.123 min-1 for SAM). The average kcat/Km of FlA (5.07×10-5 μM-1 min-1 for NaF and 5.86×10-4 μM-1 min-1) outnumbers 2.3 fold to the values of NobA (1.75×10-5 μM-1 min-1 for NaF and 3.3×10-4 μM-1 min-1 for SAM), and suggests that NobA is less efficient than FlA.\n\nThe production of 5′-ClDA was determined by HPLC and HRMS. The observed values (m/z [M+H]+ 35Cl = 286.0692, 37Cl = 288.0662) are consistent with the calculated values (m/z [M+H]+ 35Cl = 286.0707, 37Cl = 288.0677).\n\n(A) pH effect on NobA and FlA activity. The optimum activity of NobA at pH 6.5 and FlA at pH 7.0 were individually set as 100%. (B) Metal ion effect on NobA activity. The activity of negative control was set as 100%. (C) Kinetic analysis of the fluorination reaction. When the concentration of NaF was varied, the SAM was fixed at 0.4 mM, otherwise when the SAM was varied, the NaF was fixed at 5 mM.\n\nSimilar to FlA, NobA retains the conservation of the unique 23-residue loop, which is related to site architecture and fluoride selectivity, and lacks the critical residues for SAM hydrolysis (Figure S1)30,31. This unique feature confirms that fluorinases form a distinct group of enzymes which differ from other SAM-binding members in the DUF62 enzyme family. The overall identity of the fluorinases is very high (79% identity), and only a region between the R192 to L202 (FlA residue numbering) is distinct (Figure S2). Interestingly, in the crystal structure of FlA, this region constitutes a loop which links the N- and C-terminal domains to form an interface where the SAM and fluoride are bound11. The variety of the loop region can probably influence the domain interaction and binding affinities of the substrates, and this might be one of the reasons for the retardation observed for NobA. Moreover, the residue S158 of FlA plays critical role in fluoride binding and desolvation10,32. Interestingly, this residue also exists in the NobA (Figure S2). To confirm the essentiality of S158 for NobA activity, this residue was further mutated into an alanine (Figure 2A). The resulting recombinant protein NobA-S158A completely lost fluorination activity (Figure 2B) and demonstrates it is essential for NobA activity. This observation also indicates that NobA shares an identical catalytic mechanism employed by NobA.\n\nActinomycetoma is a zoonotically chronic, granulomatous and subcutaneous tissue infection caused by actinomycetes that may often lead to amputation or death33. N. brasiliensis is the major causative pathogen of the actinomycetoma infections in Mexico23,33. Its pathogenic mechanism currently is unclear. FAc is a highly toxic poison (LD50 = 0.1 mg/kg mouse ≈ 1.2 μM)34. In the human body it is first converted into the anabolite (-)-erythro-fluorocitrate (EFc), which exerts its toxicity by irreversibly inhibiting aconitase to cause cell death (IC50 = 0.01 μM)35,36. Interestingly, our research revealed the N. brasiliensis has the genetic potential to produce FAc from fluoride. In human blood, the fluoride concentration is normally around 0.13 μM and can increase to 3.2 μM in some fluorine-polluted areas37, which indicates N. brasiliensis can potentially produce FAc at levels that cause severe cytotoxicity and lead to development of the actinomycetoma.\n\nIn summary, we identified a latent pathway for FAc and 4-FT biosynthesis from the actinomycetoma pathogen N. brasiliensis ATCC 700358. Comparative analysis of the genomes of N. brasiliensis and S. cattleya revealed two common aldolase genes that were putatively involved in the FAc and 4-FT biosynthesis. By in vitro characterization, we confirmed a novel fluorinase, NobA, that can biosynthesize 5′-FDA from inorganic fluoride and SAM. This new fluorinase has similar substrate selectivity and characteristics to the homologue FlA, with a slightly less efficiency of reaction (2.3 fold). The lower reactivity might be attributed to the presence of a distinct loop region in the sequences of FlA and NobA, based on comparative sequence analysis. During the revision of this manuscript, O’Hagan and co-workers published the identification of the same fluorinase and two other fluorinases from S. sp MA37, Actinoplanes sp N902-10938. These fluorinases are highly conserved (76–79% identity to each other) but all have the distinct loop region which is identified by this study (Figure S3). This feature indicates that fluorinases are a unique protein family whose evolution is also unusual, majorly concentrated at the interface of N- and C- domains. Moreover, by searching the homologue of the putative S. cattleya 5-FDRibulP aldolases (YP_004910624 and YP_004919742.1, identified in this study) in the geome of A. sp N902-109 results identification of an aldolase (YP_007950157.1). It shares 65% identity to the aldolase from S. cattleya (YP_004910624) and N. brasiliensis (YP_006810507.1) indicating that this common homologue is likely responsible for the conversion of 5-FDRibulP. The discovery of new fluorinases and biosynthetic pathways increases the genetic resource of bio-fluorination and will benefit the future development of synthetic bio-pathways to produce fluorinated natural products.\n\n\nData availability\n\nData sets showing the fluorinase catalysed conversions of fluoride and chloride to 5′-FDA and 5′-ClDA are publicly available in ZENODO.\n\nZENODO: Conversion of fluoride and chloride catalysed by SAM-dependent fluorinase in Nocardia brasiliensis, doi: 10.5281/zenodo.833938.",
"appendix": "Author contributions\n\n\n\nYW, ZD and XQ conceived the study. YW designed the experiments and carried out the research. YW, ZD and XQ wrote the manuscript and all authors approved the final manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported in part by National Natural Science Foundation of China (31270119, 31322002) and program for New Century Excellent Talents in University (NCET-12-0423).\n\n\nAcknowledgements\n\nWe thank Dr. Johnathan Chang at Harvard University for critical reading and Professor Hong-Yu Ou at Shanghai Jiaotong University for kindly providing the S. cattleya strains.\n\n\nSupplementary materials\n\nThe residues highlighted in yellow are critical for SAM hydrolysis. The unique loops of fluorinase are highlighted in bright green. Accession numbers for DUF62 proteins are SalL from Salinispora tropica (ABP73643), PhDUF62 from Pyrococcus horikoshii OT3 (NP_142440) and SaDUF62 from S. arenicola CNS-205 (ZP_01648926).\n\nThe secondary structure of FlA (PDB No. 2V7V) is shown on the top. Critical residues (D16, D21, S23, W50, Y77, F156, N215, S269, R270 and A279) for SAM and fluoride binding are labeled by blue triangle). Residue S158 for fluoride desolvation is labeled by red pentagram. The distinct loop region (R192-L202, FlA numbering) which is bracketed by rectangle.\n\nConserved residues are indicated by bright yellow. The distinct loop region (R192-L202, FlA numbering) is highlighted by rectangle. FlA and NobA are from this study; FlA1 is from Streptomyces sp. MA-37 and FlA3 is from Actinoplanes sp. N902-10939.\n\n\nReferences\n\nBerger R, Resnati G, Metrangolo P, et al.: Organic fluorine compounds: A great opportunity for enhanced materials properties. Chem Soc Rev. 2011; 40(7): 3496–3508. 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J Am Chem Soc. 2007; 129(47): 14597–14604. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCobb SL, Deng H, McEwan AR, et al.: Substrate specificity in enzymatic fluorination. The fluorinase from Streptomyces cattleya accepts 2’-deoxyadenosine substrates. Org Biomol Chem. 2006; 4(8): 1458–1460. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDeng H, Cobb SL, Gee AD, et al.: Fluorinase mediated C-(18)F bond formation, an enzymatic tool for PET labelling. Chem Commun (Camb). 2006; (6): 652–654. PubMed Abstract | Publisher Full Text\n\nWinkler M, Domarkas J, Schweiger LF, et al.: Fluorinase-coupled base swaps: Synthesis of [18F]-5'-deoxy-5'-fluorouridines. Angew Chem Int Ed Engl. 2008; 47(52): 10141–10143. PubMed Abstract | Publisher Full Text\n\nLi X, Domarkas J, O'Hagan D: Fluorinase mediated chemoenzymatic synthesis of [18F]-fluoroacetate. Chem Commun (Camb). 2010; 46(41): 7819–7821. 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PubMed Abstract\n\nWang Y, Deng Z, Qu X: Conversion of fluoride and chloride catalized by SAM-dependent fluorinase in Nocardia brasiliensis. Data Source\n\nDeng H, Ma L, Bandaranayaka N, et al.: Identification of Fluorinases from Streptomyces sp MA37, Norcardia brasiliensis, and Actinoplanes sp N902-109 by Genome Mining. ChemBioChem. 2014; 15(3): 364–368. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "3786",
"date": "05 Mar 2014",
"name": "Changsheng Zhang",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting article dealing with the characterization of a fluorinase from Norcardia brasiliensis. This research was obviously carried out independently and met publishing scientific standard, so it deserves publication in F1000Research, although similar work has been published (Deng et al., 2014). This work mainly focused on the comparative studies on the fluorination of SAM to form 5’-FDA by two fluorinases NobA and FlA. Most of the experiments were well executed, except for the kinetic determination for these two enzymes. I suggest the authors reinvestigate the kinetic parameters, since it was not appropriate to determine the Km for NaF with a “maintaining” concentration of SAM at only 0.4 mM (the author stated that Kms for SAM of FlA and NobA were 0.2 mM and 0.4 mM, respectively). A saturation concentration is normally around 5 X Km.",
"responses": []
},
{
"id": "4207",
"date": "24 Mar 2014",
"name": "Cormac Murphy",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper confirms the very recent work of Deng et al. (2014) reporting a fluorometabolite biosynthetic gene cluster in the genome of Nocardia brasiliensis, the expression of a codon-optimised fluorinase gene (nobA) in E. coli, and the in vitro assay of fluorination/chlorination activity with SAM and fluoride/chloride. Overall the paper is well written, the experimental approach is appropriate, with the exception of the kinetic assays as described by Changsheng Zhang, and the conclusions justified. The kinetics of both the enzymes investigated differ somewhat from those reported by Deng et al., and the authors should account for these differences, particularly in the Km values for SAM.",
"responses": []
}
] | 1
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https://f1000research.com/articles/3-61
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https://f1000research.com/articles/2-187/v1
|
16 Sep 13
|
{
"type": "Research Article",
"title": "Depth-dependent mortality of reef corals following a severe bleaching event: implications for thermal refuges and population recovery",
"authors": [
"Tom C. L. Bridge",
"Andrew S Hoey",
"Stuart J Campbell",
"Efin Muttaqin",
"Edi Rudi",
"Nur Fadli",
"Andrew H Baird",
"Andrew S Hoey",
"Stuart J Campbell",
"Efin Muttaqin",
"Edi Rudi",
"Nur Fadli",
"Andrew H Baird"
],
"abstract": "Coral bleaching caused by rising sea temperature is a primary cause of coral reef degradation. However, bleaching patterns often show significant spatial variability, therefore identifying locations where local conditions may provide thermal refuges is a high conservation priority. Coral bleaching mortality often diminishes with increasing depth, but clear depth zonation of coral communities and putative limited overlap in species composition between deep and shallow reef habitats has led to the conclusion that deeper reef habitats will provide limited refuge from bleaching for most species. Here, we show that coral mortality following a severe bleaching event diminished sharply with depth. Bleaching-induced mortality of Acropora was approximately 90% at 0-2m, 60% at 3-4 m, yet at 6-8m there was negligible mortality. Importantly, at least two-thirds of the shallow-water (2-3 m) Acropora assemblage had a depth range that straddled the transition from high to low mortality. Cold-water upwelling may have contributed to the lower mortality observed in all but the shallowest depths. Our results demonstrate that, in this instance, depth provided a refuge for individuals from a high proportion of species in this Acropora-dominated assemblage. The persistence of deeper populations may provide a critical source of propagules to assist recovery of adjacent shallow-water reefs.",
"keywords": [
"Coral bleaching",
"climate change",
"Acropora",
"Aceh",
"Coral Triangle"
],
"content": "Introduction\n\nMass bleaching events causing extensive mortality of reef-building corals have become more frequent and widespread in recent decades and have affected almost all coral reef regions1–3. Coral bleaching is a generalised stress response resulting from numerous causes including sedimentation, freshwater exposure or disease4; however, the most geographically extensive and severe events are correlated with sustained periods of elevated sea water temperatures and high light irradiance5. The bleaching response is caused by the expulsion of a symbiotic dinoflagellate Symbiodinium that occur within the coral tissue and allow corals to harness energy from sunlight, thus providing a significant portion of the energy requirements. The sensitivity of this symbiosis to elevated sea temperature is well-documented5,6, suggesting that many coral species will be highly vulnerable to the effects of global warming7,8.\n\nDespite this apparent sensitivity, reef corals have persisted through numerous large-magnitude and sometimes rapid changes in sea surface temperatures over the past 240 million years3,9. One mechanism by which a species can cope with changing local climate is to move to a more favourable area, and tropical reef corals have repeatedly shifted their distribution to higher latitudes in response to past climate warming10,11. Alternatively, populations may persist in microrefugia, defined as small areas of suitable habitat within regionally unfavourable environmental conditions12,13. Despite increasing recognition of their importance for conservation planning in terrestrial ecosystems14–16, microrefugia are less considered in the marine realm.\n\nThe severity of coral bleaching is often spatially heterogeneous due to both historical17,18 and environmental19–21 factors. Coral bleaching is caused by a synergistic effect between heat and light, and therefore microrefugia from bleaching are likely to occur in regions where oceanographic or atmospheric conditions reduce water temperatures or light irradiance relative to surrounding areas22. Light irradiance declines with depth and ambient temperatures are often lower in deeper waters, therefore the incidence of bleaching and/or subsequent mortality is likely to be lower at greater water depths1,5,22. Warm-water coral bleaching is occasionally reported to depths of 50 m, however, such observations are rarely followed up in order to estimate bleaching-induced mortality. Typically the incidence of bleaching is substantially lower at greater depths and in the few cases it has been measured, so is bleaching-induced mortality23–25. For example, mortality rates of corals at a depth of 6 m were only a third of those in 2 m across several turbid inshore reefs on the Great Barrier Reef (GBR)24. A transition from high to low mortality with increasing depth was observed at numerous sites in the western Indian Ocean during 1998, the most severe and widespread bleaching event on record26. This transition often occurred across a fairly sharp depth boundary at intermediate depths of 10–15 m26, therefore species with depth ranges that straddle this transition from high to low bleaching mortality will have a refuge from bleaching in deeper water. However, most assessments of coral reefs consider only shallow habitats, and reductions in mortality with increasing depth may go unnoticed. Furthermore, recent studies of deep-water reefs have indicated that many corals may occur over a wider depth range than currently thought27,28.\n\nIn May-June 2010, a sustained increase in seawater temperatures in the Andaman and South China Seas resulted in extensive coral bleaching and caused high mortality of many coral species29. Six weeks after the peak seawater temperatures, 45% of all corals and 94% of Acropora colonies were dead in shallow waters (1–2 m) around Pulau Weh, Sumatra, Indonesia29. Here, we assess the effects of this severe thermal bleaching event at Pulau Weh over a depth gradient from 2–27 m to investigate 1) whether severe mortality of reef corals observed in shallow water (0–2 m) extended into deeper habitats; and 2) whether depth provided a refuge from bleaching mortality. We concentrate on the corals of the genus Acropora because they are the most diverse and abundant genus in the Indo-Pacific, and are important ecosystem engineers on most Indo-Pacific coral reefs. They are also often amongst the most susceptible taxa to bleaching-induced mortality, and bleaching events often result in shifts from Acropora – dominated communities towards communities dominated by more bleaching resistant taxa (e.g. Porites and the family Merulinidae)26,30. Change in Acropora cover before and after a bleaching event is therefore a useful indicator of bleaching severity.\n\n\nMaterials and methods\n\nPulau Weh (5° 50’N, 95° 20’E) is located in the province of Aceh off the northwest coast of Sumatra, Indonesia. The region’s reefs have received little attention from scientists, but support similarly diverse coral communities to the rest of the Indo-Australian Archipelago31. Northwest Sumatra was the epicentre of the December 2004 Indian Ocean tsunami, and although Pulau Weh’s coral communities were relatively unaffected by this event32, they suffered substantial mortality in the 2010 Andaman Sea bleaching29. To examine the influence of depth on bleaching mortality, we compared both total coral cover and Acropora cover collected before (November 2009 to February 2010) and after (July 2011) the bleaching event at three depths (0–2 m, 3–4 m and 6–8 m) at four sites on the northern and western sides of Pulau Weh (Batee Gla, Ba Kopra, Rubiah Sea Garden, Rubiah Channel – see31). Coral cover was estimated along 6–10 replicate 10 m line intercept transects at 0–2 m, and 3–6 replicate 50 m point intercept transects at 3–4 and 6–8 m (see Data File). Any live hard coral (i.e. scleractinian or hydrozoan coral) underlying each survey point was recorded to genus level. Changes in total live coral cover and Acropora cover between 2009 and 2011 were compared using two-factor ANOVA’s. Assumptions of the ANOVA’s were examined using residual analysis and no transformation was necessary. The analyses were based on the proportion of total coral or Acropora cover per 50 m transect.\n\nTo determine the proportion of the Acropora assemblage afforded a depth refuge from this bleaching event, we conducted species-level surveys of Acropora assemblages in 0 to 2 m and then at 5 m intervals from 3–27 m in February 2012 at five sites on the northern and western sides of Pulau Weh (Batee Gla, Ba Kopra, Rubiah Sea Garden, Rubiah Channel and Tokong). Sites were chosen based on their bathymetry profiles, with accessible deep sites only present on the steeply-sloping, ocean-facing northern and western coasts. Data were collected at 5 m depth intervals using replicate 10-minute timed swims, where the species identity of every living Acropora colony was recorded. Post-bleaching surveys were compared to shallow-water (0–2 m) surveys conducted in November 2009 before the bleaching event using 40 min timed swims31 at these same sites. Corals were identified using taxonomic references provided in “Staghorn corals of the world” by Wallace CC and “Corals of the world”, by Veron JEN33,34. Analysis of Similarities (ANOSIM), a multivariate approximation of ANOVA35, was performed on a square root-transformed Bray-Curtis similarity matrix to determine any significant difference in the Acropora assemblage among sites.\n\n\n\n\nResults and discussion\n\nA total of 40 Acropora species were observed during the study, confirming the high diversity previously reported on Acehnese reefs31. ANOSIM revealed no significant difference in assemblage structure among sites, which were therefore pooled for further analysis. Bleaching mortality was very high in the shallows, however, mortality diminished rapidly with increasing depth (Figure 1). Total coral cover declined by 75% at 0–2 m but only 20% at 3–4 m, while there was no significant change at 6–8 m (Figure 1a; 2-way ANOVA depth by year interaction; F2,123 = 21.2, p < 0.001). The decline in mortality was even more pronounced in the Acropora, with cover declining by approximately 90% at 0–2 m and 60% at 3–4 m, with no change detected at 6–8 m (Figure 1b; 2-way ANOVA depth x year; F2,123 = 17.9, p < 0.001).\n\n(a) total live coral cover; and (b) live Acropora cover.\n\nA high proportion of this diverse Acropora assemblage was afforded a refuge from bleaching mortality by depth. Of the 29 Acropora species occurring in shallow waters < 7 m, 19 (66%) also occurred below the approximate depth of transition from high to low mortality (Figure 2). However, the refuge effect would be diminished if mortality had reached into deeper waters. If, for example, the transition between high and low bleaching mortality had occurred at 12 m, 14 (48%) of the species affected would have had a refuge in depth. Similarly, if bleaching mortality extended to 22 m, only 6 species from the shallow assemblage (21%) would have had colonies persisting below the transition depth.\n\nShaded (grey) panel indicates the depth range where bleaching mortality was high. Of the 29 species occurring in 0–7 m depth, 19 (66%) also occurred below 7 m.\n\nDoubts regarding the potential significance of depth as a refuge for corals from warm-water bleaching have previously been raised because (1) bleaching has been observed in the deeper areas of reefs, (2) there is limited overlap of species between deep and shallow reef areas, and (3) genetic partitioning within species among depths suggests that deeper population cannot provide an effective source of recruits for shallow populations36,37. Firstly, while bleaching often extends to the lower depth limits of some shallow water species, both bleaching frequency and, most importantly, mortality, is often strongly depth dependent (Figure 3)24,26. Indeed, a transition from high to low mortality occurred at depths of ≤ 15 m ~50% of sites surveyed in the Indian Ocean in 199826– see Table 1). Secondly, our results indicate that even with a pronounced depth zonation in the Acropora assemblage, two-thirds of species occurring in shallow depths had a depth range that straddled the transition in bleaching mortality. The depth zonation of coral assemblages is one of the most consistent and predictable patterns in nature38,39 and therefore our results are not an anomaly. Thirdly, the genetic divergence between populations above and below the transition in mortality at between 4 and 8 m is unlikely to be sufficient to prevent larval migration in either direction. For example, larvae of the coral Seriatopota hystrix migrate among sub-populations over a 30 m depth range40. Furthermore, connectivity modelling in two Caribbean coral species indicates demographically significant larval subsidy from deep to shallow reef habitats over a much greater depth range (5–40 m) even when deep-water fertilisation rates and post-settlement survival are greatly reduced41.\n\n(a) Reef crest at 2 m depth prior to bleaching (16 November 2009); (b) the same reef crest six weeks after the peak of bleaching (26 July 2010); (c) upper reef slope community at 6 m depth largely unaffected by the bleaching event, 25 February 2012.\n\nOur results indicate that bleaching mortality can vary considerably over a small depth range. Consequently, surveys conducted only in shallow waters may greatly overestimate the proportion of coral populations killed by coral bleaching29. Conversely, surveys conducted in deeper areas are likely to underestimate the effects of bleaching. For example, long-term, large-scale monitoring of coral cover on reef slopes (6–9 m depth) on the GBR suggests that bleaching has been a comparatively minor source of coral mortality over the last few decades42,43, despite two mass bleaching events in 1998 and 200244. However, in the 1998 bleaching event on the inshore GBR, bleaching mortality was on average 3-times higher at 2–4 m when compared to 5–8 m24. Clearly, ecosystem assessments considering only a single depth may provide a biased view of the relative importance of the many different agents of coral mortality, and should therefore be conducted over a range of depths to accurately assess the relative importance of multiple stressors.\n\nIdentifying areas or conditions that consistently provide refuges for corals from thermal stress is critically important for coral reef conservation under future climate change. In 1998, lower mortality and a shallower transition depth was often associated with sites that experienced episodic upwelling of cold water26,45,46. Although environmental data are not available from Pulau Weh, pulses of cold water were regularly experienced during data collection, and rapid upwelling-driven temperature plunges of up to 10°C are recorded from the west coast of the nearby Similan Islands47. Interestingly, Acehnese reefs appeared unaffected by the 1998 bleaching event29, despite the coral bleaching extending across virtually the entire Indian Ocean from east Africa and north-western Australia26,48,49. These cold-water upwelling events may explain the lack of mortality in 1998 and the shallow transition depth during 2010 despite very high sea surface temperatures. If so, this region may provide a consistent refuge for many corals against rising sea temperatures and thermal anomalies. In summary, our results show that coral bleaching mortality can diminish rapidly even where shallow-water corals experience severe mortality, and modest depths can provide a refuge for a significant proportion of coral species. Identifying sites where oceanographic conditions reduce the effects of thermal anomalies should be a priority for coral reef conservation.",
"appendix": "Author contributions\n\n\n\nTCLB and AHB conceived the study. TCLB, AHB and ASH designed the study. All authors were involved in data collection. TCLB, AHB and ASH analysed these data. TCLB, AHB and ASH wrote the manuscript with additional contributions from all other authors.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nFunding for this study was provided by the Australian Research Council Centre of Excellence for Coral Reef Studies, the Wildlife Conservation Society Indonesia Marine Program, and the Kerzner Marine Foundation.\n\n\nAcknowledgements\n\nThe activities for this study were conducted under a Memorandum of Understanding (MoU) between the Wildlife Conservation Society (WCS) and the Indonesian Ministry of Forestry, and a MoU between the ARC Centre of Excellence for Coral Reef Studies, James Cook University, Australia and Syiah Kuala University, Banda Aceh, Indonesia. No flora or fauna were collected or manipulated during this research and all surveys were conducted on public land. We thank Ismayudi Dodent and Rubiah Tirtah Divers for their field support.\n\nThis paper is dedicated to the late Dr Edi Rudi, a pioneer of coral research in Aceh and a great friend.\n\n\nReferences\n\nHughes TP, Baird AH, Bellwood DR, et al.: Climate change, human impacts, and the resilience of Coral Reefs. Science. 2003; 301(5635): 929–933. PubMed Abstract | Publisher Full Text\n\nBaker AC, Glynn PW, Riegl B: Climate change and coral reef bleaching: an ecological assessment of long-term impacts, recovery trends, and future outlook. Estuar Coast Shelf Sci. 2008; 80: 435–471. Publisher Full Text\n\nPandolfi JM, Connolly SR, Marshall DJ, et al.: Projecting coral reef futures under global warming and ocean acidification. Science. 2011; 333(6041): 418–422. PubMed Abstract | Publisher Full Text\n\nBaird AH, Bhagooli R, Ralph PJ, et al.: Coral bleaching: The role of the host. 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Publisher Full Text\n\nWoesik R, Houk P, Isechal AL, et al.: Climate-change refugia in the sheltered bays of Palau: analogs of future reefs. Ecol Evol. 2012; 2(10): 2474–2484. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRiegl B, Piller WE: Possible refugia for reefs in times of environmental stress. Int J Earth Sci. 2003; 92: 520–531. Publisher Full Text\n\nRowan R, Knowlton N, Baker AC, et al.: Landscape ecology of algal symbionts creates variation in episodes of coral bleaching. Nature. 1997; 388(6639): 265–269. PubMed Abstract\n\nMarshall PA, Baird AH: Bleaching of corals on the Great Barrier Reef: differential susceptibilities among taxa. Coral Reefs. 2000; 19: 155–163. Publisher Full Text\n\nGlynn PW, Mate JL, Baker AC, et al.: Coral bleaching and mortality in Panama and Ecuador during the 1997–1998 El Nino-Southern Oscillation event: spatial/temporal patterns and comparisons with the 1982–1983 event. Bull Mar Sci. 2001; 69: 79–109. Reference Source\n\nSheppard CRC, Obura D: Corals and reefs of the Cosmoledo and Aldabra atolls: Extent of damage, assemblage shifts and recovery following the severe mortality of 1998. J Nat Hist. 2005; 39: 103–121. Publisher Full Text\n\nBridge TCL, Fabricius KE, Bongaerts P, et al.: Diversity of Scleractinia and Octocorallia in the mesophotic zone of the Great Barrier Reef, Australia. Coral Reefs. 2012; 31: 179–189. Publisher Full Text\n\nBridge TCL, Hughes TP, Guinotte JM, et al.: Call to protect all coral reefs. Nat Clim Chang. 2013; 3: 528–529. Publisher Full Text\n\nGuest JR, Baird AH, Maynard JA, et al.: Contrasting patterns of coral bleaching susceptibility in 2010 suggest adaptive response to the thermal stress. PLoS One. 2012; 7(3): e33353. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLoya Y, Sakai K, Yamazato K, et al.: Coral bleaching: the winners and losers. Ecol Lett. 2001; 4: 122–131. Publisher Full Text\n\nRudi E, Campbell SJ, Hoey AS, et al.: The Coral Triangle Initiative: What are we missing? A case study from Aceh, Indonesia. Oryx. 2012; 46, 482–485. Publisher Full Text\n\nBaird AH, Campbell SJ, Anggoro AW, et al.: Acehnese reefs in the wake of the Asian tsunami. Curr Biol. 2005; 15(21): 1926–1930. PubMed Abstract | Publisher Full Text\n\nWallace CC: Staghorn corals of the world. CSIRO Publishing, Collingwood, Victoria. 1999. Reference Source\n\nVeron JEN: Corals of the world. Australian Institute of Marine Science, Townsville. 2000. Reference Source\n\nClarke KR, Gorley RN: PRIMER v6: user manual/tutorial. PRIMER-E: Plymouth. 2006. Reference Source\n\nBongaerts P, Riginos C, Ridgway T, et al.: Genetic divergence across habitats in the widespread coral Seriatopora hystrix and its associated Symbiodinium. PLoS One. 2010; 5(5): e10871. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrazeau DA, Lesser MP, Slattery M: Genetic Structure in the Coral, Montastraea cavernosa: Assessing Genetic Differentiation among and within Mesophotic Reefs. PLoS One. 2013; 8(5): e65845. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDone TJ: Coral zonation: its nature and significance Perspectives on Coral Reefs. Brian Clouston, Manuka, A.C.T. 1983; pp95–147. Reference Source\n\nHughes TP, Baird AH, Dinsdale EA, et al.: Assembly rules of reef corals are flexible along a steep climatic gradient. Curr Biol. 2012; 22(8): 736–741. PubMed Abstract | Publisher Full Text\n\nvan Oppen MJ, Bongaerts P, Underwood JN, et al.: The role of deep reefs in shallow reef recovery: an assessment of vertical connectivity in a brooding coral from west and east Australia. Mol Ecol. 2011; 20(8): 1647–1660. PubMed Abstract | Publisher Full Text\n\nHolstein DM: Vertical connectivity in mesophotic coral ecosystems. Open Access Dissertations. Paper 1064. 2013. Reference Source\n\nOsborne K, Dolman AM, Burgess SC, et al.: Disturbance and the dynamics of coral cover on the Great Barrier Reef (1995–2009). PLoS One. 2009; 6(3): e17516. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDe’ath G, Fabricius KE, Sweatman H, et al.: The 27-year decline of coral cover on the Great Barrier Reef and its causes. Proc Natl Acad Sci U S A. 2012; 109(44): 17995–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBerkelmans R, De’ath G, Kininmonth S, et al.: A comparison of the 1998 and 2002 coral bleaching events on the Great Barrier Reef: spatial correlation, patterns and predictions. Coral Reefs. 2004; 23: 74–83. Publisher Full Text\n\nTeleki KA, Spencer T: Reef systems of the islands of the southern Seychelles. In: Souter D, Obura D, Linden O, editors. Coral Reef Degradation in the Indian Ocean. Kalmar, Sweden: CORDIO. 2000; p 87–93. Reference Source\n\nGoreau T, McClanahan T, Hayes R, et al.: Conservation of coral reefs after the 1998 global bleaching event. Conserv Biol. 2000; 14: 5–15. Publisher Full Text\n\nSchmidt GM, Phongsuwan N, Jantzen C, et al.: Coral community composition and reef development at the Similan Islands, Andaman Sea in response to strong environmental variations. Marine Ecolology Progress Series. 2012; 456: 113–126. Publisher Full Text\n\nSheppard CR: Predicted recurrences of mass coral mortality in the Indian Ocean. Nature. 2003; 425(6955): 294–297. PubMed Abstract | Publisher Full Text\n\nGilmour JP, Smith LD, Heyward AJ, et al.: Recovery of an isolated coral reef system following severe disturbance. Science. 2013; 340(6128): 69–71. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "1823",
"date": "20 Sep 2013",
"name": "Tyler Smith",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is of great interest considering the impact of thermal stress on coral reefs globally, and the pressing need to identify refugia that might support coral diversity in a warming ocean. The article is basic, in that it only attempts to directly answer two questions: (1) did acroporid corals do better at deeper depths (8m) versus shallower depths (2 and 4m), and (2) is there a significant proportion of acroporid diversity that would be protected by the identified depth refuge in 2010 (i.e., how many acorporid species have a sufficiently wide depth range). I believe that they answer these questions well, but I would have liked to see more thorough investigation of the patterns in the data and I think there are some anomalous parts of the data that I cannot explain. For example, while most sites followed a pattern with increasing bleaching mortality at shallower depths, plotting of the data provided in table 1 shows that one site, Ba Kopra at 4m, showed no change or even an increase in total and acroporid cover, respectively. The authors never discuss this site to site variability, which might be important for \"Identifying sites where oceanographic conditions reduce the effects of thermal anomalies should be a priority for coral reef conservation\". As for anomalous parts of the data, I can't understand why the absolute cover change of acroporid corals seems to surpass that of total coral cover at some sites. For example, at Rubiah Channel the acroporid cover drops an absolute amount of 38.1%, by my calculation. That is a simple calculation of final cover - initial cover, not standardized to the initial coral cover (which would be relative cover change). Therefore, the total absolute coral cover change has to be at least 38.1%, yet it is only 23.6%. This is not that the total cover is really the total cover excluding acropora, since the prior to bleaching acropora + total cover = 102%. Can the authors explain this? In the methods it would also be necessary to know if the pre- and post-bleaching transects are the same (i.e., fixed permanent transects) or whether they are randomly placed. In either case, but particularly for the latter, it is also necessary to know the method by which the placement of the transect was determined at a site. How are we to know potential biases in the pre- and post-assessments without this knowledge. There is also no information given on the exact location of monitoring sites, which is important for replication of the study. Perhaps coordinates and a map would be appropriate. One further addition that would be nice, though an addition to the two primary questions of the manuscript, is what is the relative importance of the depth refuge to the in situ shallow coral survival? They mention \"recovery\" in the title, but this isn't really addressed much in the manuscript, and improving the discussion of potential recovery process would help on that point. I.e., what absolute amount of cover for each species survived deep, and could contribute to shallow water recovery via larval recruitment, versus the amount that survived shallow and could contribute to direct asexual recovery and larval recruitment? Also, any speculation on the relative importance of the processes in recovery (deep to shallow larval recruitment versus shallow to shallow larval and asexual recruitment) would be a good addition and set up future research.I have a feeling all these questions are easily answerable and that the conclusions are justified, and I think the manuscript is an important addition to a rather sparse body of knowledge concerning reef refuges and refugia.",
"responses": [
{
"c_id": "592",
"date": "27 Oct 2013",
"name": "Tom Bridge",
"role": "Author Response",
"response": "Our response to the issues raised by the reviewer are summarised below:We acknowledge some variability among sites in the depth of transition from high to low mortality, but the overall trend of decreasing mortality with increasing depth is consistent among sites. The reviewer noted that the ‘absolute cover change of Acroporid corals seems to surpass that of total coral cover’, and we note three possible reasons for this observation. Firstly, such variations are commonly observed on coral reefs after a disturbance event such as coral bleaching due to ‘canopy effects’, whereby removal of canopy-forming taxa (in this case Acropora) can cause apparent increases in taxa which were concealed underneath the canopy. Canopy effects have been well documented in the coral reef literature (e.g. Goatley CHR & Bellwood DR, 2011). In Pulau Weh, the increases in encrusting/massive Porites and Faviids suggest canopy effects were a likely cause of these observations. Secondly, surviving corals will have grown from 2009 to 2011. Thirdly, transects were haphazardly placed and therefore some variability within a site is possible from year to year. We have included a map of study sites, and also stated in the text that transects were haphazardly placed within a site. Coral cover data from 2009 to 2011 was collected only to genus level, therefore it was not possible to address species-specific changes in abundance before and after the bleaching."
}
]
},
{
"id": "1825",
"date": "02 Oct 2013",
"name": "John Rooney",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper makes a valuable contribution, highlighting the marked differences in bleaching-induced coral mortality associated with changes in depth of just a few meters. It highlights the need to conduct surveys over a range of depths to characterize bleaching events and, in particular, to identify “microrefugia” - sites where oceanographic conditions reduce the effects of thermal anomalies - as a priority for coral reef conservation.\nIn their introduction the authors mention the importance of both irradiance and temperature, but no further mention of irradiance's possible role in the observed coral bleaching and mortality patterns is made. Additionally, the author’s state that “pulses of cold water were regularly experienced during data collection” as evidence that upwelling of cold water was the mechanism responsible for the reduced bleaching-induced coral mortality at their deeper survey sites. Although it may not be possible to reconstruct temperature differences during the 2010 bleaching event at their study sites, even temperature records from the different survey depths on the Acehnese reefs well after the event may provide some insight into the possible magnitude of temperature differences that were sufficient to reduce coral mortality. Some discussion of the specific parameters that might distinguish microrefugia, e.g. the frequency and magnitude of differences in temperature or irradiance relative to surrounding waters, would greatly enhance the paper’s utility, and provide an important addition to further work on this topic.",
"responses": [
{
"c_id": "593",
"date": "27 Oct 2013",
"name": "Tom Bridge",
"role": "Author Response",
"response": "Unfortunately, no data are currently available to quantify the local oceanographic patterns at this time, and to address this question without such data would be overly speculative. However, we plan to collect temperature data at Pulau Weh as part of ongoing monitoring of reefs in the region."
}
]
}
] | 1
|
https://f1000research.com/articles/2-187
|
https://f1000research.com/articles/2-225/v1
|
28 Oct 13
|
{
"type": "Case Report",
"title": "Bispectral index detects intraoperative cerebral ischaemia during balloon assisted cerebral aneurysm coiling",
"authors": [
"Zoe Harclerode",
"John Andrzejowski",
"Stuart Coley",
"Richard Dyde",
"Zoe Harclerode",
"Stuart Coley",
"Richard Dyde"
],
"abstract": "Bispectral index (BIS) is a monitoring modality designed and used for monitoring depth of anaesthesia. We wish to report a case where BIS monitoring may have alerted us to a potential adverse neurological event during angiographic coiling of a cerebral aneurysm.",
"keywords": [
"Bispectral index (BIS) is a monitoring modality designed and used for monitoring depth of anaesthesia. However",
"it may also have other advantages in detecting intracranial haemodynamic events1–3. We wish to report a case where BIS monitoring may have alerted us to a potential adverse neurological event during angiographic coiling of a cerebral aneurysm."
],
"content": "Case\n\nBispectral index (BIS) is a monitoring modality designed and used for monitoring depth of anaesthesia. However, it may also have other advantages in detecting intracranial haemodynamic events1–3. We wish to report a case where BIS monitoring may have alerted us to a potential adverse neurological event during angiographic coiling of a cerebral aneurysm.\n\nA 56 year old, right handed male was listed for coiling of an unruptured intracranial aneurysm. The aneurysm was discovered as an incidental finding on a CT scan of the head performed for the investigation of a previous episode of confusion. Cerebral angiography demonstrated an approximately 12mm wide necked aneurysm at the termination of the intracranial segment of the left internal carotid artery (ICA) (Figure 1). The patient’s past medical history included hypertension and heavy smoking and he had a BMI of 32.\n\nA small premedication dose of 2mg intravenous midazolam was given whilst an arterial line was inserted to allow continuous invasive intra-arterial blood pressure monitoring. Standard AAGBI monitoring was supplemented with BIS (Aspect Medical Systems, Newton MA, USA) that was positioned over the forehead and left temporal area before induction. An infusion of TCI (target controlled infusion) remifentanil was then commenced with an effect site (Cet) of 2ng/ml increasing to 4ng/ml before induction, which consisted of 140mg propofol, with rocuronium 60mg given to facilitate tracheal intubation.\n\nAnaesthesia was maintained with sevoflurane (end tidal concentration 1.2–1.4) and TCI remifentanil continued at Cet of 4ng/ml, targeting a BIS range of 40–55. A metaraminol infusion was used to maintain a systolic blood pressure between 110–130mmHg. End tidal CO2 was maintained in the range of 4.5–5.1kPa.\n\nCoiling was performed with vascular access obtained via a right femoral arterial puncture. Using a guide catheter placed in the extra-cranial segment of the left ICA, a balloon catheter was navigated into the intracranial circulation. The inflatable/deflatable 4mm balloon was positioned across the neck of the aneurysm and during inflation there was simultaneous occlusion of the neck of the aneurysm and the proximal segment of the left middle cerebral artery (MCA). The balloon was intermittently inflated across the neck of the aneurysm to prevent coil prolapse during placement of 11 platinum microcoils into the aneurysm. Each inflation lasted in the order of 30–120 seconds. An intravenous bolus of 5000 units of Heparin was given to minimize the risk of thromboembolism associated with the endovascular devices.\n\nThe photograph of the intraoperative BIS trend (Figure 2) shows that the first time the balloon was inflated, the BIS value fell to approximately 25 over a 60 second period. Upon balloon deflation, BIS returned more rapidly to its pre-inflation value. The anaesthetic team alerted the neuro-radiologists to these changes. They were able to limit the duration of subsequent balloon inflations resulting in demonstrably shorter subsequent BIS falls.\n\nPost procedure, the patient was woken up and on return to the ward had a GCS of 15 with no neurological deficit. He was discharged home after 48 hours and has recovered well at home with no problems.\n\n\nDiscussion\n\nThis case clearly illustrates that intraoperative Bispectral Index (BIS) monitoring may allow the detection of cerebral hypoperfusion. An abrupt decrease in BIS values not associated with changes in anaesthetic technique or haemodynamics may alert the anaesthetist to such an event. Early communication with the surgeon or interventionist can potentially avoid procedure related neurological injury.\n\nThere have been previous case reports of sudden decreases in BIS value during other neurosurgical and neuro-radiological interventions. These include rupture of cerebral aneurysms during coiling1, embolization of arterio-venous malformations2 and intraventricular haemorrhage during third ventriculoscopy3. Rapid increases in intracranial pressure and vasospasm have been postulated as possible causes for the decrease in BIS values.\n\nThe aneurysm in this case was on the ICA at the termination of the vessel. The angiographic balloon was placed via the ICA into the proximal MCA. Balloon inflation with simultaneous occlusion of the distal ICA and proximal middle cerebral arteries is more hazardous than the same procedure confined to the ICA as complete occlusion of the carotid tip prevents collateral flow from the contralateral circulation to the MCA (via the circle of Willis). Repeated balloon inflation within a vessel may lead to platelet aggregation or vessel wall injury with subsequent neurological injury. However, in this case the repeated acute change in the BIS values suggests a haemodynamic, rather than a thromboembolic, insult to the distal circulation.\n\nIn this patient, the BIS sensor was positioned to primarily monitor the cerebral hemisphere undergoing intervention. Studies investigating the effects of cerebral ischaemia (such as during carotid endarterectomy) on BIS have been contradictory with some demonstrating a correlation with ischaemia whilst others show none4,5. The advent of a bilateral BIS sensor for general anaesthesia6 may shed more light on these discrepancies and allow more subtle ischaemic changes (possibly resulting in left to right BIS differences) to be detected.\n\nNot all balloon assisted neuro-radiological procedures will result in potential ischaemia, however those involving vascular territories with poor collateral circulation might benefit from the use of bilateral BIS as a tool to detect early and potentially avoidable adverse neurological events.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.",
"appendix": "Author contributions\n\n\n\nJA and ZH conceived the idea. SC and RD were the neuro-radiologists involved with the case in question.\n\nZH prepared the first draft of the manuscript, with all authors contributing to revisions of the draft and all authors have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nPrabhakar H, Ali Z, Rath GP, et al.: Zero bispectral index during coil embolization of an intracranial aneurysm. Anesth Analg. 2007; 105(3): 887–8. PubMed Abstract | Publisher Full Text\n\nUnnikrishnan KP, Sinha PK, Sriganesh K, et al.: Case report: alterations in bispectral index following absolute alcohol embolization in a patient with intracranial arteriovenous malformation. Can J Anaesth. 2007; 54(11): 908–11. PubMed Abstract | Publisher Full Text\n\nEapen G, Andrzejowski J: Sudden decrease of Bispectral index during endoscopic neurosurgery. J Neurosurg Anesthesiol. 2009; 21: 274–82.\n\nEstruch-Pérez MJ, Ausina-Aguilar A, Barberá-Alacreu M, et al.: Bispectral index changes in carotid surgery. Ann Vasc Surg. 2010; 24(3): 393–9. PubMed Abstract | Publisher Full Text\n\nBonhomme V, Desiron Q, Lemineur T, et al.: Bispectral index profile during carotid cross clamping. J Neurosurg Anesthesiol. 2007; 19(1): 49–55. PubMed Abstract | Publisher Full Text\n\nSmith M, Wiles M, Andrzejowski J: Interhemispheric EEG variability measured using a bilateral Bispectral Index (BIS) sensor. J Neurosurg Anesthesiol. 2012; 24: 244."
}
|
[
{
"id": "2214",
"date": "05 Nov 2013",
"name": "Ehab Farag",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors should mention that BIS has a very limited ability to detect ischemia as it only monitors the frontal lobe. Therefore it cannot be compared to traditional 16 channels EEG or NIRS to detect cerebral ischemia. This case should not be used as standard for care for using BIS as a monitor for cerebral ischemia.",
"responses": [
{
"c_id": "615",
"date": "13 Nov 2013",
"name": "John Andrzejowski",
"role": "Author Response",
"response": "I agree with Dr Farag's comments. I would be happy to change the discussion to read: BIS has a limited ability to detect ischemia since, similarly to Near Infra Red Spectroscopy (NIRS), in its usual montage it only monitors a frontal lobe. It cannot be compared to a traditional 16 channel EEG that monitors the whole brain, and should not be considered as a standard of care for detecting cerebral ischaemia. In this patient, the BIS sensor was positioned to primarily monitor the cerebral hemisphere undergoing intervention.Studies investigating the effects of cerebral ischaemia (such as during carotid endarterectomy) on BIS have been contradictory with some demonstrating a correlation with ischaemia whilst others show none4,5. The advent of a bilateral BIS sensor for general anaesthesia6 may shed more light on these discrepancies and allow more subtle ischaemic changes (possibly resulting in left to right BIS differences) to be detected."
}
]
},
{
"id": "2216",
"date": "27 Dec 2013",
"name": "Luzius A Steiner",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting case report. However, the discussion is somewhat superficial. I have two comments:Please make it clear that the sensitivity to detect cerebral ischemia by EEG is not perfect and that the BIS monitor has not been developed for this purpose. Please explain why you think that an ischemic event would lead to a decrease in the BIS value from approximately 50 to 25 in your case (but not in all cases e.g. your references). What is the temporal relationship between the balloon inflation and the BIS value. You state that it \"fell to approximately 25 over a 60 second period\". Theoretically, I would expect some delay before the BIS reacts (already because it samples over 15 or 30 seconds to calculate the BIS value; which sampling interval did you use?). Is there an alternative explanation you could offer (e.g. did you check the EMG value)? Did you administer neuromuscular blockers or other drugs prior to balloon inflation, is it possible that you recorded an artifact of some type? Please expand your case description and the discussion.",
"responses": []
},
{
"id": "2943",
"date": "30 Jan 2014",
"name": "Mark Coburn",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe conclusions which the authors draw from this case report are far too strong and need to be adapted.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-225
|
https://f1000research.com/articles/3-1/v1
|
03 Jan 14
|
{
"type": "Commentary",
"title": "A letter to the Master Clinician",
"authors": [
"Kenar D. Jhaveri"
],
"abstract": "In this commentary, the author writes a letter to the Master Clinician about his concerns regarding the teaching responsibilities of current faculty members during ward rounds. This short essay highlights the transition that has been noticed in medical training in the last decade.",
"keywords": [
"Dear Master Clinician"
],
"content": "\n\nDear Master Clinician,\n\nRe: Invitation\n\nI am writing this letter to ask you to please return to us. In your absence, we have felt your loss.\n\nToday, when I walk down the halls of my academic hospital, I feel an emptiness. The constant flow of work onto the secretary’s desk begins at 7 am. The transporter brings a patient back from the ultrasound room. The residents hurry to gather data from the chart before their attending arrives. The nurses scurry to change shifts. These long halls are busy with secretaries working hard, nurses doing their jobs, and nurse practitioners and physician assistants writing notes on the chart. In the midst of all this busy life for our residents, fellows and medical students, there is something now missing.\n\nAs a consultant on the floor, I see a crowd of physicians making their rounds. The medical students are easily recognizable by their short white coats. Then I stop, I see someone presenting data: Ahhh! that must be the intern. There are residents discussing patients with a hospitalist. The endocrine consultant team walks onto the floor, and the cycle starts all over again. I have noticed in the last few years that the physician-in-charge is usually inexperienced, and probably has just 1–3 years more experience than the third year resident. What has changed? What is missing? Few now stay on to continue to build their experience but rather join a fellowship program or outpatient practice. Soon, they are replaced by new fresh group of them just graduating from residency. Have we lost the Master Clinician?\n\nThe people who inspired us to become who we are today were the great Master Clinicians of their time and they shared with us their wisdom, knowledge and wealth of experience. It would be wrong to say that we are losing the art of physical exam and diagnosis, but rather that we have lost the art. You shared your wisdom with the team and taught the fellows and residents not only bedside manners, but also told us about your experiences. Now you have been replaced with inexperienced faculty, textbooks have been replaced by Google and stethoscopes have been replaced by handheld devices.\n\nHow did this happen? While I support the advent of technology and the advancement of knowledge in medicine, medicine is also an art which cannot be taught by Google, Up-To-Date or Twitter.\n\nYou have the experienced hands of a Master Clinician who carries the wisdom of diagnostic skills and carries knowledge.\n\nWhere have you been? Now you only occasionally sit with fellows and residents and give a lecture and share your wisdom. It is always an honor and pleasure to meet someone of your caliber but one might never see you on the hospital floor showing your magic. Why is that? You have taken on extensive administrative roles, spend more time in the laboratory and have less time to come and join us on the wards. While we understand your needs and desires to do other tasks, I wish that you would come back and share your wealth with us on the floor more often. Once a year, I see you come and do some time on the wards; I get very excited that you are able to give us that time. But that time is fragmented by meetings. You have cut short what you do best: teaching, caring for patients and inspiring young professionals. When we lose you to administrative duties, we lose the enthusiasm that you share with young and up-and-coming physicians that creates their passion for medicine. We lose the art of medicine. We lose doctoring…\n\nWhy is this Master important in the making of a good clinician? The role of such a person is enormous. A 67 year old male with prostate cancer is admitted for severe metabolic alkalosis, hypokalemia and new onset hypertension. A medical student can spend hours taking histories and performing physical exams, memorize a long list of differential diagnoses but yet not come up with the right diagnosis. This Master Clinician arrives at the correct diagnosis in a few minutes of meeting the same patient. “This is Cushing’s disease-ACTH production from the prostate cancer; start ketaconazole now!” Medical school teaches us the science of medicine and post graduate training showcases us the art of medicine. From being a good teacher and a great clinician, Master Clinicians such as yourself will demonstrate the art of medicine, the bedside teaching that medical students, residents and fellows should be learning. This individual will bring to the bedside their years of experience and thoughtful discussions of tough cases to make us all understand the basics of disease. You can inspire and create many more such Masters by your aura and presence on the floor.\n\nIn my career as a student and physician-in-training, the teachers who inspired me to become an Internist and a Nephrologist were all Master Clinicians and spent a lot of time with us - showing us physical examination skills, ways to think through tough cases and how to balance family and residency life. As a community of young physicians, we would like to extend an invitation to you to return and show to us your skills and enthusiasm. We understand the competing interests you have from administration, research and education. We can devise technological and novel ways of educating in the 21st century to enable you to return. Advances in technology can aid in teaching clinical reasoning. As I walk through the hallways of the hospital, I realize what is absent… it is the “Master Clinician”. We miss you!\n\nSincerely,\n\nThe Apprentice in search of a Guru",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nAcknowledgements\n\nSincere thanks are offered to Dr. Steven Fishbane and Dr. Kedar Sankholkar for their critical comments on the manuscript."
}
|
[
{
"id": "2957",
"date": "09 Jan 2014",
"name": "Laura Maursetter",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nA very nice idea to use this avenue to encourage the master clinician to spend more time where they will make a big impact. That is great. To me this type of publication needs to be short and sweet, to keep the interest in the topic and get the reader to continue to the end. I would suggest:Combine the first two paragraphs together.Keep the third paragraph as it is written - strong.The last 3 paragraphs seem to be repetitive - I would think through the ideas you are trying to convey. Spend time, inspire learner, teach skill-set to others - there are so many examples that I feel the message gets lost.Overall, very nice. Thanks for the read.",
"responses": []
},
{
"id": "3569",
"date": "10 Feb 2014",
"name": "Ritu Soni",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nVery well written. I can identify with the ideas expressed in this article. In fact, it reminds me of my medical schooling in India, during which we focused on honing our clinical skills rather than relying on diagnostic imaging/procedures, due to limited resources. Our bedside rounds were more often than not painfully elaborate, with emphasis on physical examination. For instance, examination of the tongue used to be an hour-long lecture in itself. So were topics like variants of Babinski's reflex, types of pulses and their causes, dermatological findings in various diseases... and the list goes on. One would be fortunate to present his/her case beyond the physical examination part. But looking back, I miss that kind of learning. As much as I value my resourceful training in the U.S, I miss the mentorship of old-school clinicians, very appropriately addressed as Master Clinicians in the article. Hoping that these Master Clinicians don't go completely extinct in the future, and help us keep the art of medicine alive and vibrant.My suggestions for revision:Consider deleting the lines- \"While I support the advent of technology and the advancement of knowledge in medicine, medicine is also an art which cannot be taught by Google, Up-To-Date or Twitter. You have the experienced hands of a Master Clinician who carries the wisdom of diagnostic skills and carries knowledge.\" The same message is well conveyed in the paragraphs that follow. Consider consolidating the role of master clinicians into one paragraph and limiting the last paragraph to the 'invitation' alone, such that each paragraph conveys a distinct message.An issue of concern, very creatively drafted.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-1
|
https://f1000research.com/articles/3-55/v1
|
13 Feb 14
|
{
"type": "Commentary",
"title": "BioJS: an open source standard for biological visualisation – its status in 2014",
"authors": [
"Manuel Corpas",
"Rafael Jimenez",
"Seth J Carbon",
"Alex García",
"Leyla Garcia",
"Tatyana Goldberg",
"John Gomez",
"Alexis Kalderimis",
"Suzanna E Lewis",
"Ian Mulvany",
"Aleksandra Pawlik",
"Francis Rowland",
"Gustavo Salazar",
"Fabian Schreiber",
"Ian Sillitoe",
"William H Spooner",
"Anil S. Thanki",
"José M Villaveces",
"Guy Yachdav",
"Henning Hermjakob",
"Rafael Jimenez",
"Seth J Carbon",
"Alex García",
"Leyla Garcia",
"Tatyana Goldberg",
"John Gomez",
"Alexis Kalderimis",
"Suzanna E Lewis",
"Ian Mulvany",
"Aleksandra Pawlik",
"Francis Rowland",
"Gustavo Salazar",
"Fabian Schreiber",
"Ian Sillitoe",
"William H Spooner",
"Anil S. Thanki",
"José M Villaveces",
"Guy Yachdav"
],
"abstract": "BioJS is a community-based standard and repository of functional components to represent biological information on the web. The development of BioJS has been prompted by the growing need for bioinformatics visualisation tools to be easily shared, reused and discovered. Its modular architecture makes it easy for users to find a specific functionality without needing to know how it has been built, while components can be extended or created for implementing new functionality. The BioJS community of developers currently provides a range of functionality that is open access and freely available. A registry has been set up that categorises and provides installation instructions and testing facilities at http://www.ebi.ac.uk/tools/biojs/. The source code for all components is available for ready use at https://github.com/biojs/biojs.",
"keywords": [
"In a recent press release (http://www.nih.gov/news/health/dec2013/od-09.htm December 9th 2013) on the occasion of the naming of Dr. Philip E. Bourne as the US NIH’s first Associate Director for Data Science",
"NIH director Francis S. Collins",
"said that “the era of ‘Big Data’ has arrived",
"and it is vital that the NIH play a major role in coordinating access to and analysis of many different data types that make up this revolution in biological information”. We predict that one of Dr. Bourne’s main priorities will be dissemination and visualisation of biological data through the web. Web pages are ideal tools for the dissemination of results and data. Dynamic interactivity is crucial in the discovery process",
"particularly for data-rich applications",
"as is the case of many websites that provide interfaces to biological databases. Databases storing genomic and other types of data have proliferated in the biological sciences",
"making them a data-rich",
"data-intensive set of disciplines. The visualisation of these data plays a crucial role in their interpretation as it permits the ability to hide or to focus on a particular detail",
"enabling the researcher to shed light on specific hypotheses or to create new ones based on observed patterns. The sheer complexity of biological data",
"however",
"requires more complex technologies than the usual static pages when accessing them. They require dynamic visualisation tools to allow real-time interactions and the usability of Web 2.0-based technologies."
],
"content": "Commentary\n\nIn a recent press release (http://www.nih.gov/news/health/dec2013/od-09.htm December 9th 2013) on the occasion of the naming of Dr. Philip E. Bourne as the US NIH’s first Associate Director for Data Science, NIH director Francis S. Collins, said that “the era of ‘Big Data’ has arrived, and it is vital that the NIH play a major role in coordinating access to and analysis of many different data types that make up this revolution in biological information”. We predict that one of Dr. Bourne’s main priorities will be dissemination and visualisation of biological data through the web. Web pages are ideal tools for the dissemination of results and data. Dynamic interactivity is crucial in the discovery process, particularly for data-rich applications, as is the case of many websites that provide interfaces to biological databases. Databases storing genomic and other types of data have proliferated in the biological sciences, making them a data-rich, data-intensive set of disciplines. The visualisation of these data plays a crucial role in their interpretation as it permits the ability to hide or to focus on a particular detail, enabling the researcher to shed light on specific hypotheses or to create new ones based on observed patterns. The sheer complexity of biological data, however, requires more complex technologies than the usual static pages when accessing them. They require dynamic visualisation tools to allow real-time interactions and the usability of Web 2.0-based technologies.\n\nThe JavaScript language as implemented in browsers is today’s language for the web and has transformed modern applications into client-side browser-based. JavaScript offers common Application Programming Interfaces (API) purposely built for retrieval of remote data via RESTful services, making real-time interactivity possible. JavaScript has thus drastically changed the developer/application environment, gaining competitive advantage compared to other languages. To date, there have been successful initiatives for other languages to unify open source efforts, including BioPerl (Stajich et al., 2002), BioJava (Prlic et al., 2012) or BioRuby (Goto et al., 2010). Non-language specific communities like the GMOD (Generic Model Organism Database; http://www.gmod.org/) have also enjoyed wide adoption. These initiatives provide a centralised location in which to discover available functionality, facilitating the task of finding the desired functionality.\n\nTo cater to the burgeoning JavaScript community of developers for life sciences, we created BioJavaScript (Gómez et al., 2013). BioJavaScript, or BioJS for short, is a framework designed for the development and sharing of biological component visualisation on the web using JavaScript. BioJS provides a catalogue or registry to enable the user or developer to find existing functionality for reuse. Components in the registry show their current maintenance status, the contact name of the main author and a guide showing how to install, customise and extend the component. BioJS, however, is not just a repository with a set of libraries; it is also a standard set of minimum guidelines for developers to reutilise and create functionality in JavaScript as applied to biological concepts. BioJS thus provides a way for developers to build, extend and share functionality. Functionality in BioJS can be thought of as LEGO® pieces that can be connected to construct more complex applications.\n\n\nThe BioJS project\n\nBioJS allows developers and users alike to i) discover functionality through its registry, currently hosted at the European Bioinformatics Institute, ii) test in the registry itself the functionality of available components, iii) reuse components in different projects and applications, iv) combine components through a common set of predefined interactions and APIs, v) customise options for each component, vi) extend the functionality of available components in a standard manner - once the developer learns how to extend a component, extension of other components is consistent, vi) maintain components via community support and documentation and vii) develop new functionality following a predefined structured architecture common to all components. The typical component contains several layers of abstraction, including a style sheet, occasionally some dependencies to libraries such as D3.js, jQuery or Raphäel, the JavaScript layer and the representation layer (Figure 1a). Via common JavaScript event functions, several components can be integrated in a single web page, allowing for interactivity among them. For example, a three-component frame (Figure 1b) can have a network visualisation component, with nodes representing proteins and edges representing interactions. Similarly, an alpha helix may be highlighted in a second component on the webpage that may cause a third component to highlight where the alpha helix is located in the tertiary structure of the same protein.\n\n1a shows the different layers that a BioJS component is divided into. The representation layer sits on top of the JavaScript layer, which similarly possesses a layer of dependencies and a style. 1b presents an example of interactivity between three components, a protein-protein interaction network viewer, a secondary structure viewer and a tertiary structure viewer. Proteins in the network are represented as nodes and their interactions as edges. Clicking on a node makes the secondary and tertiary structure viewers retrieve the same protein. It is possible to select a secondary structure element in the 2D viewer and see where it is located in the 3D visualisation component.\n\n\nThe BioJS registry\n\nAt the time of writing (12-02-2014), the BioJS registry contains 39 components. The registry (http://www.ebi.ac.uk/tools/biojs/) constitutes one of the main BioJS access portals. It contains links to documentation, the community, tutorials and the list of components. A ‘components’ page in the registry contains a current list of components available. This list includes details of the component functionality, author and current version. By clicking on one of the component links, a new page appears with a series of tabs and interactive widgets to allow the user or developer to get a feel for how the component works and looks and to find details of its installation procedure, customisation and the methods that are part of the component’s architecture, dependencies and events. Event functions can be tested on the page by using the text-box field provided. This is ideal to get a quick look and feel for the component, and it is one of the most appreciated features of the BioJS project as it makes it possible for users to test a component without needing to install it.\n\nMany of the components available in the registry have been developed for particular databases. The ExpressionAtlasBaselineSummary component is an example of this (Figure 2). This component was developed for the Expression Atlas database (http://www.ebi.ac.uk/gxa/home) and subsequently deposited in the BioJS registry for reutilisation. One of the great attractive features of BioJS component visualisation is that, by sharing the component through the registry, the Expression Atlas database will ensure that other users who utilise this component will be able to enjoy a visualisation of the data exactly as intended. Having a component that can be reutilised by remote websites allows the expression data contained in this database to always be shown in the same way. This consistency in the visualisation of the same biological concepts in the same manner by different resources facilitates discovery and ease of learning by end users.\n\nShown here is the transcription profiling by high throughput sequencing of RNA from individual and a mixture of 16 types of human tissues (E-MTAB-513 – Illumina Body Map). By default the ‘Overview’ tab is selected when accessing a registry component. On the right hand panel the prospective developer can test the main event functions (e.g., onError) and methods (e.g., setQuery). Dependencies are also shown. The installation tab provides the snippets of code required to install the component. Just copying and pasting this code should be sufficient for the component to work. The remaining tabs provide a more detailed explanation on the different options, methods and events specified for this component’s usage.\n\n\nThe BioJS community\n\nThere are different degrees of involvement that users may have in BioJS. Most will utilise the web components and the registry. For those who are involved in developing biological JavaScript applications, we expect that many may find the BioJS community an attractive place to meet like-minded developers and the right environment to share their work and seek feedback and/or support. There are several mail lists currently available that reflect different levels of involvement, such as those for developers and for the Steering Committee. We also have a Twitter account (@BiojsLibrary) that informs users of news and developments. We encourage anyone interested to become involved in the way that best suits them. A number of tutorials and workshops have been organised at the European Bioinformatics Institute and elsewhere, such as the VizBi conference. We plan to organise tutorials wherever they are of service and welcome BioJS developers to become tutors whenever they wish. We have a monthly call where the Steering Committee meets, and task forces are established to reflect the needs of developers and users. Examples of task forces that have been organised include those to discuss licensing issues, compatibilities between different dependencies, and funding and usability issues. We have strong ties with the Software Sustainability Institute (SSI), a UK-based organisation that aims to provide open source community support and sustainability. SSI has recently awarded the BioJS project several months of free consultancy to make it a more robust, well-maintained and useful resource.\n\n\nDiscussion\n\nThe main motivation behind BioJS is to facilitate the creation, reutilisation and sharing of JavaScript functionality across the biological domain. BioJS provides a set of minimal common guidelines and a code architecture that makes creation of new functionality more efficient and consistent. The modular structure of components makes it possible for a web framework to isolate the visualisation aspect, and thereby facilitate maintainability. The documentation that each component is required to have is based on an automatic API generator, which transforms embedded code comments (required) into a structured document. The common architecture of components makes it possible to extend them in a consistent manner: once the developer learns how to create or extend a component, the generation or extension of new ones should be straightforward. BioJS allows developers to share the development of components by making use of the community’s support. Components, once they are developed, can also be shared through the registry, so their exposure is likely to be increased. BioJS ensures that identical biological entities are visualised in the same manner and avoids different resources displaying the same content slightly differently. This reduces end-user confusion and ensures consistency across different biological domains. Having a common way in which biological entities are shown makes it easy for users to interpret the data in a more intuitive manner. BioJS can be particularly attractive to institutions that might want to have a common “flavour” for how they visualise a particular type of data. BioJS also encourages developers to aim at developing common implementations by following common guidelines on how to implement code, and makes people aware of what components are being developed by an organisation. This may constitute a competitive advantage for showcasing development contributions carried out by a particular research group or institution.\n\n\nFuture directions\n\nBioJS is a thriving community that so far has been able to attract plenty of voluntary contributions in the same spirit as other biologically-inspired open-source communities. We aim to start a series of Google Summer of Code projects to attract students interested in developing their JavaScript skills while working on life-sciences research projects. A collaborative research project has been established with the bioinformatics consultancy Eagle Genomics to develop functionality that support visualisation of genome data without a reference assembly. Many important projects and institutions have already shown commitment to the project by developing components (i.e. SwissProt (UniProt Consortium, 2014), Genome3D (Lewis et al., 2013), Reactome (Croft et al., 2011), Expression Atlas (Petryszak et al., 2014), TGAC Browser (http://tgac-browser.tgac.ac.uk/), etc.), and the time looks ripe to take the project to a new phase. Many challenges remain, however. The BioJS project is planning to establish a sustainable future with both robust institutional and community financial support. As the number of users increases, the need for support increases, both in terms of support to help contributors deliver but also to keep track of the state of maintenance of deposited components. Currently most of the work is done by volunteers who help fix bugs and improve the quality of submitted work. BioJS has worked well as a prototype for many projects where simple components have been created. Our first-stage mission of developing a common framework has thus been achieved. Whether we are able to meet the expectations raised by the potential of the project will only be guaranteed by the explicit commitment of important players in the bioinformatics arena.",
"appendix": "Author contributions\n\n\n\nAll authors have participated in the development of the BioJS community through provision of code, meeting attendance or writing of grants.\n\n\nCompeting interests\n\n\n\nNo competing interests have been disclosed.\n\n\nGrant information\n\nWe are grateful to the NHLBI Proteomics Center Award (HHSN268201000035C), the UK’s Biotechnology and Biological Sciences Research Council (BBSRC) and European Commission grant PSIMEx (FP7-HEALTH-2007-223411).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe would like to thank Teresa K. Attwood for comments on the manuscript.\n\n\nReferences\n\nCroft D, O'Kelly G, Wu G, et al.: Reactome: a database of reactions, pathways and biological processes. Nucleic Acids Res. 2011; 39(Database issue): D691–D697. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGoto N, Prins P, Nakao M, et al.: BioRuby: bioinformatics software for the Ruby programming language. Bioinformatics. 2010; 26(20): 2617–2619. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLewis TE, Sillitoe I, Andreeva A, et al.: Genome3D: a UK collaborative project to annotate genomic sequences with predicted 3D structures based on SCOP and CATH domains. Nucleic Acids Res. 2013; 41(Database issue): D499–D507. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPetryszak R, Burdett T, Fiorelli B, et al.: Expression Atlas update--a database of gene and transcript expression from microarray- and sequencing-based functional genomics experiments. Nucleic Acids Res. 2014; 42(1): D926–D932. PubMed Abstract | Publisher Full Text\n\nPrlic A, Yates A, Bliven SE, et al.: BioJava: an open-source framework for bioinformatics in 2012. Bioinformatics. 2012; 28(20): 2693–2695. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStajich JE, Block D, Boulez K, et al.: The Bioperl toolkit: Perl modules for the life sciences. Genome Res. 2002; 12(10): 1611–1618. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUniProt Consortium: Activities at the Universal Protein Resource (UniProt). Nucleic Acids Res. 2014; 42(1): D191–D198. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "3819",
"date": "20 Feb 2014",
"name": "Philip E. Bourne",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI am supportive of the BioJS registry with regards to the technology used and its principles. To give you a flavour of what I will be looking for, from an NIH perspective, is really how much the components are used and why. What findings have resulted in their use? If there is non-use of components and why etc. In short, I will be looking more at the degree of software projects that are making a difference to the scientific community. That said, I very much look forward to seeing how the BioJS community develops.",
"responses": []
},
{
"id": "3706",
"date": "17 Mar 2014",
"name": "Reinhard Schneider",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nVisualization is one of the bottlenecks we are facing in modern biology. With bigger and bigger data sets originating from various heterogeneous sources that span several concepts, dimensions and timelines, it is becoming an art to condense the wealth of information into good and easy ways to capture visual pictures.For non-expert users it can be quite a challenge to come up with a \"publication ready\" visualization of complex relationships. The BioJS community tries to tackle this challenge by employing an open modular architecture, which allows a broad audience to use and further develop the components in the registry. The community already provides a wide spectrum of available components and it will be very interesting to see the future development of this open source community.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-55
|
https://f1000research.com/articles/3-54/v1
|
13 Feb 14
|
{
"type": "Software Tool Article",
"title": "DNAContentViewer a BioJS component to visualise GC/AT Content",
"authors": [
"Anil S. Thanki",
"Shabhonam Caim",
"Manuel Corpas",
"Robert P. Davey",
"Shabhonam Caim",
"Manuel Corpas",
"Robert P. Davey"
],
"abstract": "Summary: Compositional GC/AT content of DNA sequences is a useful feature in genome analysis. GC/AT content provides useful information about evolution, structure and function of genomes, giving clues about their biological function and organisation. We have developed DNAContentViewer, a BioJS component for visualisation of compositional GC/AT content in raw sequences. DNAContentViewer has been integrated in the BioJS project as part of the BioJS registry of components. DNAContentViewer requires a simple configuration and installation. Its design allows potential interactions with other components via predefined events. Availability: http://github.com/biojs/biojs; doi: 10.5281/zenodo.7722.",
"keywords": [
"DNA"
],
"content": "Introduction\n\nGeneral compositional features of DNA sequences (e.g., GC/AT content) are important to understand the evolution, structure and function of genomes1. Genes are often characterised by having a higher GC content than the rest of the genome. It has also been shown that the length of the coding sequence is directly proportional to a higher GC content2. Stop codons, however, have a bias towards A and T nucleotides and are thus indicative of shorter gene sequences3.\n\nGC content is usually expressed as a percentage value and sometimes as a ratio (GC ratio). The AT and GC content percentages and ratios can be measured in different ways (e.g., simple arithmetic or using a free online AT/GC calculator4).\n\nTo calculate the GC content percentage the following formula can be used5:\n\n\n\nThe AT content percentage can be calculated in a similar manner:\n\n\n\n\nThe DNAContentViewer component\n\nDNAContentViewer has been developed as a part of the BioJS project6. It is available via GitHub or the BioJS registry7, a centralised repository of BioJS components hosted at the European Bioinformatics Institute (EBI).\n\nDNAContentViewer uses the JavaScript library D3.js8 for generating graphs. It reads the DNA sequence in raw format and plots it as a Scalable Vector Graphics (SVG) object. To our knowledge, this is the first client-side modular BioJS component that visualises GC/AT content directly from a raw sequence.\n\nThe way DNAContentViewer works is by calculating GC/AT content for dynamically generated slices of the sequence. This dynamic feature makes it fully scalable, working with sequences of any length. DNAContentViewer represents AT and GC content in separate graphs, allowing toggling of both to show and hide.\n\nDNAContentViewer shows a percentage marker, which can be set on the left (by default), in the middle or on the right side of the graph. To adjust the visual aspect of the DNA content, DNAContentViewer contains simple controls for zooming and panning. DNAContentViewer can interact with other components via events and methods. Both events and methods are demonstrated through the component’s page in the BioJS registry. Following the BioJS standard recommendations, documentation is available giving more details on how to implement the extension of the component via the registry.\n\nThe code below shows how to initialize DNAContentViewer with some minimal input. The name given to YourOwnDivId constitutes the component container identifier and file is defined absolute or relative path to the file containing the raw DNA sequence.\n\n\n\nAn example is shown in Figure 1, depicting GC/AT content from a genomic sequence in scaffold 1 of an assembly of the Chinese Hamster Ovary (CHO) genome. The GC/AT density data calculated from the raw sequence file are presented as bins, where the Y-axis indicates the percentage of GC/AT content for each bin. The overall AT content is higher than the GC content. The 13K region shows a much higher AT content than average. This may be indicative of a stop codon and a poly A tail present in the region. The figure also shows a dramatic change in the GC/AT content in the 23K-32K and 284K regions, showing that in these locations GC/AT content does not sum up to 100% of the total base content. This is indicative of regions that contain unidentified nucleotides (X or N bases) instead of A, T, G or C.\n\nThe top controls are designed to scroll sideways and to zoom; radio buttons can be used to set the position of the marker to the left (default), centre or to the right. Checkboxes can be used to toggle the GC/AT graph. Peaks show the GC/AT percentage density of dynamically generated bins. Differences can be observed around the 13K mark, with greater density of AT content, indicative of a stop codon and a poly A tail.\n\n\nConclusion\n\nThe DNAContentViewer component provides the functionality to visualise GC/AT content of DNA sequences within the BioJS framework. DNAContentViewer can be easily integrated with other BioJS web components or extended to provide new functionality. Like any other BioJS component, DNAContentViewer requires little technical knowledge for its reutilisation.\n\n\nSoftware availability\n\nZenodo: DNAContentViewer: a BioJS component to visualise DNA Content, doi: 10.5281/zenodo.77229.\n\nGitHub: BioJS, http://github.com/biojs/biojs.",
"appendix": "Author contributions\n\n\n\nAT developed the code for DNAContentViewer and AT, MC and RD wrote the paper. SC designed the algorithm and wrote the initial version.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nAT, SC, MC and RD were supported by the UK Biotechnology and Biological Sciences Research Council (BBSRC) National Capability Grant (BB/J010375/1) at TGAC.\n\n\nAcknowledgements\n\nWe are grateful to all BioJS developers who have contributed their work under an open source license.\n\n\nReferences\n\nGao F, Zhang CT: GC-Profile: a web-based tool for visualizing and analyzing the variation of GC content in genomic sequences. Nucleic Acids Res. 2006; 34(Web Server issue): W686–W691. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPozzoli U, Menozzi G, Fumagalli M, et al.: Both selective and neutral processes drive GC content evolution in the human genome. BMC Evol Biol. 2008; 8(1): 99. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWuitschick JD, Karrer KM: Analysis of genomic G + C content, codon usage, initiator codon context and translation termination sites in Tetrahymena thermophila. J Eukaryot Microbiol. 1999; 46(3): 239–247. PubMed Abstract | Publisher Full Text\n\nWikipedia. GC-content. [Online; accessed 27–Dec-2013]. Reference Source\n\nMadigan MT, Martinko JM, Parker J: Brock, Biologia de Los Microorganismos (Spanish Edition). Pearson Educacion. 2004. Reference Source\n\nGómez J, García LJ, Salazar GA, et al.: BioJS: an open source JavaScript framework for biological data visualization. Bioinformatics. 2013; 29(8): 1103–1104. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBioJS: registry. http://www.ebi.ac.uk/Tools/biojs/registry/, 2013. Reference Source\n\nD3.js data-driven documents. http://d3js.org, 2012. Reference Source\n\nThanki AS, Caim S, Corpas M, et al.: DNAContentViewer: a BioJS component to visualise DNA content. Zenodo. 2014. Data Source"
}
|
[
{
"id": "4051",
"date": "24 Mar 2014",
"name": "Dan Schwartz",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThanki et al. describe DNAContentViewer, a component of the BioJS collection aimed at visualizing GC and AT content in a string of nucleotides. While we are wholly supportive of the BioJS project, we have some important concerns with the DNAContentViewer manuscript/tool as it presently stands.ManuscriptThe manuscript starts with an introduction that is vague and difficult to read. The first sentence sets up the importance of GC content, but references an alternate web tool for visualizing GC content (a primary reference should be used). It appears that the authors took the remainder of their introduction directly from an inaccurate and poorly worded Wikipedia entry (complete with the identical references).Compare the Wikipedia entry for \"GC-content\":\"Within a long region of genomic sequence, genes are often characterised by having a higher GC-content in contrast to the background GC-content for the entire genome. Evidence of GC ratio with that of length of the coding region of a gene has shown that the length of the coding sequence is directly proportional to higher G+C content (Pozzoli et al., 2008). This has been pointed to the fact that the stop codon has a bias towards A and T nucleotides, and, thus, the shorter the sequence the higher the AT bias (Wuischick et al., 1999)\".to the DNAContentViewer introduction:\"Genes are often characterised by having a higher GC content than the rest of the genome. It has also been shown that the length of the coding sequence is directly proportional to a higher GC content (Pozzoli et al., 2008). Stop codons, however, have a bias towards A and T nucleotides and are thus indicative of shorter gene sequences (Wuischick et al., 1999).\"The authors then go on to cite Wikipedia itself in the following sentence:\"The AT and GC content percentages and ratios can be measured in different ways (e.g., simple arithmetic or using a free online AT/GC calculator (improper reference to Wikipedia \"Talk\" tab)).\"Wikipedia version:\"the GC-content can be accurately calculated by simple arithmetic or by using the free online GC calculator (web link to http://www.basic.northwestern.edu/biotools/oligocalc.html).\"Finally, for reference 5 in the manuscript (the reference to the GC content formula), the authors again use the same reference as noted in the Wikipedia entry (Brock Biology of Microorganisms, 2003), but this time deciding to go with the Spanish Edition (Brock, Biologia de Los Microorganismos, 2004).Other concerns with the manuscript:Despite the limited computation necessary for creating the visualization, the authors have omitted some key information that is essential to understanding how the tool works. For example, the manuscript does not describe how the window size for the calculation is determined. This is particularly important given that it is not a parameter of the existing tool. How was the bin size chosen? How will shorter sequences be affected (i.e., is there a recommended lower bound for use)? Why were bins used rather than a sliding average? While we were able to partially answer some of these questions by digging through the code base, this is important information for users and should be described in the manuscript.The example given in Figure 1 suggests that the 13K mark increase in AT content \"may be indicative of a stop codon and a poly A tail\". First, it is impossible to discern 2-3 nucleotides in a bin size of 4,600 (the authors should note that the 3' UTR typically sits between the stop codon and poly(A) tail in eukaryotes). Second, is there a poly(A) tail in this region? The authors should either definitively answer this question, or preferably use a real example that highlights the utility of the tool in answering a real biological question (i.e., how does this visualization allow us to see something that is difficult to detect otherwise).Code/ToolThe DNAContentViewer component code is overall well-organized and well-documented. We were able to get a working demo up and running on a local server in roughly 20 minutes. That said, several issues should be corrected:If one accesses the demo page at https://www.ebi.ac.uk/Tools/biojs/registry/Biojs.DNAContentViewer.html via HTTPS (which seems to be the default if you get there via Google) the demo will not load due to D3.js being loaded via HTTP and therefore blocked on HTTPS pages.The provided <script> and <link> tags in the installation instructions (http://www.ebi.ac.uk/Tools/biojs/registry/Biojs.DNAContentViewer.html#) contain relative URLs. Changing these to absolute URLs would make installation quicker and simpler.The provided <script> tags include jQuery UI before including jQuery itself, causing a “ReferenceError: jQuery is not defined” error in the JavaScript console of both Chrome and Firefox. Although it seems this error does not prevent the component from working, there is no need for it to be there. Simply switching the order of the <script> tags removed this error.When using the viewer, two errors are thrown in the console whenever a user clicks on the left control arrow. The component still appears to work normally despite these errors and the graph will still shift to the left and render (assuming the user is not already as far left as the graph can go).At most zoom levels, panning left and right actually changes the resultant GC and AT lines (i.e., they do not stay fixed). This seems to be an issue with how the system chooses to bin the data upon panning, and should be corrected. Additionally, a more fluid panning and zooming capability would be preferable (e.g., panning by dragging and zooming with a slider).",
"responses": []
},
{
"id": "4001",
"date": "13 May 2014",
"name": "Matúš Kalaš",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHere, the authors publish the BioJS component DNAContentViewer JavaScript widget for visualizing the GC and AT content of an input DNA sequence. This widget is part of the BioJS community effort which I strongly support.ArticleThe title and the abstract are appropriate. The rest of the article is short, what I certainly appreciate. It has, however, both some major and some minor issues.“raw sequence(s)” is an unnecessary expression, and possibly misleading.A more appropriate reference than Gao et al. 2008 for the importance of GC content should be included.Reference number 4 is inappropriate, not because it is Wikipedia, but because it does not point to the mentioned calculator or to a proper Wikipedia article.Reference number 5 is unnecessary and not particularly useful.The window for the content calculations is not described.The algorithm is not described.The events and methods of DNAContentViewer are listed in the BioJS registry, but I am not convinced that they are demonstrated as is claimed.I am not convinced that details on how to implement an extension of this component are given in its documentation.“This is indicative of regions that contain unidentified nucleotides (X or N bases) instead of A, T, G or C.” from a practical point of view is a somewhat ridiculous sentence. The purpose of the visualization is not to enable users to hypothesize about unidentified nucleotides which are obvious from the input sequence. Instead, the unidentified nucleotides should be visualized within the graph, in order to enable users to focus on the other, relevant indications.\"DNAContentViewer can be easily integrated with other BioJS web components or extended to provide new functionality\" isn’t really justified. The following sentence may also not be.It would be nice if the DNAContentViewer were used in production, and in that case a production usage example were mentioned in the article. Production usage would serve crucial feedback to the design of the component. On the other hand, I understand that despite its usefulness, production usage is not essential for designing a good component.WidgetDNAContentViewer is a very simple JavaScript component for visualisation of GC and AT content of an input DNA sequence. I can imagine scenarios where its simplicity would be appreciated. In its current state, I would consider it a prototype, as I am not sure it is completely ready for publication. To consider the DNAContentViewer ready for production, I suggest addressing a substantial part of the following points:Despite its simplicity, it is quite slow when zooming or panning.Zooming in is not functional below max resolution but still requires all the clicks to zoom out.Unnecessary animation when changing the y axis position, while zooming and panning is far from convenient.It would be much more usable with mouse wheel to zoom (and centralize), and mouse drag to pan. See e.g. http://mbostock.github.io/d3/talk/20111018/area-gradient.html.Reasonable positions on the x axis would be more useful, instead of random-looking ones like now, and not only in kbp range. They would ideally only extend and scroll when zooming or panning, but not change otherwise.Maybe show the sequence when zoomed-in enough.The 'nnnnn' bits of the sequence should be shown in the view.An option for inverting the y axis upside-down for one of the GC or AT would be useful.Selection of a subsequence by dragging mouse over a different part of the widget than for panning (e.g. at the x axis area while above the axis would pan - or vice versa - in any case indicated by cursor change).Enable zooming in to see the subsequence selected with mouse dragging.Showing the used sliding window size as a scale, and maybe even allowing customizing it if that would be useful.The sharp edges of the graphs – or the sparsity of points with values if that is the case – look a bit suspicious to me. What are the ‘bins’ and where do they come from? And could they be shown in the visualization, e.g. like in http://mbostock.github.io/d3/talk/20111018/area-gradient.html?Details of the input format are not documented (e.g. character case and allowed characters).Enabling more sequence formats, including a JS string without the need of a file.I don't understand the 'selection' options. Which selected text do they concern, one on the axes or in the \"menu\" (why is that useful to do)?Example of integration with another BioJS widget, naturally for example BioJS Sequence or BioJS FeatureViewer.It would be nice to enable at least testing with another sequence, without a need for local installation of Apache etc.BioJS registryI don’t know how involved the authors are in the design and the future evolution of the BioJS registry and BioJS itself, and although this may thus not be a suitable place, I have a couple of general suggestions related to the BioJS registry:I haven't found an easier way to view the source code of the components, that would be without the need for copying & pasting pieces of the URL, or navigating through GitHub.I find pagination at http://www.ebi.ac.uk/Tools/biojs/registry/components.html both useless and inconvenient. In addition it didn't always work well for me with sorting by a column versus the pagination.Examples of integration of multiple components, such as the Sequence and Protein3D example that is only described in the spec - or the even fancier example at http://cdn.f1000r.com.s3.amazonaws.com/manuscripts/3850/22cef3a8-64b5-4189-8c1c-71b545883363_figure1.gif - would be both useful, and justifying the interoperability claims of BioJS. It would be awesome to have an integrated example for each BioJS component easily accessible from the registry.I'd love being able to test out at least such simple components as this Biojs.DNAContentViewer in my own HTML, without having to install Apache with PHP locally. Is that possible? If it isn't due to the BioJS itself, at least some interpreter like http://www.w3schools.com/js/tryit.asp?filename=tryjs_lib_jquery would be very nice to have.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-54
|
https://f1000research.com/articles/3-52/v1
|
13 Feb 14
|
{
"type": "Web Tool",
"title": "Sequence, a BioJS component for visualising sequences",
"authors": [
"John Gomez",
"Rafael Jimenez",
"Rafael Jimenez"
],
"abstract": "Summary: Sequences are probably the most common piece of information in sites providing biological data resources, particularly those related to genes and proteins. Multiple visual representations of the same sequence can be found across those sites. This can lead to an inconsistency compromising both the user experience and usability while working with graphical representations of a sequence. Furthermore, the code of the visualisation module is commonly embedded and merged with the rest of the application, making it difficult to reuse it in other applications. In this paper, we present a BioJS component for visualising sequences with a set of options supporting a flexible configuration of the visual representation, such as formats, colours, annotations, and columns, among others. This component aims to facilitate a common representation across different sites, making it easier for end users to move from one site to another.Availability: http://www.ebi.ac.uk/Tools/biojs; http://dx.doi.org/10.5281/zenodo.8299",
"keywords": [
"Visualising biological data on the web is a common practice on sites providing bio-oriented services and resources. A wide variety of JavaScript libraries are being used to build pieces of software capable of representing bio-entities such as DNA sequences1",
"protein sequences (http://www.uniprot.org)",
"protein structures (http://www.wwpdb.org)",
"ontology trees2",
"protein-protein interactions (http://www.ebi.ac.uk/intact/)3",
"and others. Therefore",
"a variety of possible visual representations for the same bio-entity can be found as a result of its multiple implementations. In many cases",
"such implementations are difficult to maintain",
"test",
"and reuse as they are developed only with one use case in mind. Furthermore",
"user experience (UX) and usability across different sites may be compromised."
],
"content": "Introduction\n\nVisualising biological data on the web is a common practice on sites providing bio-oriented services and resources. A wide variety of JavaScript libraries are being used to build pieces of software capable of representing bio-entities such as DNA sequences1, protein sequences (http://www.uniprot.org), protein structures (http://www.wwpdb.org), ontology trees2, protein-protein interactions (http://www.ebi.ac.uk/intact/)3, and others. Therefore, a variety of possible visual representations for the same bio-entity can be found as a result of its multiple implementations. In many cases, such implementations are difficult to maintain, test, and reuse as they are developed only with one use case in mind. Furthermore, user experience (UX) and usability across different sites may be compromised.\n\nOne particular type of data commonly affected by multiple representations is the sequence, either a DNA or protein sequence. A sequence is a common bio-entity present in most sites offering biological data resources. Figure 1 shows different visual representations of a protein sequence as it can be found in Uniprot (http://www.uniprot.org), Dasty4 (http://www.ebi.ac.uk/dasty) and Ensembl (http://www.ensembl.org), among others5,6. Multiple features are identified across the entire set of sequences. Features such as formatting, indexing numbers, annotations, marks, colouring tags, and even the capability of user interaction are not integrated in one reusable piece of code. Instead, multiple representations prevail. Furthermore, web developers often make their own isolated efforts to reproduce those views for their sites and, in most cases, the representation is not identical, no documentation is available, and often they are not portable to other sites.\n\nIn this paper, a reusable component to visualise sequences is presented under the BioJS set of minimum standards for visualisation of biological components. BioJS is a community-driven standard to develop visualisation functionality7. The library is developed using well-established methodologies and object-oriented design with inheritance that facilitates rapid development, reuse, extension, integration and deployment of web applications.\n\n\nThe Sequence component\n\nExploring sequence visualisation across different sites reveals a set of features that should be supported by a single, reusable, and well documented piece of code, capable of painting sequences on the web in a consistent manner. In this sense, BioJS provides a baseline for Javascript coding and development to create pieces of reusable code, called components. Creating a new Sequence component consists of extending a core BioJS class and defining three core concepts: options, methods and events. Options are the data required by the component for initialisation, while methods and events are actions supported in execution time. Methods are fired externally while events are triggered in the component and exposed to external listeners.\n\nMethods and events allow the component to communicate with others components as well as web applications. Figure 2 shows a working example implemented within the Biotea project8. This example shows a communication between two component instances, the Sequence component and the Protein3D component. When a region (highlighted in yellow) on the sequence is selected, automatically a selection action is fired in the Protein3D. Additionally, Sequence supports a set of options to change the visual representation of the sequence by using different formats, colours, indexing numbers, annotations and more. It helps deployment because the component can be easily fitted to the particular need. Figure 3 shows an example of the Sequence component displaying the protein P918283 in CODATA format.\n\nThe part highlighted in yellow denotes the current selection, the black pop-up box indicates what the interval is with every move of the pointer. Green highlight denotes an annotation on that interval. Multiple annotations are supported.\n\nAs any other BioJS component, the Sequence component is well documented and has been tested during development, not only for functionality but also for usability. BioJS makes it easier to document the code by adding annotations that are later exposed as a web page. Thus, human-friendly documentation is generated without any additional effort. BioJS web pages for components are compiled in a registry that acts as a showcase of working examples extracted from the component annotations. The registry makes it easier for both developers and end users to understand components and their functionality. Once a component has met the BioJS guidelines, it becomes a candidate to be submitted and publicly shared in the common repository of components, the EBI BioJS registry (http://www.ebi.ac.uk/Tools/biojs/registry/). There, it is possible to find more information about options, installation, methods, and events (http://www.ebi.ac.uk/Tools/biojs/registry/Biojs.Sequence.html).\n\n\nFuture work\n\nCurrently, the Sequence component supports the visualisation of a single strand. However, in some cases, it should be more interesting to display similarities between two or multiple sequences. Another possible extension is using this component as a base for multiple aligned sequences visualisation. Aligner algorithms9 could be run on the server side or consumed from a web service10 while the component would be in charge of painting the similarities, taking advantage of already developed features such as colouring, highlighting, and tagging.\n\nCollaborative work and social networking is nowadays a mechanism for knowledge construction. Such features can be integrated into the Sequence component so end users can submit sequences and annotations to public sequence databases such as UniProt. Comments and references could also be added, adding valuable information for a researcher during his/her investigation.\n\n\nSoftware availability\n\nZenodo: Sequence BioJS component for visualising sequences, doi: 10.5281/zenodo.829911.\n\nGitHuB: BioJS, http://www.ebi.ac.uk/Tools/biojs.",
"appendix": "Author contributions\n\n\n\nThe work presented here was carried out in collaboration between both authors. RJ collected the component requirements across several EBI teams and collaborated with JG in the visual design, UX and usability tests. JG implemented all functionality in JavaScript following the guidelines of BioJS. This manuscript was written and revised by both authors.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nNHLBI Proteomics Center Award HHSN268201000035C.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors thank Henning Hermjakob for his support to the project, and Leyla Garcia for his comments on the component. We also acknowledge Sangya Pundir for helpful UX and usability testing and invaluable feedback.\n\nThe authors thank all researchers who have deposited information into publically available datasets as well as developers who have provided their work as open source: our work stands upon their shoulders and would not have been possible without them.\n\n\nReferences\n\nRutherford K, Parkhill J, Crook J, et al.: Artemis: sequence visualization and annotation. Bioinformatics. 2000; 16(10): 944–945. PubMed Abstract | Publisher Full Text\n\nCote RG, Jones P, Apweiler R, et al.: The Ontology Lookup Service, a lightweight cross-platform tool for controlled vocabulary queries. BMC Bioinformatics. 2006; 7(1): 97. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKerrien S, Aranda B, Breuza L, et al.: The IntAct molecular interaction database in 2012. Nucleic Acids Res. 2012; 40(Database issue): D841–D846. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVillaveces JM, Jimenez RC, Garcia LJ, et al.: Dasty3, a WEB framework for DAS. Bioinformatics. 2011; 27(18): 2616–2617. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIlyin VA, Pieper U, Stuart AC, et al.: ModView, visualization of multiple protein sequences and structures. Bioinformatics. 2003; 19(1): 165–166. PubMed Abstract | Publisher Full Text\n\nO'Shea JP, Chou MF, Quader SA, et al.: pLogo: a probabilistic approach to visualizing sequence motifs. Nat Methods. 2013; 10(12): 1211–1212. PubMed Abstract | Publisher Full Text\n\nGómez J, García LJ, Salazar GA, et al.: Biojs: an open source JavaScript framework for biological data visualization. Bioinformatics. 2013; 29(8): 1103–1104. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGarcia A, Garcia LJ, Gómez J: Conceptual exploration of documents and digital libraries in the biomedical domain. In volume 952 of Semantic Web Applications and Tools for Life Sciences,. Adrian Paschke et al., editor. 2012, Springer: France. Reference Source\n\nLi H, Homer N: A survey of sequence alignment algorithms for next-generation sequencing. Brief Bioinform. 2010; 11(5): 473–483. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLarkin MA, Blackshields G, Brown NP, et al.: Clustal w and clustal x version 2.0. Bioinformatics. 2007; 23(21): 2947–2948. PubMed Abstract | Publisher Full Text\n\nGomez J, Jimenez R: Sequence BioJS component for visualising sequences. Zenodo. 2014. Data Source"
}
|
[
{
"id": "3694",
"date": "17 Mar 2014",
"name": "Christoph Gille",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneralThe authors present the first re-usable JavaScript based sequence component. It can be used in web applications dealing with bio-polymers like proteins and nucleotide sequences and can also interact with other parts of the website via events. Previously, Java applets have been used for interactive web content. However, Java constitutes an additional layer of software and thereby carries an own set of technical problems and risks. For this reason,JavaScript is being increasingly used at the client side. In this respect, the development of BioJS components follows a general trend.The BioJS registry is the first and only framework plus standard for interactive web components, and Sequence will be one of the most important components following the BioJS specification. Therefore, I expect that the Sequence component will be widely used in bioinformatics web services. Even if current features might not satisfy all needs, the BioJS format allows for extensions and incorporation of new features with the source code clear and well documented, allowing developers to change it to their requirements.ManuscriptI would suggest replacing the word \"compiled\" with another word (in the figure 1 legend and in the third paragraph of the Sequence component section) as it might be mistaken for source code getting compiled on a server like on the Debian Linux server.The manuscript does not provide answer to some important questions:Is the length of the sequence limited?Is the sequence immutable? Or could it change like alternative splicing? Can parts of the sequences be hidden like cutting of signal peptide?\"Indexing numbers\" - does the numbering support PDB insertion codes?It would be good if these points could be clarified in the manuscript.ExampleFor demonstration, the authors have coupled the sequence view with a BioJS 3D component.With the newest Java, the JMol applet fails to start with the message: \"Your security system has blocked an untrusted ...\". I expect that the line Permissions: sandbox in the jar-file manifest and signing the jar-file will fix the problem.The authors should also consider using a JavaScript based 3D visualization.APIOn events like 'Annotation Clicked', there is no parameter indicating whether the context pop-up trigger (right click, long touch) is active and what modifier keys like Shift and Ctrl are pressed - this should be made clearer for ease of use.",
"responses": []
},
{
"id": "3802",
"date": "17 Mar 2014",
"name": "Jeremy Goecks",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHere, the authors present Sequence, a web-based visualization component for biological sequence data implemented in JavaScript. Investigators can use Sequence to visualize both DNA and protein sequences, either as a standalone visualization or together with other visualizations.Strengths of Sequence include (a) the ability to customize sequence using options and (b) integration of sequence via events. These features ensure that Sequence can be used in a wide variety of applications.What is missing from this manuscript is a description of how well Sequence scales to large sequences and whether a Sequence visualization can be updated dynamically in response to events from other components.Overall, Sequence is a solid contribution to web-based visualization that is useful as it is and forms the foundation for more complex web-based sequence visualization in the future.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-52
|
https://f1000research.com/articles/3-51/v1
|
13 Feb 14
|
{
"type": "Web Tool",
"title": "BioJS DAGViewer: A reusable JavaScript component for displaying directed graphs",
"authors": [
"Alexis Kalderimis",
"Radek Stepan",
"Julie Sullivan",
"Rachel Lyne",
"Michael Lyne",
"Gos Micklem",
"Alexis Kalderimis",
"Radek Stepan",
"Julie Sullivan",
"Rachel Lyne",
"Michael Lyne"
],
"abstract": "Summary: The DAGViewer BioJS component is a reusable JavaScript component made available as part of the BioJS project and intended to be used to display graphs of structured data, with a particular emphasis on Directed Acyclic Graphs (DAGs). It enables users to embed representations of graphs of data, such as ontologies or phylogenetic trees, in hyper-text documents (HTML). This component is generic, since it is capable (given the appropriate configuration) of displaying any kind of data that is organised as a graph. The features of this component which are useful for examining and filtering large and complex graphs are described.Availability: http://github.com/alexkalderimis/dag-viewer-biojs; http://github.com/biojs/biojs; http://dx.doi.org/10.5281/zenodo.8303.",
"keywords": [
"The graph abstract data type is an important concept in mathematics and computer science",
"and is the most appropriate representation for several classes of real world phenomena and scientific constructions. Some examples of these include phylogenetic trees",
"protein-protein interaction networks and scientific ontologies such as the Gene Ontology1 and the Sequence Ontology2. One feature of this type of data structure is that they are much easier for humans to understand when presented as a graphical network which preserves the structured nature of the data",
"than when they are displayed flattened in tabular or list format. The component described here is capable of displaying graphs of data",
"in particular Directed Acyclic Graphs (DAGs)",
"efficiently using JavaScript to calculate the layout",
"and features of modern web browers for rendering",
"and is designed to integrate with other components in the BioJS3 project."
],
"content": "Introduction\n\nThe graph abstract data type is an important concept in mathematics and computer science, and is the most appropriate representation for several classes of real world phenomena and scientific constructions. Some examples of these include phylogenetic trees, protein-protein interaction networks and scientific ontologies such as the Gene Ontology1 and the Sequence Ontology2. One feature of this type of data structure is that they are much easier for humans to understand when presented as a graphical network which preserves the structured nature of the data, than when they are displayed flattened in tabular or list format. The component described here is capable of displaying graphs of data, in particular Directed Acyclic Graphs (DAGs), efficiently using JavaScript to calculate the layout, and features of modern web browers for rendering, and is designed to integrate with other components in the BioJS3 project.\n\nTwo commonly used approaches for representing graphs in two dimensions, allowing display in HTML documents, are the force-directed layout, and the Sugiyama layout. These differ in the way that they represent the hierarchical organisation of elements within a graph, and are each suitable for different kinds of data.\n\nForce-directed layouts distribute nodes throughout the available co-ordinate space, placing related nodes closer to each other, and unrelated nodes further away from each other. A typical method of achieving this is to model the layout as a two-dimensional particle simulation, where nodes exert a repulsive force upon each other, and edges between nodes exert an attractive force. Stable layouts are those representing local energy minima of the simulation.\n\nThis method is straightforward to implement (see Cytoscape4 and D3 project5 for example JavaScript implementations), and is a suitable representation of graphs where we care more about the existence of edges than their directions, and more about identifying clusters of nodes than elucidating the internal structure of such clusters. For example in a protein-protein interaction network (see Figure 1) force-directed layouts are often used since they are good at indicating highly connected interactors and clusters of interactors, thus highlighting centrally significant parts of the graph.\n\nThe other commonly-used approach to rendering graphs visually is Sugiyama-style graph drawing6. This method also attempts to group related elements, but in addition it assigns significance to the structure of relationships by introducing the concept of root and rank. Rank is defined as the number of edges in the shortest path from a node to a root. A root is defined as a node of rank 0. When rendered, nodes of the same rank within a graph are aligned visually, either horizontally or vertically, producing a structured hierarchical layout of the graph.\n\nThis method requires edges to have a direction that indicates which side of the relationship is closer to the root. Such graphs are typically described as trees, and the nodes furthest from the root as leaves. This kind of representation is suitable for graphs in which the structure of relationships is important, which is a feature of several types of graphs, such as ontologies, and phylogenetic trees. Singly rooted, acyclic trees are the most straightforward structures to lay out and display, but this method can be applied to multiply rooted directed graphs with cycles (such as biochemical pathways).\n\nUntil now, using the Sugiyama method has required the generation of image files, either on demand or through batch preprocessing, and then sending them out over the network to a suitable display device. Several tools exist for this purpose, including GraphViz7, which is used by several projects for rendering Gene Ontology graphs. This requires any group wishing to employ this method for graphical network analysis to have access to the resources and expertise to manage either a server capable of dynamically generating such images, or to produce the images required in advance. In either case, user interaction is very limited.\n\nWhat is new about the DAGViewer component is the use of a JavaScript Sugiyama layout engine to eliminate the image file generation, which cannot be done within a browser. Modern web browsers have advanced to the point where it is now practicable to calculate layouts for graphs of moderate size (in the order of around 200 to 500 nodes, depending on the density of connections) and render them in a dynamic hyper-text page, using tools such as JavaScript and Scaled Vector Graphics (SVG). This accounts for the great majority of networks that one might want to visualise, particularly since networks of greater information densities are very difficult for humans to interpret when rendered. We have taken advantage of the opportunity afforded by modern browser tools to produce a generic network display tool that does not require any server-side resources, and that is suitable for a variety of scientific purposes. This approach provides a much greater degree of customisation, interaction and flexibility than approaches based on image generation.\n\nThe graph viewer presented here uses a collection of publically available, open-source JavaScript tools, including the Backbone8 framework, the dagre-d39 layout engine, and D35 data-binding and presentation library. The combination of these tools make it possible to build a tool in JavaScript and running in modern browsers that provides rich interaction and graphical analysis possibilities, allowing users to focus on the data, e.g. in the Gene Ontology Annotation displayer in Figure 2.\n\nThe current implementation allows a JavaScript component to be placed on any page and be provided with any kind of linked network data; the data are rendered to the screen in the familiar box and line style of a Sugiyama graph drawing. Unlike static images, this graph can be zoomed, panned, reorientated and rescaled, allowing users to make sense of dense networks. Since the graph is rendered with SVG technology, rescaling does not lower graphical resolution, and text legibility is preserved over a wide range of zoom levels.\n\nThe user can interact more deeply with this representation than they could with a standard fixed image. Individual nodes and edges can each have their own styles and behaviour, allowing contextual tooltips and mousehover effects to provide information even when zoomed out. Since the information composing the graph is available to the page at runtime as a data-structure, it can be searched and filtered, and the graph can been zoomed and scaled to highlight particular nodes and edges that interest the user.\n\nA control panel element (see Figure 2) provides access to this functionality, allowing users to search for nodes within the graph, and filter the graph to focus on relevant sub-sets of the available information. Figure 3 illustrates the display of one particular subgraph of the information presented in Figure 2, reorientated to make the best use of the available screen space. This particular subgraph is defined as those nodes reachable from one particular high-level ontology term, developmental process.\n\nAs a BioJS JavaScript component, the intended audience is web developers aiming to provide functionality for life scientists. It is expected to be deployed within HTML pages and rendered in modern browsers. As such, installation means indicating which resources a page needs to load. The DAGViewer tool is a modular javascript component, making use of other existing resources (Supplementary materials A); these dependencies need to be included on the page before the component itself can be used. Once these are loaded the BioJS DAGViewer component itself can be included (see code sample 1). This should be downloaded from the BioJS repository10 and hosted locally.\n\nListing 1. Loading the DAG-Viewer Library\n\n\n\nWith these elements available, a user is then able to instantiate a new DAGViewer component pointing at a defined element in the document object model (DOM), or page:\n\nListing 2. Instantiating a new DAGViewer Component\n\n\n\nThere are a large number of configurable parameters that can be provided at instantiation (or indeed, later). These mostly relate to configuring how to interpret the graph data provided. It is accepted that data may come in different formats, and rather than requiring users to convert their node and edge data to a predefined format, users can provide adapters that allow this component to read and display different kinds of graph data, while providing sensible defaults. More detail is provided on the BioJS registry documentation pages, but as an example consider a graph (representing a protein interaction network) which has nodes of the form:\n\nListing 3. Example Nodes\n\n\n\nHere we will want to identify each node by its accession number (here from Uniprot) and label it by its name, if it has one, or by its accession if it does not. This behaviour can be defined by passing a couple of parameters:\n\nListing 4. Node Adaptor Example\n\n\n\nHere the nodeLabels parameter indicates which fields should be read to obtain a label for this node, and the nodeKey parameter is a function that takes a node and returns an identifier (possibly computed). Similar configuration options exist for interpreting edges, determining the list of graph roots, providing style classes to nodes and edges and other functions.\n\nOnce configured, the component must be given the definition of the graph it is meant to visualise. A graph here is defined as two collections, one of nodes, and the other of edges between nodes. These can be unconnected data structures, such as loaded from JSON files, without interior references, or they may be circular self-referential data-structures, with edges pointing to their nodes. A small graph that represents a (grossly simplified) portion of the H. sapiens family tree, and the viewer to display it, could be configured as follows:\n\nListing 5. H. sapiens phylogenetic tree sample graph\n\n\n\nAs well as defining the data model, this component allows applications to respond to user input. An example of this is responding when a user clicks on a node in the graph. In the case of our human ancestry graph, that might look like this:\n\nListing 6. Listening for Events\n\n\n\n\nDiscussion\n\nUntil recently it has been difficult to find freely available, open-source libraries for efficiently rendering Sugiyama graph diagrams in the browser. The Cytoscape project4 includes a hierarchical tree layout in its Cytoscape Web JavaScript package; this is however rather less configurable and flexible than this library. Furthermore, The publication of this library as part of the BioJS project explicitly encourages interaction between multiple components of different types. This enables a number of applications that are currently very difficult to implement correctly, such as rendering sets of annotations from the Gene Ontology and allowing user interaction. The DAGViewer component is aimed at a need that is particularly relevant for developers in the life sciences, where there is frequent need to represent directed graphs, e.g. when dealing with phylogeny, pathways, developmental stages or ontologies.\n\nBeyond simplifying this task for developers wishing to get started in graphical network visualisation and analysis, by being built from open web-standard technologies this tool can be used to interoperate with existing and future applications in ways impossible for static image rendering tools. The graph definition can be fetched from a remote networked web service, for example, thus integrating with a large number of existing browser accessible tools.\n\nThe original use case for this tool was to create a graph viewer that would work well with InterMine web-services11, and was generic as to data type. The wide variety of InterMine web-services now available as part of the InterMOD project12, leads us to expect that this component would be broadly useful to a wide section of the bio-informatics developer community. While it in no way depends on InterMine services, the design of this tool makes it straightforward to load from any of the available data-warehouses.\n\nBecause of its flexible data definition, this component is able to consume data from a wide variety of different sources with minimal parsing effort. Since the standard node and edge representation is generally in the form of subject-predicate-object, this component would integrate very well into semantic web tools serving triples as their data representation.\n\n\nConclusion\n\nThis component addresses an important need in the bioinformatics community for an effective, attractive and usable visualisation tool for a broad variety of directed acyclic graphs. It is therefore anticipated that this tool will be of use to those developing tools for researchers in the life sciences. A great deal of effort has gone into creating, curating and integrating high quality data sets, and there already exist many services which expose these data-sets to the world through networked web services. This tool is designed to plug in seamlessly with existing technologies, helping to maximise the value of existing and future curated data sets by bringing enhanced visualisation and exploration functionality. By publishing this component freely within the BioJS project we expect that a great deal of duplicated effort can be avoided, saving significant amounts of time and money for researchers and their funding bodies.\n\n\nSoftware availability\n\nZenodo: BioJS DAG-Viewer Component, doi: 10.5281/zenodo.830313.\n\nGitHub:, BioJS, http://github.com/biojs/biojs.",
"appendix": "Author contributions\n\n\n\nAlex Kalderimis wrote the manuscript and implemented the component, under the supervision of Gos Micklem, to a set of user specifications supplied by Julie Sullivan. Rachel Lyne, Radek Štĕpán and Mike Lyne contributed to the component design and revised the manuscript. All authors have approved the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nInterMine has been developed with the support of the following grants, awarded to Dr. G. Micklem: the Wellcome Trust (Grant number: 090297), and the National Human Genome Research Institute (Grant number: R01HG004834). The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding bodies.\n\n\nAcknowledgements\n\nThe authors thank Manuel Corpas for useful feedback.\n\n\nSupplementary materials\n\n\n\n\nReferences\n\nAshburner M, Ball CA, Blake JA, et al.: Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000; 25(1): 25–29. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEilbeck K, Lewis SE, Mungall CJ, et al.: The Sequence Ontology: a tool for the unification of genome annotations. Genome Biol. 2005; 6(5): R44. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGomez J, Garcia LJ, Salazar GA, et al.: BioJS: an open source JavaScript framework for biological data visualization. Bioinformatics. 2013; 29(8): 1103–1104. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShannon P, Markiel A, Ozier O, et al.: Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2013; 13(11): 2498–2504. PubMed Abstract | Publisher Full Text | Free Full Text\n\nD3. Reference Source\n\nShojiro T, Mitsuhiko T, Kozo S: Methods for Visual Understanding of Hierarchical System Structures. IEEE Trans Syst Man and Cybernetics. 1981; 11(2): 109–125. Publisher Full Text\n\nGraphviz. Reference Source\n\nBackbone. Reference Source\n\ndagre-d3. Reference Source\n\nBiojs project registry. Reference Source\n\nSmith RN, Aleksic J, Butano D, et al.: InterMine: a flexible data warehouse system for the integration and analysis of heterogeneous biological data. Bioinformatics. 2012; 28(23): 3163–3165. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSullivan J, Karra K, Moxon SA, et al.: InterMOD: integrated data and tools for the unification of model organism research. Sci Rep. 2013; 3: 1802. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlexis K, Gos M, Radek S: BioJS DAG-Viewer Component. Zenodo. 2014. Data Source"
}
|
[
{
"id": "3693",
"date": "20 Feb 2014",
"name": "Tom C. Freeman",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe work is an interesting and well written article and reports a resource that should be of interest to numerous developers. The comments below are intended to help make the work more accessible to others: It would be useful to define what some of the graph terms mean such as acyclic/cycles for those who are not familiar with the terminology. Similarly, some background information about the InterMine web services would be useful.How does one reproduce the examples in Figures 2 and 3? Where do the graph size numbers 200-500 nodes for modern browsers come from? Maybe the authors could qualify which with browser, system RAM and which JavaScript API the numbers are for (presumably D3). I would also disagree with statement that ‘networks of greater information densities are very difficult for humans to interpret when rendered’. See our tool BioLayout Express3D.How do I run the examples at http://zenodo.org/record/8303? It might be useful to host the examples somewhere that we can just click and run the demo? (jsFiddle is a great place to host JavaScript examples).There are a few typos - paragraph 7 of the 'Current methods and implementations' section \"Scaled Vector Graphics (SVG)\" should read Scalable Vector Graphics (SVG). In paragraph 3 of the 'Features' section should \"mousehover\" read mouseover? In the same paragraph the phrase \"the graph can been zoomed\" should read the graph can be zoomed.",
"responses": []
},
{
"id": "3750",
"date": "20 Feb 2014",
"name": "Lynn Fink",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article describes a JavaScript component that allows web developers to render node/edge-type graphs in a web page, rather than in a static image, and interact with them dynamically via a tool bar and control panel. This component is a really nice addition to the world of graph visualization because it separates the definition of the graph from the displaying of it; it allows a user to programatically define the node/edge relationships and is extremely lighweight relative to most similar software tools.The Sugiyama method used by the authors to determine node and edge placement seems to work well. I only tested the demo graph, but I couldn't think of any way to improve the layout - unlike most other tools I've tried. The implementation also seems quite straightforward and clear, very human-readable for source code.There are, however, a couple issues that I would like the authors to address: Even though I didn't think moving nodes around was necessary in the demo it was still the first thing I wanted to do (e.g., clicking on node and dragging it into a new position) and I can imagine other graphs where this might feel a bit more urgent. I suspect this sort of functionality is beyond the scope of the JS component, but could the authors either convince me that I'm being old-fashioned and this is something I don't really want to do or mention that this is not an intended use of tool?I can imagine situations in which a user would like to generate a slide or publication-ready image from the graph that they've rendered and lovingly customized. Is there a way to export an image? Or is that in contradiction to the imageless nature of the HTML-based rendering?Will there be a tutorial or walk through of the control panel features? I think this would be welcomed by future users.Minor issues:In paragraph 3 of the 'Features' section the phrase \"the graph can been zoomed\" should read the graph can be zoomed.Demo code - in demo.html one of the script src links is localhost. I had to change this to match the other src link before the script would work so this needs to be corrected.Overall, this seems like a sound and useful contribution to the field of computational biology.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-51
|
https://f1000research.com/articles/3-50/v1
|
13 Feb 14
|
{
"type": "Software Tool Article",
"title": "PPI layouts: BioJS components for the display of Protein-Protein Interactions",
"authors": [
"Gustavo A. Salazar",
"Ayton Meintjes",
"Nicola Mulder",
"Ayton Meintjes",
"Nicola Mulder"
],
"abstract": "Summary: We present two web-based components for the display of Protein-Protein Interaction networks using different self-organizing layout methods: force-directed and circular. These components conform to the BioJS standard and can be rendered in an HTML5-compliant browser without the need for third-party plugins. We provide examples of interaction networks and how the components can be used to visualize them, and refer to a more complex tool that uses these components. Availability: http://github.com/biojs/biojs; http://dx.doi.org/10.5281/zenodo.7753",
"keywords": [
"mycobacteria"
],
"content": "Introduction\n\n“Proteins are the building blocks of life”. This is probably the most commonly used analogy in the molecular biology world. Despite the danger of falling into a cliché, we will elaborate on this. A brick is not a building, not a room, not even a wall; in the same way, proteins rarely perform functions in isolation, it is their interactions which form the complex networks that are responsible for almost all cellular processes.\n\nThe number of reported Protein-Protein Interaction (PPI) networks has grown considerably, partly due to advances in high-throughput experimentation and partly due to the new predictions that result from these empirical data.\n\nVarious strategies have been used to store this data for ease of retrieval and analysis of PPI networks1. For instance, IntAct2 is an open source repository for molecular interactions reported in the literature or directly deposited by the user; the STRING database3 includes physical and functional associations derived from genomic context, high-throughput experiments, coexpression analysis or the literature. Other PPI sources are described in:4–9.\n\nThe volume of data now contained in PPI repositories has made the analysis and understanding thereof a challenge that can be enriched by the use of visualization techniques. PPI networks have been found to follow a behaviour that in graph theory is referred to as scale-free, which uses a few highly connected nodes and many nodes with few interactors. This feature allows a user to predefine visualization strategies to highlight certain characteristics. This approach has been used for multiple tools, some are stand-alone applications (e.g. Cytoscape10), Java applications that are web available via Applets (e.g.VisANT11), or those developed on Flash (e.g. STITCH4). Details about other tools, comparisons between them, highlighted features and supported layouts can be found in any of the following reviews:12–14.\n\nThe web technologies covered under the umbrella term of HTML5 allow the display of some of those layouts natively on the web in a fully interactive way. We have used these technologies to implement two of the most popular layouts for PPI network visualization: Force-directed and Circle.\n\nThe components were developed following the BioJS15 standard and are freely available at its registry: http://goo.gl/064ChR, http://goo.gl/RGlRLB.\n\n\nBioJS components\n\nWe have implemented the standards defined by BioJS in the development of two components for the visualization of PPI networks. The use of BioJS gives visibility to the components so that they can be discovered by third parties interested in the visualization of protein networks on the web.\n\nWe consider the publication via the BioJS registry beneficial for both the potential user and the developer of the component. The user of the component will save time by adopting a component instead of developing it from scratch. The particular needs of the user may require the implementation of specifics which could contribute to the further development of the component in a communal effort. All this is in the nature of open source projects, but without visibility it would hardly ever happen.\n\nBoth PPI network visualizer components are implemented as wrappers of layouts included in the Data Driven Document (D3) library16, a popular JavaScript library for the processing and visualization of data. Besides making these layouts follow the BioJS structure, most of the effort has been directed at providing methods that are closer to the PPI vocabulary (e.g. addProtein(),addInteraction()). Both components follow the same API (Application Program Interface) and therefore any script developed to display on one layout can be used on the other.\n\nThe D3 library provides an implementation of the Barnes-Hut simulation in order to efficiently create a self-organized network. This algorithm performs better than others because it aggregates the forces of close nodes, to avoid individual calculation on the forces whenever possible. Its performance facilitates a smoother transition between the execution steps of the algorithm, creating an appealing visual effect of live self organization. We have used this generic network layout available in D3 to provide a set of methods and events to represent PPI networks.\n\nFigure 1 displays a subset of proteins from the organism Mycobacterium tuberculosis, that interact with furB. It includes all the interactions between these proteins with each protein color coded by reported functional class. FurB is a zinc uptake regulation protein involved in repression of many genes involved in zinc homeostasis17. FurB interacts with a number of different genes, with related functions, including the transcriptional regulator FurA, which represses transcription of katG, the catalase-peroxidase gene as well as itself, and the HTH-type transcriptional repressor SmtB, also involved in zinc homeostasis. FurB also interacts with the cAMP receptor protein, a global transcriptional regulator, although no link to metal ions has been annotated for this protein, a disruption in the protein results in slow growth. Additional interactions for FurB are with enzymes involved in amino acid biosynthesis (dapB, acn) or purine metabolism (purE, purK). One thing in common between these proteins appears to be their relation to growth.\n\nBioJS provides instructions regarding the installation of each component including their required dependencies, style files and snippets of JavaScript code to be inserted into the web page where the component will be embedded. For example, installation instructions for the force-directed layout can be found on the Installation tab of the component page in the BioJS Registry (http://goo.gl/064ChR).\n\nThe full script used to generate Figure 1 is available as a jsFiddle (a web resource for the online edition and display of snippets of JavaScript code) at this URL: http://jsfiddle.net/Bvh6k/1/. Below is a description of the main components of that code:\n\nA developer should start by creating an instance of the component:\n\n\n\nAll the proteins in the graphic should then be added. The following example shows how to add the protein with UniProt id O05839 (http://www.uniprot.org/uniprot/O05839). Note how the organism to which it belongs is included, along with which feature should be used for the label:\n\n\n\nIn the same way, the interactions should be added to the graphic, ensuring that the interactions are between proteins that have already been added. For instance:\n\n\n\nOnce all the proteins and interactions have been declared, the graphic can be restarted so it reflects the additions:\n\n\n\nThe example also includes some instructions for coloring and format. For more details on those methods see the component on the BioJS registry: http://goo.gl/064ChR.\n\nAs mentioned previously, both components have the same API with the exception of methods that help to control the force layout of the first component which do not apply to the Circle layout. Thanks to this, the script to generate a PPI visualization of the same network is very similar in both. In order to demonstrate this we have created a second script that visualizes the same network as in Figure 1 but now on a Circle layout (Figure 2). The only difference between both scripts is the declaration of the object. Instead of using the object Biojs.InteractionsD3 it uses Biojs.InteractionsBundleD3 and rest of the script is exactly the same. This can be seen in the fiddle: http://jsfiddle.net/J4CE7/1/.\n\n\nUse Case: PINV\n\nWe have used these two layouts and a few other BioJS components to create a web native application that allows the querying and visual exploration of PPI networks. It is called PINV: Protein Interactions Network Visualizer, and is freely available at http://biosual.cbio.uct.ac.za/pinv.html.\n\nWith PINV, we are looking to offer an alternative for the visualization of PPI data that takes advantage of the web to provide collaborative tools.\n\nCurrently PINV can display the PPI networks in three ways: the two layouts discussed above and through a table of the raw data. The addition of other layouts in the future is not complicated, thanks to the standards defined by BioJS and the adoption of the same API.\n\nThe same example used for Figure 1 and Figure 2 is available on PINV under this link: http://goo.gl/r4XpOS.\n\n\nConclusions\n\nOne of the limitations of current web-based methods for PPI visualization is that they require third-party browser plugins. We demonstrate that the HTML5 standard provides enough functionality to render these networks in compliant browsers, without the need to install additional browser components. Adoption of the BioJS standard has the advantage of greater exposure to potential users, and an established set of features such as testing and documentation. Finally, the components abstract much of the details of rendering complex scalable vector graphics (SVG) behind an intuitive API, allowing users to focus on building richer applications.\n\n\nSoftware availability\n\nZenodo: BioJS components for the display of Protein-Protein Interactions, doi: 10.5281/zenodo.775318.\n\nGitHub: BioJs, http://github.com/biojs/biojs.",
"appendix": "Author contributions\n\n\n\nCritical revision of the manuscript for important intellectual input: GS, AM and NM. Supervision: NM. Study concept: GS, AM and NM. Software development: GS. Drafting of the manuscript: GS and AM. All authors have read and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis project was funded by the Computational Biology Group at the University of Cape Town.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe want to thank J. Holtzhausen for her contribution to the editorial matters of this article.\n\n\nReferences\n\nMathivanan S, Periaswamy B, Gandhi TK, et al.: An evaluation of human protein-protein interaction data in the public domain. BMC Bioinformatics. 2006; 7(Suppl 5): S19. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKerrien S, Aranda B, Breuza L, et al.: The IntAct molecular interaction database in 2012. Nucleic Acids Res. 2012; 40(Database issue): D841–D846. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFranceschini A, Szklarczyk D, Frankild S, et al.: String v9.1: protein-protein interaction networks, with increased coverage and integration. Nucleic Acids Res. 2013; 41(Database issue): D808–D815. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKuhn M, von Mering C, Campillos M, et al.: STITCH: interaction networks of chemicals and proteins. Nucleic Acids Res. 2008; 36(Database issue): D684–D688. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrown KR, Jurisica I: Unequal evolutionary conservation of human protein interactions in interologous networks. Genome Biol. 2007; 8(5): R95. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKeshava Prasad TS, Goel R, Kandasamy K, et al.: Human protein reference database—2009 update. Nucleic Acids Res. 2009; 37(Database issue): D767–D772. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMathivanan S, Ahmed M, Ahn NG, et al.: Human proteinpedia enables sharing of human protein data. Nat Biotechnol. 2008; 26(2): 164–167. PubMed Abstract | Publisher Full Text\n\nLicata L, Briganti L, Peluso D, et al.: Mint, the molecular interaction database: 2012 update. Nucleic Acids Res. 2012; 40(Database issue): D857–D861. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChen JY, Mamidipalli S, Huan T: HAPPI: an online database of comprehensive human annotated and predicted protein interactions. BMC Genomics. 2009; 10(Suppl 1): S16. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShannon P, Markiel A, Ozier O , et al.: Cytoscape: A software environment for integrated models of biomolecular interaction networks. Genome Res. 2003; 13(11): 2498–2504. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHu Z, Chang YC, Wang Y, et al.: VISANT 4.0: Integrative network platform to connect genes, drugs, diseases and therapies. Nucleic Acids Res. 2013; 41(Web Server issue): W225–W231. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSuderman M, Hallett M: Tools for visually exploring biological networks. Bioinformatics. 2007; 23(20): 2651–2659. PubMed Abstract | Publisher Full Text\n\nGehlenborg N, O'Donoghue SI, Baliga NS, et al.: Visualization of omics data for systems biology. Nat Methods. 2010; 7(3 Suppl): S56–68. PubMed Abstract | Publisher Full Text\n\nAgapito G, Guzzi PH, Cannataro M: Visualization of protein interaction networks: problems and solutions. BMC Bioinformatics. 2013; 14(Suppl 1): S1. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGómez J, García LJ, Salazar GA, et al.: BioJS: An open source javascript framework for biological data visualization. Bioinformatics. 2013; 29(8): 1103–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBostock M, Ogievetsky V, Heer J: D3; data-driven documents. IEEE Trans Vis Comput Graph. 2011; 17(12): 2301–2309. PubMed Abstract | Publisher Full Text\n\nMilano A, Branzoni M, Canneva F, et al.: The mycobacterium tuberculosis rv2358–furb operon is induced by zinc. Res Microbiol. 2004; 155(3): 192–200. PubMed Abstract | Publisher Full Text\n\nSalazar GA, Meintjes A, Mulder N: Biojs components for the display of protein-protein interactions. Zenodo. 2014. Data Source"
}
|
[
{
"id": "3688",
"date": "10 Mar 2014",
"name": "Laurent Gatto",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript PPI layouts:BioJS components for the display of Protein-Protein Interactions by Salazar and colleagues briefly illustrate the BioJS component for the interactive html5-based visualisation of PPI.The manuscript is easy to read and provides a quick, yet useful introduction to the BioJS PPI visualisation tools. The component described in the manuscript and the BioJS project in general are a welcome initiative. The PPI interaction tools was straightforward to assess thanks to support for the online edition, simple implementation, and its distribution to the bioinformatics community under an open access license.Below, I have a few suggestions:BioJS component sectionIn the first paragraph the sentence \"The use of BioJS gives visibility to the components so that they can be [discovered] by third parties...\". Do the authors mean that the individual component will benefit from the BioJS visibility, or is there a specific discovery mechanism?M. tuberculosis example networkWhile I appreciate that it is not easy to find the right balance between a simple yet biologically relevant illustration of the tool, I believe that the paragraph that describes the interaction network could be improved. For example, some proteins are named and described while others are not.While reading the description of the code generating the network in Figure 1 and 2 and using the jsFiddle instance, several questions arose that could be addressed in the manuscript.the authors provide a UniProt identifier. Could the nodes be links to the relevant protein UniProt pages? Could other identifiers be used and how would the links to other arbitrary pages be defined?What is the use of the species name in the protein instance definition? Is it mandatory? Are there examples of PPIs with multiple species?Graph annotation. Is it possible to use different symbols for the nodes? How to annotate the edges (through colours and/or labels)? Any comments on the use of node colours as a means to illustrate additional meta-data: are specific colour palettes (for continuous data for example) or transparency available?PINVThe PINV application is a step further than the examples described in the manuscript, that possibly deserves a bit more description in the text and could guide the reader on how to build up from simple cases to more complex applications.Before Figure 2Instead of using the object Biojs.InterationsD3 it uses Biojs.InteractionsBundleD3 and [the] rest of the script is exactly the same.Compliant browsersThe authors repeat the requirement for compliant browsers. I was wondering if examples of compliant browsers could be provided to guide users new to html5 and issues related to browser compatibility.",
"responses": []
},
{
"id": "4104",
"date": "28 Mar 2014",
"name": "Hagen Blankenburg",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe two JavaScript components presented in this article facilitate the visualization of interaction networks in HTML5-enabled web browsers without requiring any third-party plugins like Flash. As they follow the BioJS standard, they are listed in the BioJS registry and should be interoperable with other components. The manuscript is comprehensibly written and, despite the length limitations, successfully introduces both tools and their basic usage. The availability of the source code and particularly the jsFiddle live demo setup enable the interested reader to explore the software without complicated installation procedures. In the following, I will list a few comments that might help to assure that the tools unlock their full potential. Note that while there is an overlap to the review by Laurent Gatto, I will repeat certain aspects for the sake of completeness. Interoperability with other BioJS components / PINV:The ability to show additional information about protein interactors (e.g., database identifiers, cross-references) and their interactions (e.g. links to source databases and publications, confidence scores) is what distinguishes a truly interactive network display from a nice visualization. In contrast to the two basic examples currently provided, PINV nicely demonstrates that this can be accomplished. Given that BioJS is essentially about modularity and re-usability of code, it would be great if the authors could provide more detailed examples that illustrate how their layout components can be combined with other BioJS components, e.g., to visualize additional information in popups. If manuscript space permits, additional commented source code files or a jsFiddle setup would be great and would further ease the application of the tools. M. tuberculosis example:As part of the BioJS collection, the manuscript generally seems to be written for computer scientists / developers rather than biologists. As such, the paragraph describing the M. tuberculosis interaction network example is too extensive and does not fit the focus of the remaining article. This example could be shortened and moved into the corresponding figure legends, making room for the aforementioned, more thorough description of other parts. Cytoscape.js:While the authors mention similar software like Cytoscape and VisANT, the HTML5 library Cytoscape.js (http://cytoscape.github.io/cytoscape.js/) and its predecessor Cytoscape Web (http://cytoscapeweb.cytoscape.org/) are omitted, although they are likely the closest competitors. Source code examples:From the manuscript or the source code examples it is not obvious why the UniProtKB accession numbers and the organism information is provided, as neither is used or shown in the examples. It should be clarified, if tools require this information or if this might be used for connecting with other BioJS components. Title:Looking only at the title and the abstract, it appears that “PPI layouts” is the name of the BioJS component that is described in the article. As this is not the case and the name “PPI layouts” is never used again, it could be removed from the title to prevent confusion.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-50
|
https://f1000research.com/articles/3-49/v1
|
13 Feb 14
|
{
"type": "Web Tool",
"title": "treeWidget: a BioJS component to visualise phylogenetic trees",
"authors": [
"Fabian Schreiber"
],
"abstract": "Summary: Phylogenetic trees are widely used to represent the evolution of gene families. As the history of gene families can be complex (including lots of gene duplications), its visualisation can become a difficult task. A good/accurate visualisation of phylogenetic trees - especially on the web - allows easier understanding and interpretation of trees to help to reveal the mechanisms that shape the evolution of a specific set of gene/species. Here, I present treeWidget, a modular BioJS component to visualise phylogenetic trees on the web. Through its modularity, treeWidget can be easily customized to allow the display of sequence information, e.g. protein domains and alignment conservation patterns.Availability: http://github.com/biojs/biojs; http://dx.doi.org/10.5281/zenodo.7707",
"keywords": [
"A phylogenetic tree is a branching diagram showing the inferred relationships of genes or species. Reconstructing a phylogenetic tree is a routine task in most evolutionary-related analyses and a number of databases exist containing precomputed phylogenetic trees (e.g. TreeFam1",
"Ensembl Gene Trees2",
"Panther3). These reconstructed trees can vary considerably in the number of gene/species shown and in their complexity. While there are many offline tools available to visualise phylogenetic trees (e.g. ETE4)",
"the number of online tools for this purpose is rather limited. Some of them are written in Java and tend to become slow when the number of nodes/edges increases (e.g. Archaeopteryx5). Others written in JavaScript",
"are therefore faster and more scalable",
"but do not allow the additional display of useful sequence annotation",
"e.g. protein domains and alignments. (e.g. phyloWidget6",
"jsPhyloSVG7). Yet other tools offer that functionality but are not available for download",
"customization or to be embedded into the users’ own websites (e.g. iTol8). Despite its widespread use in Bioinformatics",
"biological web applications for viewing phylogenetic trees are usually implemented with no standard reutilisation guidelines in mind."
],
"content": "Introduction\n\nA phylogenetic tree is a branching diagram showing the inferred relationships of genes or species. Reconstructing a phylogenetic tree is a routine task in most evolutionary-related analyses and a number of databases exist containing precomputed phylogenetic trees (e.g. TreeFam1, Ensembl Gene Trees2, Panther3). These reconstructed trees can vary considerably in the number of gene/species shown and in their complexity. While there are many offline tools available to visualise phylogenetic trees (e.g. ETE4), the number of online tools for this purpose is rather limited. Some of them are written in Java and tend to become slow when the number of nodes/edges increases (e.g. Archaeopteryx5). Others written in JavaScript, are therefore faster and more scalable, but do not allow the additional display of useful sequence annotation, e.g. protein domains and alignments. (e.g. phyloWidget6, jsPhyloSVG7). Yet other tools offer that functionality but are not available for download, customization or to be embedded into the users’ own websites (e.g. iTol8). Despite its widespread use in Bioinformatics, biological web applications for viewing phylogenetic trees are usually implemented with no standard reutilisation guidelines in mind.\n\nHere I present treeWidget, a BioJS9 component written in JavaScript to visualise phylogenetic trees on the web. treeWidget can be easily integrated into websites and customized. To my knowledge, this is the first modular tree visualisation component.\n\n\nThe treeWidget component\n\nThe treeWidget component has been developed as part of the TreeFam project and follows the standards set by the BioJS registry (1, see Figure 2 for an example).\n\nThe BioJS registry is a centralised repository of BioJS components hosted at the European Bioinformatics Institute. treeWidget uses the JavaScript library D3 for building trees10. It reads trees in JSON format (http://www.json.org/) and plots them as scalable vector graphics (SVG, see the treeWidget component documentation for a working example of a JSON-formatted tree). Additionally, treeWidget can plot annotation in a separate SVG. This way the tree diagram stays fixed whenever the annotation changes (e.g. switching from a domain to a sequence alignment view). treeWidget automatically scales the SVG according to the tree’s height and so will work with small trees containing only 3 leaves up to trees with several hundreds of leaves by plotting the tree size according to the tree height (number of internal nodes).\n\nTo make the leaf names of gene trees more meaningful, users can add additional information (e.g. gene name, source species, common name, gene function, etc) to the JSON file. treeWidget will then plot this information next to the tree leaves (see Figure 2). treeWidget can either draw an ultrametric tree (all leaves have the same branch length) or a tree with estimated branch lengths and inner nodes can be labelled with taxon names and bootstrap values.\n\nIn cases where the gene trees contain too many leaves, it is useful to start looking at the gene tree by focussing on a specific part of the tree. This could be, for example, a pig gene that the user is particularly interested in. treeWidget allows highlighting of a specific gene and collapsing of sister clades to hide less relevant parts of the tree display (see Figure 1). These collapsed clades can be expanded/collapsed by a mouse click. To make the identification of related species in a gene tree easier, treeWidget colours pre-selected taxonomic clades (see Figure 2).\n\nThe surrounding sister clades (Carnivora, Cetartiodactyla, Laurasiatheria) are collapsed. They can be expanded by clicking on the node.\n\ntreeWidget can be used to visualise the evolution of species but also that of genes as it is done on the TreeFam website (http://www.treefam.org). TreeFam’s main goal is to present phylogenetic trees of gene families across the animal tree of life. TreeFam also predicts orthology/parology relationships: we speak of orthologs when two genes in different species are the result of a speciation event, whereas paralogs are genes stemming from a duplication event. The treeWidget component allows the display of this information by labelling the internal nodes of each gene tree as either speciation or duplication events. Additionally, the treeWidget component displays patterns of alignment conservation as well as matches of Pfam11 protein domains on each sequence in the database. In Figure 2 the conserved alignment pattern from the underlying protein sequence alignment is shown. The white alignment parts represent gaps and green parts are aligned parts. Visualising alignment conservation in conjunction with Pfam domains along a gene tree gives useful insights about the evolution of domain architectures12. Furthermore, this view can be used to spot problems with assembled genes (split genes or falsely assembled genes).\n\n\nConclusions\n\nThe treeWidget component provides a platform for the exploration of complex phylogenetic trees depicting the evolution of large gene families with many duplication/speciation events as well as displaying sequence annotation features. Visualising such complex data allows researchers to see interesting features for further study. I expect this component to be particularly useful to developers and users alike, requiring little technical knowledge for its full functioning.\n\n\nSoftware availability\n\nZenodo: BioJS TreeWidget component, doi: 10.5281/zenodo.775113\n\nGitHub: BioJS, http://github.com/biojs/biojs",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nWellcome Trust [WT077044/Z/05/Z] to FS.\n\n\nAcknowledgements\n\nThe author thanks Mateus Patricio, Miguel Pignatelli, Matthieu Muffato as well as Alex Bateman for useful feedback.\n\n\nReferences\n\nSchreiber F, Patricio M, Muffato M, et al.: TreeFam v9: a new website, more species and orthology-on-the-fly. Nucleic Acids Res. 2014; 42(Database issue): D922–D925. PubMed Abstract | Publisher Full Text\n\nVilella AJ, Severin J, Ureta-Vidal A, et al.: EnsemblCompara GeneTrees: Complete, duplication-aware phylogenetic trees in vertebrates. Genome Res. 2009; 19(2): 327–335. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMi H, Muruganujan A, Thomas PD: PANTHER in 2013: modeling the evolution of gene function, and other gene attributes, in the context of phylogenetic trees. Nucleic Acids Res. 2013; 41(Database issue): D377–D386. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuerta-Cepas J, Dopazo J, Gabaldón T: ETE: a python Environment for Tree Exploration. BMC Bioinformatics. 2010; 11(1): 24. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHan MV, Zmasek CM: phyloXML: XML for evolutionary biology and comparative genomics. BMC Bioinformatics. 2009; 10: 356. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJordan GE, Piel WH: PhyloWidget: web-based visualizations for the tree of life. Bioinformatics. 2008; 24(14): 1641–1642. PubMed Abstract | Publisher Full Text\n\nSmits SA, Ouverney CC: jsPhyloSVG: a javascript library for visualizing interactive and vector-based phylogenetic trees on the web. PLoS One. 2010; 5(8): e12267. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLetunic I, Bork P: Interactive Tree Of Life v2: online annotation and display of phylogenetic trees made easy. Nucleic Acids Res. 2011; 39(Web Server issue): W475–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGómez J, García LJ, Salazar GA, et al.: BioJS: an open source JavaScript framework for biological data visualization. Bioinformatics. 2013; 29(8): 1103–1104. PubMed Abstract | Publisher Full Text | Free Full Text\n\nD3.js - Data-Driven Documents. Reference Source\n\nPunta M, Coggill PC, Eberhardt RY, et al.: The Pfam protein families database. Nucleic Acids Res. 2012; 40(Database issue): D290–D301. PubMed Abstract | Publisher Full Text | Free Full Text\n\nForslund K, Sonnhammer EL: Evolution of protein domain architectures. Methods Mol Biol. 2012; 856(Chapter 8): 187–216. PubMed Abstract | Publisher Full Text\n\nSchreiber F: BioJS TreeWidget component. Zenodo. 2014. Data Source"
}
|
[
{
"id": "3713",
"date": "25 Feb 2014",
"name": "Jaime Huerta-Cepas",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTreeWidget is a handy JavaScript component for the visualization of phylogenetic data. It is currently used to display trees in the TreeFam database and allows for inline representation of rectangular trees, alignments and sequence domains. After testing the application with trees of different sizes I came out with the following comments that may serve the author to improve this publication: MajorUsability Apart from domain and alignment annotation, the documented functionality of TreeWidget looks quite limited as compared with other JavaScript components. In my tests, I could only represent static trees with tip labels and motif structure. I have the impression that the application is capable of much more, but I could not find any reference to it in the help pages. For instance: Would it be possible to add or modify OnClick and MouseOver events? Search for nodes in large trees? What about zooming or showing bootstrap and branch length values? These are basic features present in the TreeFam version and in most tree viewers, but not in the current version of TreeWidget. Note that although Figure 1 shows bootstrap support values in the tree, I could not find that option in the documentation. It would be useful to have the example code that generates the figures in the paper. JSON is not a common format for phylogenetic data, but it seems to be the only way to load trees into the application. Can TreeWidget read the newick or extended-newick formats? If so, please document it. In my case, I had to write a little parser to convert regular trees into treewidget/d3 format (gist.github.com/jhcepas/9205262) to test the software, but it would be great to have this feature as a built-in option. The same applies for alignments. I think that providing Newick and Fasta files would be much more convenient for end users than pointing them to create a new JSON structure on top of the original data. Manuscript Although I acknowledge the potential usefulness of TreeWidget, I think the text goes a bit too far when states that this is the \"first modular tree visualization component\". Even if we restrict to the JavaScript context, several libraries do provide online tree visualization from some time ago. In fact, the same d3 back-end library used by TreeWidget is a JavaScript modular library that supports tree and network visualization by itself (bl.ocks.org/mbostock/4063570) . Of note, the nice performance and responsiveness obtained when drawing large trees is a d3 feature available for the representation of any type of hierarchical data. Similarly, jsPhyloSVG is a modular JavaScript library that allows online representation of phylogenetic trees, providing interesting features such as newick support and circular tree drawing. This should not prevent TreeWidget from being published in F1000Research, but the emphasis should be put on the possibility of representing phylogenetic information (i.e. duplication and speciation events) together with alignments and domain structure, in a very easy way.There are also several mistakes regarding the literature cited in the introduction: 1) The ETE package is not only an offline tool, but a programming library offering a webplugin module that can be used for online interactive visualization of custom phylogenetic data (i.e. the PhylomeDB database uses ETE to render interactive tree images with alignments and PFam domain annotations). 2) To my knowledge, PhyloWidget is not JavaScript but Java. 3) I would not say that Archeopteryx is that slow for medium/large trees, and it also offers web integration and alignment and domain visualization. MinorDocumentation While testing the library I had the feeling that the documentation was not very clear. First, I could not find the relevant code, documentation or examples within the github repository provided as the main link in manuscript. It seems that the so called 'registry installation' step is necessary to start using the library. This will install the whole BioJS package, making the code and examples available at ./biojs/target/registry/, which is in fact the base path assumed by the examples provided in the online example. This is not a big deal, but I had to guess it by browsing different pages and files of the BioJS project. It would be useful to point readers to the specific installation and help pages of TreeWidget, clarifying whether it can be used independently or requires the BioJS project to be downloaded and compiled as a whole. Note, that it is also not clearly mentioned in the manuscript, that the examples and documentation of this component can be found at https://www.ebi.ac.uk/Tools/biojs/registry/Biojs.Tree.html.",
"responses": []
},
{
"id": "4248",
"date": "24 Mar 2014",
"name": "Stephen A. Smith",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\ntreeWidget is a nice feature full JavaScript tree viewer. This is a relatively straightforward announcement of the tool and I see no major problems. Below are some comments for the author's consideration.MajorThe discussion of the license is not in the manuscript. I believe it is Apache 2.0 but it would be good to mention that. Also, I understand it is open source but I might also mention that clearly as well.I feel like the author overstates the originality of the library. There is mention of jsphyloSVG which of course is a modular tree visualisation component (negating the last sentence of the introduction). Furthermore, there are others. In particular, Roderic Page has been developing a number of different options that are simple and perhaps not as feature-full but serve to have trees in the BioNames project (http://bionames.org/). In fact, this has been around for a few years and available for experimentation (here is one of the early posts about this http://iphylo.blogspot.com/2012/12/viewing-phylogenies-on-web-javascript.html). Of course not all of these would be considered more than experimentation, but I would note that it has been an area of long discussion in the bioinformatics field for a while now.The documentation is pretty difficult to navigate. There is a fair bit of (also unclear at this point) documentation of the BioJS library with some specific language that is not well defined (what is the registry for exactly?). If treeWidget is in fact going to be particularly useful for web developers to drop into a website, I would suggest making a tutorial specifically designed to show a developer how to incorporate just treeWidget and not the entire BioJS package.I was unable to generate dynamic examples. This is related to the above comment, but I would generate some more examples and tutorials that explore these options in more detail. Please distribute the code used to make the figures as part of the distribution or in another package. That would be helpful.MinorI feel like a few edits would improve the manuscript quite a bit. For example, in the introduction, you state:\"Reconstructing a phylogenetic tree is a routine task in most evolutionary-related analyses and a number of databases exist containing precomputed phylogenetic trees (e.g. TreeFam1, Ensembl Gene Trees2, Panther3). These reconstructed trees can vary considerably in the number of gene/species shown and in their complexity\"For the first sentence, I am not entirely sure I understand the relevance of the existence of precomputed trees and if that is important, this is an odd assortment of those resources. Of course there are many more. For the second sentence, I am not certain what you mean by varying in their complexity. I don't think you mean in their complexity in computational sense (but perhaps you do?).Because JSON is not often used in phylogenetics packages, I suspect that you will attract more users if you can take newick formats. You can see some examples using other packages here: http://bl.ocks.org/rdmpage/raw/4224658/.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-49
|
https://f1000research.com/articles/3-48/v1
|
13 Feb 14
|
{
"type": "Web Tool",
"title": "HeatMapViewer: interactive display of 2D data in biology",
"authors": [
"Guy Yachdav",
"Maximilian Hecht",
"Metsada Pasmanik-Chor",
"Adva Yeheskel",
"Burkhard Rost",
"Maximilian Hecht",
"Metsada Pasmanik-Chor",
"Adva Yeheskel",
"Burkhard Rost"
],
"abstract": "Summary: The HeatMapViewer is a BioJS component that lays-out and renders two-dimensional (2D) plots or heat maps that are ideally suited to visualize matrix formatted data in biology such as for the display of microarray experiments or the outcome of mutational studies and the study of SNP-like sequence variants. It can be easily integrated into documents and provides a powerful, interactive way to visualize heat maps in web applications. The software uses a scalable graphics technology that adapts the visualization component to any required resolution, a useful feature for a presentation with many different data-points. The component can be applied to present various biological data types. Here, we present two such cases – showing gene expression data and visualizing mutability landscape analysis.Availability: https://github.com/biojs/biojs; http://dx.doi.org/10.5281/zenodo.7706.",
"keywords": [
"Biological data are often organized into matrices in which the rows signify different items of interest (a gene",
"a subject",
"a probe or a position in a sequence)",
"while the columns describe different experiments",
"variations",
"or samples. Matrices are easy to process by algorithms. In contrast",
"the details in large matrices are often",
"at best",
"challenging for experts who want to “understand” the data. The information in matrices is usually better digested if presented by 3D plots or heat maps. Heat maps are essentially simplified versions of 3D plots that replace the 3rd dimension with color gradients",
"thereby conveniently displaying the information contained in matrices. Such heat maps allow for easy visual differentiation between high and low values in a matrix."
],
"content": "Introduction\n\nBiological data are often organized into matrices in which the rows signify different items of interest (a gene, a subject, a probe or a position in a sequence), while the columns describe different experiments, variations, or samples. Matrices are easy to process by algorithms. In contrast, the details in large matrices are often, at best, challenging for experts who want to “understand” the data. The information in matrices is usually better digested if presented by 3D plots or heat maps. Heat maps are essentially simplified versions of 3D plots that replace the 3rd dimension with color gradients, thereby conveniently displaying the information contained in matrices. Such heat maps allow for easy visual differentiation between high and low values in a matrix.\n\nSuch heat maps are, for example, commonly used to display microarray data as they quickly show which genes (rows) are differentially expressed under some conditions (columns). Microarray technologies utilize arrays of probes located on different exons for each gene and can be helpful in determining gene function by measuring transcription and translation levels under certain experimental conditions. The expression values for the differential expression may be presented at the exon level, correlated with protein domains, and may help to decipher a complex gene expression pattern.\n\nHeat maps can also display the effect of point mutations (single amino acid substitutions, or non-synonymous Single Nucleotide Polymorphisms – nsSNPs). Through the application of methods that predict the impact of mutations1–4 we can expand from the view of single variants to the level of sketching the entire mutability landscape5. This mutability landscape is defined by the impact of substituting every residue at each position in a protein by each of the 19 non-native amino acids. The resulting predictions can then be shown in a heat map in order to visualize the impact of each substitution. Regions where mutations have a high average effect (i.e. where almost every substitution is predicted to alter protein function) are especially interesting as these are likely to be of particular and direct importance for protein function.\n\nWe developed HeatMapViewer as a BioJS component that can easily be used, reused and, if needed, extended to display matrix data. BioJS6 is an open source JavaScript library of components for visualization of biological data on the web. As a JavaScript component, the HeatMapViewer is very flexible, interactive and webready. Previous libraries generating graphical HeatMaps render either static images7 or are highly specialized and cannot be reused8. To the best of our knowledge, this is the first client-side modular component to visualize matrices that can be integrated into other web applications in a standard manner.\n\n\nThe HeatMapViewer component\n\nHeatMapViewer uses the D39 JavaScript library to render Scalable Vector Graphics (SVG) objects. SVG technology is now standardized and native to modern web browsers (e.g. Mozilla, Chrome, Safari). The component accepts a simple JSON object containing the data matrix that will be rendered. A secondary JSON object contains configuration directions such as the target DIV element onto which the component will be rendered, the data range to be shown, the color scheme to be used for the component, the size of the canvas showing the component and the minimum cell size (by default these last two options can be computed automatically).\n\nThe HeatMapViewer component automatically renders a heat map based on the input data object and the pre-set color-scheme. Positioning and layout are automatically calculated given the available browser window size. If presenting the entire heat map requires individual cells to be smaller than a given threshold, a secondary panel is automatically rendered to show a zoomed-in version of a local segment in the heat map. This zoom-in panel is presented right under the main heat map panel. The labels for the X-axis and Y-axis are laid out above the top row and next to the left column. The component provides a user control in the form of a frame that can be dragged along the main heat map to determine which area of the heat map should appear in the zoom-in panel. Additionally, a scale bar is presented to show the value ranges and which colors correspond to those values. Finally, each cell in the heat map is associated with a mouse-over event that pops-up tooltips showing the data-value of the cell.\n\nThe HeatMapViewer component can be obtained from the BioJS registry at https://github.com/biojs/biojs. For users wishing to test the component’s capabilities to generate heat map plots for their data without downloading and installing the component, we have set up a server: http://www.rostlab.org/services/heatmap-viewer. The server allows users to upload their data in Comma Separated Values (CSV) format and then renders a heat map on the screen. The server also allows exporting the resulting graphics rendering it into an image.\n\n\nApplication use-cases and examples\n\nThe rhodopsin gene encodes a protein of the outer photoreceptor segment that is essential for the visual transduction cascade. Since 1989, many mutations in the rhodopsin gene have been found to be involved in the eye disease Retinitis Pigmentosa (also known as Retinopathia pigmentosa or simply RP10). RP is a hereditary disease causing retinal degeneration and thereby destroying photoreceptors; this results in severe vision impairment or even blindness.\n\nA typical study of such a hereditary disease might begin with a protocol as follows. According to the UCSC genome browser11, human rhodopsin (RHO, RefSeq: NM_000539.3) consists of 5 exons (located on chr3:129,247,482-129,254,187). The total gene length is 6706 bps (base pairs/nucleotides). The coding region (chr3:129,247,577-129,252,561; i.e. extending over 4985 bps), is translated into a gene-product/protein with 348 residues (UniProt identifier: P0810012, SwissProt identifier: OPSD_HUMAN13). This protein has a single large domain (Pfam identifier: PF0000114) that is dominated by a \"standard\" 7-transmembrane receptor region (rhodopsin family), which spans most of the coding region (residues 55 - 306). The human rhodopsin is highly expressed in the heart, liver, skeletal muscle, thyroid and the eye retina.\n\nIt is interesting to locate the array probes intensities on the various protein domain regions. We map the expression profiles of the RHO (from GEO43134) to the structural protein regions through visualization with the HeatMapViewer component (Figure 1). The different experimental conditions are presented on the rows, while the probes for the RHO gene are shown on the columns, annotated with exon and trans-membrane (TM) location. Probes with high expression are marked in red; those with low expression are colored green. The differences in color of the same probe along the different conditions provides useful information concerning the expression intensity of the various probes, and possible variations in alternative splicing patterns and region conservation across the different samples.\n\nIn this experiment, a mutation in a splicing factor that causes Retinitis Pigmentosa (RP) was shown to have an effect on mRNA splicing. Moreover, mutations in the rod photoreceptor-specific protein rhodopsin (RHO) are known to cause RP. Log2 expression values for the 8 probes of human RHO were obtained and located to each of its 5 exons and the 7 trans-membrane (TM) regions (columns). It is interesting to note that the different probes (located on the various regions of RHO), are differentially expressed (high expression colored red and low expression in green). Moreover, we can observe that some RHO probes are expressed differently in the control than in the treatment (case, rows). These results may indicate the effect of the mutated splicing factor on RHO gene in RP disease.\n\nSince RP is caused by mutations in the rhodopsin gene, researchers have extensively investigated different variations of the gene. Thus, up to now over 100 mutations have been identified and associated with RP. More generally: single nucleotide variations constitute most of the genetic variation among humans and therefore play an important role when studying hereditary diseases or differential drug response. In this context, we show another possible application of the HeatMapViewer, again using the 7TM human rhodopsin (SwissProt identifier: OPSD_HUMAN13). The HeatMapViewer provides a fast and easy way to represent high dimensional data in a visually comprehensible way that immediately conveys where mutations are likely to be deleterious. Without using a tool such as the HeatMapViewer, we could hardly obtain an overview of the protein mutability landscape5. Mutability landscape studies involve predicting the effect of all possible nsSNPs through computational methods, visualizing the predictions in heat maps and cross-linking these predictions with additional sources of information (such as secondary structure, active sites and correlated mutational behavior). Such regions might highlight important aspects of RP. To this end, heat maps (Figure 2b, 2c) can easily distinguish between low effect regions (represented in blue) and high effect regions (represented in red) while additional information (such as the secondary structure; Figure 2a) can simply be over-laid. These two components already perfectly convey the information that high effect regions are mainly found in the transmembrane helices and in close proximity of the binding sites. Displaying this simple fact without a heat map would be daunting due to the high dimensionality of the underlying data.\n\nPanel a) sketches the secondary structure (helices in red, beta strands in blue). Panel b) shows the predictions of effects for each amino acid substitution. Effects are depicted as color intensities ranging from dark blue (high probability of no or little effect) over white (effect can not be predicted or only with very low reliability) to dark red (high probability of strong effects). Black depicts wildtype residues. The blue box marks the zoomed-in region shown in panel c).\n\n\nConclusions\n\nThe HeatMapViewer component provides a new, powerful way to generate and display matrix data in web presentations and in publications. The use of scalable graphics enables the rendering of high-resolution images as the interactive nature of the component permits those graphics to be scaled on-demand. Furthermore the component can be applied to different cases highlighting various points of interest from gene expression levels to the effects of mutability on protein function. Finally, to make the HeatMapViewer component widely accessible, we set up a public web server to which users can upload their matrix data and use the resulting code to show an interactive heat map.\n\n\nSoftware availability\n\nZenodo: HeatMap Viewer, doi: 10.5281/zenodo.770615\n\nGitHub: BioJS, https://github.com/biojs/biojs",
"appendix": "Author contributions\n\n\n\nGY developed the original HeatMapViewer component and maintains the HeatMapViewer service. MH contributed the SNP analysis use case. MPC and AY contributed the gene expression data use case. BR contributed the initial idea for the component and helped with its design.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nGY and MH are supported by a grant from the Alexander von Humboldt foundation through the German Ministry for Research and Education (BMBF: Bundesministerium fuer Bildung und Forschung).\n\n\nAcknowledgements\n\nThanks to Tim Karl and Laszlo Kajan (TUM) for invaluable help with hardware and software; to Marlena Drabik (TUM) for administrative support; to thanks Edda Kloppman and Tatyana Goldberg (TUM) for helpful discussions and invaluable feedback.\n\n\nReferences\n\nBromberg Y, Rost B: SNAP: predict effect of non-synonymous polymorphisms on function. Nucleic Acids Res. 2007; 35(11): 3823–35. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBromberg Y, Overton J, Vaisse C, et al.: In silico mutagenesis: a case study of the melanocortin 4 receptor. FASEB J. 2009; 23(9): 3059–69. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSunyaev S, Ramensky V, Koch I, et al.: Prediction of deleterious human alleles. Hum Mol Genet. 2001; 10(6): 591–7. PubMed Abstract | Publisher Full Text\n\nPauline CNg, Steven H: SIFT: Predicting amino acid changes that affect protein function. Nucleic Acids Res. 2003; 31(13): 3812–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHecht M, Bromberg Y, Rost B: News from the protein mutability landscape. J Mol Biol. 2013; 425(21): 3937–48. PubMed Abstract | Publisher Full Text\n\nGómez J, García LJ, Salazar GA, et al.: BioJS: an open source Javascript framework for biological data visualization. Bioinformatics. 2013; 29(8): 1103–4 2013. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPavlidis P, Noble WS: Matrix2png: a utility for visualizing matrix data. Bioinformatics. 2003; 19(2): 295–6. PubMed Abstract | Publisher Full Text\n\nXia J, Lyle NH, Mayer ML, et al.: INVEX–a web-based tool for integrative visualization of expression data. Bioinformatics. 2013; 29(24): 3232–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHeer J, Bostock M, Ogievetsky V: D3: Data-Driven Documents. IEEE Trans Vis Comput Graph. 2011; 17(12): 2301–9. PubMed Abstract | Publisher Full Text\n\nDryja TP, McGee TL, Reichel E, et al.: A point mutation of the rhodopsin gene in one form of retinitis pigmentosa. Nature. 1990; 343(6256): 364–6. PubMed Abstract | Publisher Full Text\n\nKent WJ, Sugnet CW, Furey TS, et al.: The human genome browser at ucsc. Genome Res. 2002; 12(6): 996–1006. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUniProt Consortium. Update on activities at the universal protein resource (uniprot) in 2013. Nucleic Acids Res. 2013; 41(Database issue): D43–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBoeckmann B, Blatter MC, Famiglietti L, et al.: Protein variety and functional diversity: Swiss-Prot annotation in its biological context. C R Biol. 2005; 328(10–11): 882–99. PubMed Abstract | Publisher Full Text\n\nPunta M, Coggill PC, Eberhardt RY, et al.: The Pfam protein families database. Nucleic Acids Res. 2012; 40(Database issue): D290–301. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYachdav G: HeatMap Viewer. Zenodo. 2014. Data Source"
}
|
[
{
"id": "3752",
"date": "27 Feb 2014",
"name": "Jordi Deu-Pons",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting article and piece of software. I think it contributes towards further alternatives to easily visualize high dimensionality data on the web. It’s simple and easy to embed into other web frameworks or applications.Minor revisionsa) About the software1 - CSV format. It was hard to guess the expected format. The authors need to add a syntax description of the CSV format at the help page.2 - Simple HTML example. It will be easy to test HeatmapViewer (HmV) if you add a simple downloadable example file with the minimum required HTML-JavaScript to set up a HmV (without all the CSV import code).3 - Color scale. HmV only implements a simple three point linear color scale. For me this is the major weakness of HmV. It will be very convenient that in the next HmV release the user can give as a parameter a function that manages the score to color conversion.b) About the paper1 - Introduction (4th paragraph): There are many alternatives to explore a dataset using heat maps. The author only cites two and it’s not clear if you refer to “JavaScript” or “web” alternatives. I think that you have to emphasize the strengths of HmV in comparison to other alternatives (in my opinion, one strength is that it is a good lightweight alternative to embed heat maps in a web report). Example of alternatives that I know of (but I’m sure that there are many more) are:http://www.broadinstitute.org/gsea (desktop)http://jheatmap.github.io/jheatmap/ (website)http://www.gitools.org/ (desktop)http://blog.nextgenetics.net/demo/entry0044/ (website)http://docs.scipy.org/doc/numpy/reference/generated/numpy.histogram2d.html (python)http://matplotlib.org/api/pyplot_api.html (python)2 - Predicted protein mutability landscape: The authors say: “Without using a tool such as the HeatmapViewer, we could hardly obtain an overview of the protein mutability landscape”. This paragraph seems to suggest that you can explore the data with HmV. I think that HmV is a good tool to report your data, but not to explore it.3 - Conclusions: The authors say: “... provides a new, powerful way to generate and display matrix data in web presentations and in publications.” To use heat maps in web presentations and publications is nothing new. I think that HmV makes it easier and user-friendly, but it’s not new.",
"responses": []
},
{
"id": "3682",
"date": "05 Mar 2014",
"name": "Jon C. Ison",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is very well written and tells a nice story, with compelling real-world examples. The software should have scientific impact by providing a convenient overview of matrix data, along the lines already suggested by the authors, and certainly by saving other scientist/developers the need to develop such functionality. The software fulfills a need for an easy to use widget for heatmap rendering and there is no doubt that this is a valuable contribution to the BioJS collection. However, I believe It would help many readers if the article placed the software in the broader context of similar offerings, so it would be good if the article enumerated these, perhaps as some sort of \"feature matrix\"? I appreciated the test server very much and encourage the authors to support this service in the future and further develop the functionality. Please note that examples of the CSV format are needed (at least I could not find them).",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-48
|
https://f1000research.com/articles/3-46/v1
|
13 Feb 14
|
{
"type": "Web Tool",
"title": "BioJS InterMineTable Component: A BioJS component for displaying data from InterMine compatible webservice endpoints",
"authors": [
"Alexis Kalderimis",
"Radek Stepan",
"Julie Sullivan",
"Rachel Lyne",
"Michael Lyne",
"Gos Micklem",
"Alexis Kalderimis",
"Radek Stepan",
"Julie Sullivan",
"Rachel Lyne",
"Michael Lyne"
],
"abstract": "Summary: The InterMineTable component is a reusable JavaScript component as part of the BioJS project. It enables users to embed powerful table-based query facilities in their websites with access to genomic data-warehouses such as http://www.flymine.org, which allow users to perform flexible queries over a wide range of integrated data types.Availability: http://github.com/alexkalderimis/im-tables-biojs; http://github.com/biojs/biojs; http://dx.doi.org/10.5281/zenodo.8301.",
"keywords": [
"There are currently a number of genomics data-warehouses available which are powered by the InterMine1 platform. This set includes large curated services dedicated to the primary Model Organism database (MOD) communities as part of the InterMOD project2",
"the collected data sets of research projects such as the modENCODE project3",
"as well as a range of other resources including metabolicMine4",
"TargetMine5",
"FlyTFMine6",
"and MitoMiner7. In addition to being accessible through web-interfaces these resources also provide web-service access (to be described in a forthcoming paper)."
],
"content": "Introduction\n\nThere are currently a number of genomics data-warehouses available which are powered by the InterMine1 platform. This set includes large curated services dedicated to the primary Model Organism database (MOD) communities as part of the InterMOD project2, the collected data sets of research projects such as the modENCODE project3, as well as a range of other resources including metabolicMine4, TargetMine5, FlyTFMine6, and MitoMiner7. In addition to being accessible through web-interfaces these resources also provide web-service access (to be described in a forthcoming paper).\n\nThe InterMine system provides users with a number of benefits. A typical InterMine instance, such as FlyMine8 or YeastMine9, contains feature annotations, protein data, publications, biochemical pathways, orthology, Gene Ontology (GO), array expression results, and other kinds of data, all integrated into a single knowledge graph. This means end users are able to ask questions across different data types. InterMine’s particular data integration strategy puts minimal limitations on the kinds of queries that can be performed: any arbitrary number of data-sets can be referred to in the same query (provided links exist between them) and a wide variety of logical contraints can be added. The InterMine platform thus provides a basis for very flexible, user-defined queries over linked data sets.\n\nThe BioJS10 project seeks to provide a suite of reusable JavaScript components that members of the bioinformatics community will find useful for producing analysis and visualisation tools. The InterMineTable BioJS component contributes towards this aim by adding a data query and exploration tool to the set of BioJS components which exposes the full flexibility and power of user defined queries over integrated linked data in a clear user interface.\n\nAs a visual BioJS component, the intended audience is web-developers aiming to provide extended functionality to web-based resources for life-scientists. It is expected to be deployed within modern browser environments with access to third-party resources. With this in mind, installation comprises of including the dependencies for the InterMine table component on the page (usually added in the head section of an HTML page), see Supplementary materials A. Once the dependency on the InterMine tables library is loaded, the InterMineTable BioJS component may be included (see code listing 1).\n\nListing 1. Loading the BioJS InterMine Table library\n\n\n\nThis last resource contains the definition of the InterMine.Table BioJS component. As it is not available from a reliable third party source, it currently needs to be downloaded from the BioJS registry11, and hosted locally.\n\nThis component is used by instantiating the InterMine table component in a user-included JavaScript file, passing in the appropriate configuration for the desired source of data as well as the query over those data.\n\nOnce instantiated, the results of the query against the specified integrated data-warehouse are loaded into a component where they can be browsed and manipulated. This means that the two critical concepts for using this component are 1) the location of the data-store, defined as the uniform resource locator (URL) pointing at the root of a set of web-services, and 2) the query to be run on the data in the store, defined in a configuration object. For example, to load a table of data from FlyMine the user would want the URL to point to FlyMine’s webservices:\n\nListing 2. Specifying the Data-Store\n\n\n\nThe query can be broadly defined as a list of fields, identified by paths from a root, constrained by a (possibly empty) set of filters. There are some refinements to this (such as sort-order, optional element definition, and constraint composition) for which more detailed documentation12 exists. The concept of a path is important to the idea of a graph of linked data, as it enables chains of relationships between entities to be followed, with minimal syntactic overhead. For example the chain of relationships the names of the protein domains of the proteins encoded by the genes belonging to a biochemical pathway can be referred to as Pathway.genes.proteins.proteinDomains.name.\n\nA query is defined as a plain JavaScript object which can be simple, such as the following query, which requests the common name, scientific name and taxon ID for all organisms in the data-store:\n\nListing 3. A simple Query, see Figure 1\n\n\n\nor arbitrarily complex, such as the following query which combines information from multiple data sources (OMIM13, PANTHER14, Treefam15, KEGG16, Reactome17, FlyBase18) and across different organisms to find the Drosophila melanogaster genes in the pathways of genes which are orthologous to human genes implicated in Alzheimer’s disease:\n\nListing 4. A Complex Query, see Figure 2\n\n\n\nAn element also needs to be present on the page where the table should be loaded. This can be any element (although a DIV element is conventional), and should be uniquely identifiable (through its ID for instance).\n\nListing 5. Defining the Target Element\n\n\n\nThese values are then passed to the component constructor, which builds a new table in the page, and loads the relevant data from the configured service:\n\nListing 6. Instantiation\n\n\n\nOnce instantiated, a table will be loaded into the page displaying rows of data as specified by the query (see Figure 1, Figure 2).\n\nThe table, as well as providing a number of common dynamic features such as resorting, pagination and column rearrangement, also permits much deeper interaction than other comparable table libraries. The table allows the underlying query to be changed: the constraints of the underlying query can be edited (Figure 3); columns (including ones referring to data types not in the original query) can be added; existing columns can be removed; changes made can be undone; the data can be exported in a number of formats or sent to another application, such as a local Galaxy19–21 instance, or to a remote application such as GenomeSpace22 (Figure 4); the results can be saved as a resusable set (a list) within the originating service; individual items can also be previewed (Figure 5).\n\nOne particularly useful feature is the ability to view the contents of a single column, analysing it on aggregate and adding or editing filters. This facility is able to present summary charts for columns based on data type: binned histograms for numerical data (Figure 6), and column charts for categorical data (Figure 7, showing the user adding a filter by selecting items from the column).\n\nThe standard mechanism for communication between components in JavaScript is event signalling. As per the BioJS specification, this component supports other objects registering event listeners so they may be notified when events of interest (such as user interactions) occur.\n\nOnce loaded, the table may emit a number of different events (as listed in the API documentation23), and may be manipulated by calling methods on the instance, allowing the calling page to respond to user interactions. For example, if a developer wished to receive notifications when the user clicks on any of the cells in the table, they can register to listen for these events:\n\nListing 7. Adding an Event Listener\n\n\n\nThis integration means that the table need not be an isolated part of an application, but can be fully integrated with other components. For example, instead of just notifying the user by using alert, the information about this object could be displayed in another component. If the user clicked on a protein, this could be detected, and other suitable components could be instantiated to display protein-specific analysis (see code sample 8).\n\nListing 8. Integrating with Other Components - Example 1\n\n\n\nAs well as responding to user interaction with the table, the table component exposes an API to change the state of the table by changing the query it represents. This allows communication in the other direction. For example if a linked component, such as a protein structure displayer, emits an event indicating the user has selected a given set of protein domains, the table could be modified by adding a filter for these domains to the current query (see code sample 9):\n\nListing 9. Integrating with Other Components - Example 2\n\n\n\nIn this way, the interoperability of these components makes them of increasing utility to developers, as more of them are published and integrated into third party applications.\n\n\nDiscussion\n\nThis tool addresses an important set of needs for bioinformatics developers. While there are some other libraries that aim to make creating dynamic tables easy to develop (such as DataTables24), there are none that tightly integrate facilities such as query constraint editing, column summary analysis, data export, support for genomic data types, and list management without a great deal of development effort. Access to flexible and powerful data-warehousing tools on the InterMine platform makes these table components uniquely powerful in the life sciences sphere. Services that implement the InterMine public API are also able to make use of the features of these tables. The extremely high quality data sets curated by the principal Model Organism databases, which the the InterMOD project members make available, makes this tool unique in its immediate access to integrated life sciences data.\n\n\nConclusions\n\nIt is hoped that this component will prove useful to those developing tools for researchers in the life sciences. Various publically funded groups, such as the MODs and other data-producing consortia, have put significant effort into creating, curating and composing high quality data sets. InterMine is an effective platform to add value to this work by integrating the data and exposing a flexible query API and user interface. The recent work in exposing these resources through web-services and producing reusable web-based components allows this investment to benefit not just visitors to the sites of InterMine applications, but any developer or user who wants to include complex query tools as part of their platform. With access to a broad range of data sources meeting the needs of several diverse research communities, we expect that a great deal of duplicated effort can be avoided, saving significant amounts of time and money.\n\n\nSoftware availability\n\nZenodo: im-tables-biojs, doi: 10.5281/zenodo.830125\n\nGitHub: BioJS, http://github.com/biojs/biojs.",
"appendix": "Author contributions\n\n\n\nAlex Kalderimis wrote the manuscript and implemented the component, under the supervision of Gos Micklem, to a set of user specifications supplied by Julie Sullivan. Rachel Lyne, Radek Štěpán and Mike Lyne contributed to the component design and revised the manuscript. All authors have approved the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nInterMine has been developed with the support of the following grants, awarded to Dr. G. Micklem: the Wellcome Trust (Grant number: 090297), and the National Human Genome Research Institute (Grant number: R01HG004834). The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding bodies.\n\n\nAcknowledgements\n\nThe authors thank Manuel Corpas for useful feedback.\n\n\nSupplementary materials\n\n\n\n\nReferences\n\nSmith RN, Aleksic J, Butano D, et al.: InterMine: a flexible data warehouse system for the integration and analysis of heterogeneous biological data. Bioinformatics. 2012; 28(23): 3163–3165. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSullivan J, Karra K, Moxon SA, et al.: InterMOD: integrated data and tools for the unification of model organism research. Sci Rep. 2013; 3: 1802. PubMed Abstract | Publisher Full Text | Free Full Text\n\nContrino S, Smith RN, Butano D, et al.: modMine: flexible access to modENCODE data. Nucleic Acids Res. 2012; 40(Database issue): D1082–1088. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLyne M, Smith RN, Lyne R, et al.: metabolicMine: an integrated genomics, genetics and proteomics data warehouse for common metabolic disease research. Database (Oxford). 2013; 2013: bat060. PubMed Abstract | Publisher Full Text\n\nChen YA, Tripathi LP, Mizuguchi K: TargetMine, an integrated data warehouse for candidate gene prioritisation and target discovery. PLoS ONE. 2011; 6(3): e17844. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAdryan B, Teichmann SA: FlyTF: a systematic review of site-specific transcription factors in the fruit fly Drosophila melanogaster. Bioinformatics. 2006; 22(12): 1532–1533. PubMed Abstract | Publisher Full Text\n\nSmith AC, Robinson AJ: MitoMiner, an integrated database for the storage and analysis of mitochondrial proteomics data. Mol. Cell Proteomics. 2009; 8(6): 1324–1337. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLyne R, Smith R, Rutherford K, et al.: FlyMine: an integrated database for Drosophila and Anopheles genomics. Genome Biol. 2007; 8(7): R129. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBalakrishnan R, Park J, Karra K, et al.: YeastMine–an integrated data warehouse for Saccharomyces cerevisiae data as a multipurpose tool-kit. Database (Oxford). 2012; 2012: bar062. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGómez J, García LJ, Salazar GA, et al.: BioJS: an open source JavaScript framework for biological data visualization. Bioinformatics. 2013; 29(8): 1103–1104. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBiojs project registry. Reference Source\n\nPathquery documentation. Reference Source\n\nMcKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, MD) Online mendelian inheritance in man, omim®. 2013. Reference Source\n\nMi H, Muruganujan A, Thomas PD: PANTHER in 2013: modeling the evolution of gene function, and other gene attributes, in the context of phylogenetic trees. Nucleic Acids Res. 2013; 41(Database issue): D377–386. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi H, Coghlan A, Ruan J, et al.: TreeFam: a curated database of phylogenetic trees of animal gene families. Nucleic Acids Res. 2006; 34(Database issue): D572–580. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOgata H, Goto S, Sato K, et al.: KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 1999; 27(1): 29–34. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJoshi-Tope G, Gillespie M, Vastrik I, et al.: Reactome: a knowledgebase of biological pathways. Nucleic Acids Res. 2005; 33(Database issue): D428–432. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarygold SJ, Leyland PC, Seal RL, et al.: Fly-Base: improvements to the bibliography. Nucleic Acids Res. 2013; 41(Database issue): D751–757. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGoecks J, Nekrutenko A, Taylor J, et al.: Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol. 2010; 11(8): R86. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBlankenberg D, Von Kuster G, Coraor N, et al.: Galaxy: A web-based genome analysis tool for experimentalists. Curr Protoc Mol Biol. 2010; Chapter 19: 19.10.1–19.10.21. PubMed Abstract | Publisher Full Text\n\nGiardine B, Riemer C, Hardison RC, et al.: Galaxy: a plat-form for interactive large-scale genome analysis. Genome Res. 2005; 15(10): 1451–1455. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGenomespace. Reference Source\n\nBiojs project api documentation. Reference Source\n\nDatatables. Reference Source\n\nKalderimis A, Micklem G: im-tables-biojs. Zenodo. 2014. Data Source"
}
|
[
{
"id": "3676",
"date": "18 Feb 2014",
"name": "Arnaud Ceol",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nReusable libraries for building web applications are very welcome and allow interested groups to build modern, dynamic web tools without losing time to learning new technologies and having to study data access from various different resources.Here, the authors propose a library to query all InterMine repositories. The result is a powerful tool: it is not only the web developers who can use this library to access in theory all the InterMine repositories, but users too can build and update queries as well as table directly from the web page by selecting graphically all fields and columns.The manuscript provides some useful examples of possible queries. Starting from the example at the address http://zenodo.org/record/8301 (selected from the Software Availability section), it is easy to have a running page that can be modified.Nevertheless, starting a page from scratch is not as straight forward as it could be because the availability links and the installation description are confusing. It is not clear what the meaning of the data availability links provided are (there are three links in the abstract under the heading “availability” and two in the Software Availability section). The authors may want to add a short description to each so the user can redirect to the one he/she is interested in. Besides, in the Installation section of the manuscript it is written that the “last resource contains the definition of the InterMine.Table BioJS component. As it is not available from a reliable third party source, it currently needs to be downloaded from the BioJS registry, and hosted locally”. This is where the confusion is: what should be downloaded and from where? I didn't find references to the InterMine tables in Zenodo or from the GitHub repository. In the example (Zenodo), it looks like the code is actually inserted in the index.js file.One of the advantages of the library proposed here is that it is based on the InterMine repositories. In the manuscript the authors provide the example of the FlyMine query, but the component should also be able to query all the others InterMine repositories (I was able to find the updated code on GitHub reference to the worm repository). From what I understand from the author’s comments is that a separate article should soon be available and will describe the web service implemented by the InterMine repositories and which are used by this BioJS library. It makes it difficult to understand what queries can be done (i.e. what are the tables and fields that can be queried). Nevertheless, this can be inferred by using the example provided (using the manage columns and adding filter buttons on the example page). The authors may want to provide a supplementary table with the address of the web services that can be queried (or a link to another registry if it exists).In the Supplementary material A section, the last “<script/>” should be </script>.To summarize, I believe that this library is useful and works well, and that after a few clarifications in the manuscript it will help the user integrate straightforward InterMine tables into their own webpages.",
"responses": []
}
] | 1
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https://f1000research.com/articles/3-46
|
https://f1000research.com/articles/3-45/v1
|
13 Feb 14
|
{
"type": "Web Tool",
"title": "BioJS InterMine List Analysis: A BioJS component for displaying graphical or statistical analysis of collections of items from InterMine endpoints",
"authors": [
"Alexis Kalderimis",
"Radek Stepan",
"Julie Sullivan",
"Rachel Lyne",
"Michael Lyne",
"Gos Micklem",
"Alexis Kalderimis",
"Radek Stepan",
"Julie Sullivan",
"Rachel Lyne",
"Michael Lyne"
],
"abstract": "Summary: The InterMineTable component is a reusable JavaScript component as part of the BioJS project. It enables users to embed powerful table-based query facilities in their websites with access to genomic data-warehouses such as http://www.flymine.org, which allow users to perform flexible queries over a wide range of integrated data types.Availability: http://github.com/alexkalderimis/im-tables-biojs; http://github.com/biojs/biojs; http://dx.doi.org/10.5281/zenodo.8301.",
"keywords": [
"InterMine1 is a platform for building data warehouses which includes specialisations for the life-sciences. As part of the InterMOD2 project",
"a number of InterMine data-warehouses have been developed and released to the public containing high-quality integrated data curated by the major model organism database (MOD) organisations. In addition",
"the InterMine platform is widely used by other projects",
"such as the modENCODE project3",
"as well as a range of other resources including metabolicMine4",
"TargetMine5",
"FlyTFMine6",
"and MitoMiner7. This means that reliable integrated data sets exist for use by researchers working in a wide range of fields in the life-sciences",
"which can be accessed by a common interface."
],
"content": "Introduction\n\nInterMine1 is a platform for building data warehouses which includes specialisations for the life-sciences. As part of the InterMOD2 project, a number of InterMine data-warehouses have been developed and released to the public containing high-quality integrated data curated by the major model organism database (MOD) organisations. In addition, the InterMine platform is widely used by other projects, such as the modENCODE project3, as well as a range of other resources including metabolicMine4, TargetMine5, FlyTFMine6, and MitoMiner7. This means that reliable integrated data sets exist for use by researchers working in a wide range of fields in the life-sciences, which can be accessed by a common interface.\n\nOne of the features of the InterMine system is the ability to store named sets of entities, called lists, and refer to them in queries and other analysis. This allows a user, for example, to save a list of genes and reuse this saved collection easily. The InterMine system also allows specialised analysis to be performed taking advantage of the integrated nature of the data warehouse system. For example the system can run queries that aggregate information about relationships between data types, and provide indications of levels of statistical significance for the results (enrichment queries).\n\nUntil recently, the output of these list analysis tools was only accessible through the web-application built into the InterMine system. Recent work on the InterMine web services has enabled this functionality to be externalised into the list-widgets8 project: separate JavaScript-based components that can be used in third party websites. These developments have already been incorporated into the standard InterMine web-application configuration, meaning that users of the tools described here have access to the same query and display mechanisms in their own sites that are available through the standard InterMine web-application.\n\nInterMine supports the aims of the BioJS9 initiative to provide well-designed, robust website components to application developers in order to foster code reuse and minimise duplicated effort. This leads us to contribute to the BioJS project this set of components for running list analysis tools and displaying their output, so that they may be widely distributed, and interoperate with tools from other developers.\n\nAs a JavaScript web component, these tools are designed to be run within the JavaScript virtual machines provided by modern browsers, and render to HTML pages. Installation means indicating to the remote client (the user), which resources to load as dependencies, as well as where these are located. Typically this is done by adding references to these resources in the head section of a page through the use of script element (see code sample 1). Recent practice suggests loading these resources in at the end of the body improves page load time. The dependencies that must be loaded to use these tools are listed in Supplementary materials A.\n\nThe BioJS InterMine list analysis library needs to be downloaded from the BioJS registry10 and hosted in an accessible location.\n\nListing 1. Loading the list analysis tools library.\n\n\n\nOnce the BioJS component and its dependencies are loaded, the component itself may be instantiated, which creates a new list analysis displayer, inserts it into the document, and populates it with the appropriate data by calling to the InterMine web-services. This requires that an element exists within the document (see code listing 2) into which the component can be inserted.\n\nListing 2. The target document element\n\n\n\nThe JavaScript code to instantiate the component refers to this element as the target, and provides the other arguments required to specify which list we wish to analyse, the url of the service where that list is to be found, and which specific analysis tool we wish to run. The example below uses a list of genes encoding putative Drosophila melanogaster transcription factors made available as a public list at FlyMine11 and runs the pathway enrichment statistical analysis tool. The full list of available lists (which each user can extend by creating personal lists) and analysis tools can be accessed from the InterMine service being used.\n\nOne category of tools is the enrichment tools, which run queries that attempt to find relationships that are statistically significant for the set of entities as a whole. For example, FlyMine11 contains both genes, loaded from sources such as FlyBase12, and biochemical pathways, loaded from sources such as KEGG13 and Reactome14. The pathways enrichment tool lists pathways of which genes in the list are members, ordered by the degree of significance for the list of genes as a whole.\n\nFor example, if one gene in a list is in a particular pathway, but none of the others are, it would be considered less significant than a pathway that all or most genes in a list belonged to. Similarly, the background probability that a particular relationship exists for an item is taken into account, meaning for example that finding a publication that lists many or even all genes for a organism, such as Clark 200715, would not be considered as significant as a publication that mentions fewer genes, but with most of them being in the list of interest.\n\nThe p-values used as measures of statistical significance are calculated by modelling the relationships as a hypergeometric distribution (as Rivals 200716 and Beissbarth 200417), which determines the probability that a relationship between two entities would be selected at random given the set of items to choose from. Let n be the number of items in the list, and N be the size of the reference population, and k be the number of items in the list which are involved in the given relationship (are mentioned in the publication, for example, or belong to a particular biochemical pathway), and M be the number of items in the reference population which share that same relationship. Then for each relationship\n\n\n\nThe options made available for multiple test correction include the Bonferroni, Holm-Bonferroni, and Benjamini Hochberg18 algorithms.\n\nThe tools in this category are all prefixed with enrichment:, and can be loaded as follows:\n\nListing 3. Loading an enrichment list analysis tool.\n\n\n\nOnce run, the component should be inserted into the document (see Figure 1). The component allows the user to adjust the parameters of the analysis, including the multiple test correction method used, the p-value threshold and the background population.\n\nThe component also allows the user to interact with the results in a number of ways, specifically: by clicking on an individual item that was matched; by clicking on a button to show a set of matches; and by clicking on a button to request that the selected items be saved to some location. All these actions cause the component to emit events, which can be listened for and handled by the host JavaScript application. For example, to alert a string such as Gene - FGBN0123 when a user clicks on the corresponding element, one might attach an event listener to capture the onClickMatch event, see code listing 4.\n\nListing 4. Listening for a click event.\n\n\n\nThis enables the behaviour of the component to be integrated into the hosting application. The full listing of events and their arguments is included in the BioJS API documentation19.\n\nThe canonical example for the use of statistical enrichment in bioinformatics is enrichment of Gene Ontology (GO) terms for sequence annotations (Rivals 200716). This functionality is supported as one of the statistical analysis tools (see Figure 2), within this more generic enrichment analysis framework. The GO enrichment tool merits some further notes, however, as it supports some of the more advanced parameters.\n\nThe GO enrichment tool demonstrates the use of optional filter parameters to limit the results in some way. In the GO tool, it allows the user to select the sub-ontology they are interested in. The user can also choose to normalise the results of this tool, in this case by transcript length.\n\nThe other main category of analysis tools is the chart tools. These run aggregate queries over the items in a list, and present the information graphically in interactive charts. The InterMine system supports both numerical and categorical charting, reflected in the supported chart formats: bar charts, line charts, pie charts and scatterplots.\n\nLoading a chart analysis tool is identical to loading a statistical enrichment tool - only the name of the tool need differ (see code listing 5).\n\nListing 5. Loading a chart list analysis tool.\n\n\n\nThis code will request data for the particular tool (flyfish), as run against the given input list (PL FlyTF_putativeTFs), and then display the results in the appropriate chart format (Figure 3). The chart tools have fewer parameters; they may take a single parameter, as detailed in the tool description available from the relevant service (e.g. http://www.flymine.org/query/service/widgets).\n\nIn most cases they do not provide mechanisms for the user to change the results displayed. They do however provide several mechanisms for the user to interact with the results displayed. The user can click on the groupings or data-points represented on the chart (see Figure 4), which allows the user to trigger the same events available to enrichment tools, which can be captured the same way (see code listing 4).\n\n\nDiscussion\n\nThis tool addresses an important set of needs for bioinformatics developers: the ability to perform enrichment analysis, and the the visualisation of typed relationships between entities. The InterMine platform, and this BioJS component make performing these analyses and displaying the output straightforward. It allows the developers to focus on integrating this functionality where it is needed, and users to focus on interpreting rather than retrieving the data. It is expected that wide availability of these tools will provide significant savings in time for typically stretched developers and researchers. By providing this functionality as a BioJS component, it is hoped that integration between different tools will result in the creation of applications that are able to integrate analysis and visualisation from different platforms.\n\n\nConclusions\n\nIt is hoped that this component will prove useful to those developing tools for researchers in the life-sciences. Significant work has gone into creating, curating and combining high quality data sets. The recent work in exposing these resources through web-services and producing reusable web-based components allows this investment to benefit not just visitors to sites based on InterMine applications, but any developer or user who aims to include this kind of statistical analysis and visualisation in their platform. By providing bioinformatics web-developers, and their users, with access to a broad range of data sources meeting the needs of many diverse research communities, we expect to help reduce the development burden on projects with limited resources, and help minimise redundancy of effort.\n\n\nSoftware availability\n\nZenodo: BioJS InterMine List Analysis Widgets, doi: 10.5281/zenodo.830221.\n\nGitHub: BioJS, http://github.com/biojs/biojs.",
"appendix": "Author contributions\n\n\n\nAlex Kalderimis wrote the manuscript and implemented the BioJS wrapper, under the supervision of Gos Micklem, to a set of user specifications supplied by Julie Sullivan. Radek Štěpán implemented the list analysis component, based on designs and specification from Julie Sullivan, Rachel Lyne, Mike Lyne and Alex Kalderimis. Rachel Lyne and Mike Lyne contributed to the component design and revised the manuscript. All authors have approved the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nInterMine has been developed with the support of the following grants, awarded to Dr. G. Micklem: the Wellcome Trust (Grant number: 090297), and the National Human Genome Research Institute (Grant number: R01HG004834). The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding bodies.\n\n\nAcknowledgements\n\nThe authors thank Manuel Corpas for useful feedback.\n\n\nSupplementary materials\n\n\n\n\nReferences\n\nSmith RN, Aleksic J, Butano D, et al.: InterMine: a flexible data warehouse system for the integration and analysis of heterogeneous biological data. Bioinformatics. 2012; 28(23): 3163–3165. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSullivan J, Karra K, Moxon SA, et al.: InterMOD: integrated data and tools for the unification of model organism research. Sci Rep. 2013; 3: 1802. PubMed Abstract | Publisher Full Text | Free Full Text\n\nContrino S, Smith RN, Butano D, et al.: modMine: flexible access to modENCODE data. Nucleic Acids Res. 2012; 40(Database issue): D1082–D1088. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLyne M, Smith RN, Lyne R, et al.: metabolicMine: an integrated genomics, genetics and proteomics data warehouse for common metabolic disease research. Database (Oxford). 2013; 2013: bat060. PubMed Abstract | Publisher Full Text\n\nChen YA, Tripathi LP, Mizuguchi K: TargetMine, an integrated data warehouse for candidate gene prioritisation and target discovery. PLoS ONE. 2011; 6(3): e17844. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAdryan B, Teichmann SA: FlyTF: a systematic review of site-specific transcription factors in the fruit fly Drosophila melanogaster. Bioinformatics. 2006; 22(12): 1532–1533. PubMed Abstract | Publisher Full Text\n\nSmith AC, Robinson AJ: MitoMiner, an integrated database for the storage and analysis of mitochondrial proteomics data. Mol Cell Proteomics. 2009; 8(6): 1324–1337. PubMed Abstract | Publisher Full Text | Free Full Text\n\nList widgets project. Reference Source\n\nGómez J, García LJ, Salazar GA, et al.: BioJS: an open source JavaScript framework for biological data visualization. Bioinformatics. 2013; 29(8): 1103–1104. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBiojs project registry. Reference Source\n\nLyne R, Smith R, Rutherford K, et al.: FlyMine: an integrated database for Drosophila and Anopheles genomics. Genome Biol. 2007; 8(7): R129. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarygold SJ, Leyland PC, Seal RL, et al.: Fly-Base: improvements to the bibliography. Nucleic Acids Res. 2013; 41(Database issue): D751–757. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOgata H, Goto S, Sato K, et al.: KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 1999; 27(1): 29–34. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJoshi-Tope G, Gillespie M, Vastrik I, et al.: Reactome: a knowledgebase of biological pathways. Nucleic Acids Res. 2005; 33(Database issue): D428–432. PubMed Abstract | Publisher Full Text | Free Full Text\n\nClark AG, Eisen MB, Smith DR, et al.: Evolution of genes and genomes on the Drosophila phylogeny. Nature. 2007; 450(7167): 203–218. PubMed Abstract | Publisher Full Text\n\nRivals I, Personnaz L, Taing L, et al.: Enrichment or depletion of a GO category within a class of genes: which test?. Bioinformatics. 2007; 23(4): 401–407. PubMed Abstract | Publisher Full Text\n\nBeissbarth T, Speed TP: GOstat: find statistically overrepresented Gene Ontologies within a group of genes. Bioinformatics. 2004; 20(9): 1464–1465. PubMed Abstract | Publisher Full Text\n\nBenjamini Y, Hochberg Y: Controlling the false discovery rate: A practical and powerful approach to multiple testing. J R Stat Soc Series B Methodol. 1995; 57(1): 289–300. Publisher Full Text\n\nBiojs project api documentation. Reference Source\n\nLécuyer E, Yoshida H, Parthasarathy N, et al.: Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function. Cell. 2007; 131(1): 174–187. PubMed Abstract | Publisher Full Text\n\nKalderimis A, Micklem G, Stepan R: BioJS InterMine List Analysis Widgets. Zenodo. 2014. Data Source"
}
|
[
{
"id": "5874",
"date": "13 Oct 2014",
"name": "Clemens Wrzodek",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe Manuscript:The Article is very clearly written and formatted. It strongly focuses on the end-users that want to use the published library and describes how to include it and what possibilities it offers. The examples shown in the manuscript are nice and well-picked. The manuscript is interesting and easy-to-read.I did not found it useful to include the formula for the hypergeometric test in the manuscript. Nearly every manuscript that even mentions enrichments of lists of genes depicts this formula. It is already known very well by researchers in this area and for beginners, the information provided is rarely sufficient. Personally, I would remove it.I would recommend the authors to rethink the title. Maybe something shorter like \"BioJS InterMine List Analysis: A BioJS component for displaying InterMine analysis results\" might be less-confusing (even though it misses the information about the various possible endpoints). What-is-published-here:However, what is published here is only the approximate 400 lines-of-code long BioJS-wrapper for Intermine (available as a single JS file on GitHub). It's not the implementation of the described analysis methods, nor is it the Intermine library itself. It's just the plain BioJS wrapper for the analysis methods offered by the Intermine endpoints.Actual source code:I tested the provided Demo on GitHub:Works very well in Chrome 37.It does not work in IE10 (\"Unable to construct query: 8070000c\").Works in Firefox (although the grid on-mouse over-popup behaves different than in Chrome).It may be nice to mention some information about Browser compatibility in the manuscript and on GitHub. I further tested the automated code generation, available from a button in the upper-right corner (specifically, the Java-Code). That worked well. I extended the Demo JavaScript file and played a bit with the information, provided in the manuscript. Everything seemed to work well. Generally, the code listings in the manuscript are very helpful when working with the library. Also, the Demo file on Github helps getting started. The JavaScript code itself is well documented with comments.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-45
|
https://f1000research.com/articles/3-44/v1
|
13 Feb 14
|
{
"type": "Software Tool Article",
"title": "PsicquicGraph, a BioJS component to visualize molecular interactions from PSICQUIC servers",
"authors": [
"Jose M. Villaveces",
"Rafael C. Jimenez",
"Bianca H. Habermann",
"Jose M. Villaveces",
"Rafael C. Jimenez"
],
"abstract": "Summary: Protein interaction networks have become an essential tool in large-scale data analysis, integration, and the visualization of high-throughput data in the context of complex cellular networks. Many individual databases are available that provide information on binary interactions of proteins and small molecules. Community efforts such as PSICQUIC aim to unify and standardize information emanating from these public databases. Here we introduce PsicquicGraph, an open-source, web-based visualization component for molecular interactions from PSIQUIC services. Availability: PsicquicGraph is freely available at the BioJS Registry for download and enhancement. Instructions on how to use the tool are available here http://goo.gl/kDaIgZ and the source code can be found at http://github.com/biojs/biojs and DOI:10.5281/zenodo.7709.",
"keywords": [
"cell"
],
"content": "Introduction\n\nProteins are one of the major actors in cellular processes and perform many different functions, which are required for the survival of a cell and an organism. Depending on the cell type, a different set of proteins will be available to ensure proper functioning of the cell within a larger context, for instance an organ. Typically, a cellular process is controlled by many different proteins that form a sophisticated network of interactions. Some proteins are even part of larger complexes, so-called molecular machines and the majority of interacting members are required to carry out a specific molecular task. In Systems Biology, we can use the networks of protein interactions to help us understand highly complex cellular processes.\n\nDifferent efforts have been used to collect protein interactions. For example IntAct, an open-source, open data molecular interaction database1 contains approximately 275 000 curated binary interactions extracted from over 5000 publications. ChEMBL is another example of an open source database2 and holds more than 600 000 interactions between proteins and small molecules (chemicals).\n\nIn order to standardize access to interaction databases, the Proteomics Standard Initiative proposed the Proteomics Standard Initiative Common QUery InterfaCe (PSICQUIC)3 that defines:\n\n1. a web service with well defined methods to enable programmatic access to molecular interactions.\n\n2. a Molecular Interactions Query Language (MIQL4), that specifies a syntax to allow flexible queries.\n\n3. a registry, that lists available PSICQUIC services and enables providers of databases for molecular interactions to register.\n\nMeanwhile, 28 different databases have registered with PSICQUIC, including IntAct and ChEMBL, which altogether contain more than 150 million binary interactions.\n\nHere, we present PsicquicGraph, a web component to visualize molecular interactions from PSICQUIC services. We have realized PsicquicGraph using BioJS5, an open source JavaScript library of components for visualization of biological data on the web.\n\n\nThe PsicquicGraph component\n\nThe minimal input for PsicquicGraph is (i) the URL of a valid PSICQUIC server, (ii) a valid MIQL query, (iii) a target container (HTML tag; usually a DIV) identifier to render the interactions graph and (iv) a proxy URL to bypass the same domain policy constraint in JavaScript.\n\nUsing the MIQL query, PsicquicGraph queries the PSICQUIC server. After retrieving the interactions in PSIMITAB6 format, the interactions are parsed by PsicquicGraph and the graph is rendered using Cytoscape.js7 (Figure 1a).\n\nThe VEGF pathway is an essential regulator of vasculogenesis, as well as angiogenesis. In adults, it is up-regulated in a vast number of tumors. Solid tumors often build novel blood vessels and VEGF is one important regulator in that development. It is also a drug target in tumor medicine and several drugs directly target the VEGF receptor to block blood vessel formation in tumors. (a) Default values were used to define the layout as well as other visualization options. (b) A force-directed layout was used to render the graph and other visualization options such as node shape, node color and node label were customized.\n\nThe code below illustrates how to initialize PsicquicGraph by providing the minimal input. The query defined finds the first 100 human interactions (restricted by maxResults) and the psicquicUrl provided corresponds to the IntAct database. The name given to target constitutes the identifier of the component container.\n\n\n\nBy default, PsicquicGraph renders the graph using a circle layout. However, other layouts (force-directed, hierarchy, grid, random and preset) can be defined while initializing the component. Similarly, different visualization attributes such as node shape, color and font family can be defined (Figure 1b).\n\n\nConclusions\n\nPsicquicGraph is a publicly available web component to render interactions from PSICQUIC servers. It relies on PSICQUIC and open data databases in order to simplify the rendering of complex protein-protein interaction networks.\n\nThe adoption of the BioJS specification facilitates PsicquicGraph integration, testing and documentation in addition to the potential exposure to new users.\n\n\nSoftware availability\n\nZenodo: PsicquicGraph, a BioJS component to visualize molecular inteactions from PSICQUIC servers, doi: 10.5281/zenodo.77098.\n\nGitHub: BioJS, http://github.com/biojs/biojs",
"appendix": "Author contributions\n\n\n\nJosé M. Villaveces: Tool development, manuscript preparation. Rafael C. Jimenez: Project supervision. Bianca H. Habermann: Manuscript preparation, project supervision. All authors critically revised the manuscript and agreed to its publication.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nJMV was financed by BMBF-grant 315737 (Virtual Liver Network). This work was supported by the Max Planck Society.\n\n\nReferences\n\nKerrien S, Aranda B, Breuza L, et al.: The IntAct molecular interaction database in 2012. Nucleic Acids Res. 2012; 40(Database issue): D841–D846. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGaulton A, Bellis LJ, Bento AP, et al.: ChEMBL: a large-scale bioactivity database for drug discovery. Nucleic Acids Res. 2012; 40(Database issue): D1100–D1107. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAranda B, Blankenburg H, Kerrien S, et al.: PSICQUIC and PSISCORE: accessing and scoring molecular interactions. Nat Methods. 2011; 8(7): 528–529. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMIQL. Accessed: 2014-16-01. Reference Source\n\nGómez J, García LJ, Salazar GA, et al.: BioJS: an open source JavaScript framework for biological data visualization. Bioinformatics. 2013; 29(8): 1103–1104. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKerrien S, Orchard S, Montecchi-Palazzi L, et al.: Broadening the horizon--level 2.5 of the HUPO-PSI format for molecular interactions. BMC Biol. 2007; 5: 44. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCytoscape.js. Accessed: 2013-11-06. Reference Source\n\nJosé MV, Rafael CJ, Bianca H: PsicquicGraph, a BioJS component to visualize molecular inteactions from PSICQUIC servers. Zenodo. 2014. Data Source"
}
|
[
{
"id": "3841",
"date": "24 Feb 2014",
"name": "Christine Orengo",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis short article describes a component (PsicquicGraph) within the larger javascript library (BioJS) that assists the visualisation of molecular interactions on web pages. The component effectively provides the glue to stick the following pieces of technology together: - Cytoscape.js (a powerful tool for viewing networks on web pages) - MILQ (a language for querying molecular interactions) - BioJS (an event-driven javascript library which facilitates interaction between javascript widgets) The component described in this article is already useful - mainly in that it provides a standard way of interacting with these existing modules and provides a single point of focus for future development. However there are some obvious developments the authors should make in a following version of the article that would make the component more useful:Registering events (such as when a user selects a \"node\" or \"edge\").Providing access to the underlying \"cytoscape\" javascript instance (to allow manipulation of the network after it has been created).",
"responses": []
},
{
"id": "4210",
"date": "25 Mar 2014",
"name": "Frederic de Lamotte",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis web tool article describes a component of BioJS, a javascript library of reusable components for the presentation of biological data. This particular tool, PsicquicGraph, is a web component intended to visualize molecular interactions from PSICQUIC servers.PSICQUIC servers offer a standardised method for accessing proteomic interaction databases. From a biologists point of view, PsicquicGraph and the BioJS initiative are extremely positive as it will allow for the retrieval of information from various databases in a seamless manner. Technically speaking, PsicquicGraph cleverly takes advantage of BioJS, Cytoscape.js as well as MILQ by providing useful interactions within all these modules. The links provided in this article allows access to decent documentation of the tool and also provides a useful working example.ImprovementsI consider that this article could be improved by listing the 28 databases that the authors claim to have registered with PSICQUIC and making them available in this article. That would allow the reader to easily figure out how important this standardization is.I spent some times trying, but I was not successful in recreating the example in the window mode as advised by the authors (I tried Chrome, Firefox and Safari). I was thus not able to experience the demo example completely, which is bad for a web tool.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-44
|
https://f1000research.com/articles/3-43/v1
|
13 Feb 14
|
{
"type": "Software Tool Article",
"title": "KEGGViewer, a BioJS component to visualize KEGG Pathways",
"authors": [
"Jose M. Villaveces",
"Rafael C. Jimenez",
"Bianca H. Habermann",
"Jose M. Villaveces",
"Rafael C. Jimenez"
],
"abstract": "Summary: Signaling pathways provide essential information on complex regulatory processes within the cell. They are moreover widely used to interpret and integrate data from large-scale studies, such as expression or functional screens. We present KEGGViewer a BioJS component to visualize KEGG pathways and to allow their visual integration with functional data. Availability: KEGGViewer is an open-source tool freely available at the BioJS Registry. Instructions on how to use the tool are available at http://goo.gl/dVeWpg and the source code can be found at http://github.com/biojs/biojs and DOI:10.5281/zenodo.7708.",
"keywords": [
"insulin"
],
"content": "Introduction\n\nNetworks and network-based techniques are widely used in systems biology to model biological processes such as gene regulation, protein interactions and signaling pathways. Signaling pathways in particular, provide an understanding of cell dynamics by describing step by step the temporal interactions that a group of molecules or metabolites undergo in order to control one or more cellular functions.\n\nDifferent attempts have been made to store and aid the retrieval and analysis of signaling pathways. For example the Kyoto Encyclopedia of Genes and Genomes (KEGG)1 contains a large collection of manually curated pathway maps. Panther Pathway2, as another example, provides access to a number of mainly signaling pathways, subfamilies and protein sequences mapped to individual pathway components.\n\nKEGG is widely used by researchers to retrieve pathway information. Pathway maps in KEGG can be downloaded as static PNG images or alternatively as KEGG Markup Language (KGML) files (free of charge for academic use). KGML is an XML-like format that describes a pathway, its components and relationships and can, for instance, be used to visualize pathways3, generate systems biology models4 or perform network analysis5.\n\nLarge-scale techniques like expression arrays, deep sequencing or proteomics allow monitoring the relative or absolute level of expression for a large number of genes simultaneously. However, expression profiling by itself is not sufficient to understand the exact role of a set of genes in a biological process. In order to gain new insights into the regulatory relationships of differentially regulated genes, expression profiles from a large-scale study can be integrated with signaling pathways.\n\nHere, we present KEGGViewer, software that allows visual integration of KEGG pathways and expression profiles. We have coded KEGGViewer in BioJS6, a JavaScript library that holds components for visualizing biological data on the web. The KEGGViewer component is open source and freely available at http://goo.gl/dVeWpg.\n\n\nThe KEGGViewer component\n\nTo run KEGGViewer (i) a target DIV ID (unique identifier) to render the pathway, (ii) a KEGG pathway ID and (iii) a proxy URL to bypass the same domain policy constraint in JavaScript are required. The following code snippet illustrates how to initialize the component:\n\n\n\nWith that input, KEGGViewer queries the KEGG API7 in order to obtain the KGML-formatted KEGG pathway. Once retrieved, the KGML file is parsed by KEGGViewer and the pathway is rendered using Cytoscape.js8 Figure 1a).\n\n(a) KEGGViewer rendering of the insulin signaling pathway. Pathway components can be manually repositioned. Genes and pathways are represented as green and blue boxes respectively while purple dots represent chemical compounds. Relationships represent reactions e.g. activation, inhibtion or phosphorilation. (b) Zoomed view of the insulin signaling pathway. Condition 1 is selected in the control panel (top right) and the expression range is set to consider expression levels between -0.43 and 0.43 to be non differentially expressed. Genes PPP1CA and PYGB in red are upregulated while GYS1 in blue is downregulated. GSK3B and CALML6 in green are non differentially expressed genes. The purple dot C00369 represents Starch.\n\nTo contextualize regulatory relationships between a predefined set of genes, KEGGViewer can integrate userprovided gene expression data in a pathway (Figure 1b). For this, the expression values must be handed over to KEGGViewer. The following code shows how to initialize the component to overlay expression data:\n\n\n\nThe expression parameter defines the color to highlight up- and down-regulation, the genes affected and the different experimental conditions, in which expression values were obtained for the affected genes (Figure 1b).\n\nBy providing expression data to KEGGViewer, the tool is able to (i) highlight genes according to their expression values in each experimental condition, (ii) allow users to change the threshold parameters for up- and down-regulation, and (iii) visualize expression changes under different experimental conditions as a slideshow.\n\nMore details on how to use KEGGViewer can be obtained from the BioJS Registry in http://goo.gl/dVeWpg.\n\n\nConclusions\n\nKEGGViewer is a simple, web-based component for visualization of KEGG pathways and integration of user-provided expression data on pathway information. It follows the principles of reutilization, sharing and development behind BioJS. KEGGViewer is easy to integrate in any website and provides functionality to interact with other JavaScript components. As a BioJS component, KEGGViewer is easy to extend allowing changes to be made or new functionality to be included.\n\n\nSoftware availability\n\nZenodo: KEGGViewer, a BioJS component to visualize KEGG pathways, doi: 10.5281/zenodo.77089\n\nGitHub: BioJS, http://github.com/biojs/biojs",
"appendix": "Author contributions\n\n\n\nJosé M. Villaveces: Tool development, manuscript preparation. Rafael C. Jimenez: Project supervision. Bianca H. Habermann: Manuscript preparation, project supervision. All authors critically revised the manuscript and agreed to its publication.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nJMV was financed by BMBF-grant 315737 (Virtual Liver Network). This work was supported by the Max Planck Society.\n\n\nReferences\n\nKanehisa M, Goto S: KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000; 28(1): 27–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMi H, Muruganujan A, Thomas PD: PANTHER in 2013: modeling the evolution of gene function, and other gene attributes, in the context of phylogenetic trees. Nucleic Acids Res. 2013; 41(Database issue): D377–D386. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWrzodek C, Dräger A, Zell A: KEGGtranslator: visualizing and converting the KEGG PATHWAY database to various formats. Bioinformatics. 2011; 27(16): 2314–2315. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWrzodek C, Büchel F, Ruff M, et al.: Precise generation of systems biology models from KEGG pathways. BMC Syst Biol. 2013; 7: 15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHu Z, Mellor J, DeLisi C: Analyzing networks with visANT. Curr Protoc Bioinformatics. 2004; 8: 8.8.1–8. PubMed Abstract | Publisher Full Text\n\nGómez J, García LJ, Salazar GA, et al.: BioJS: an open source JavaScript framework for biological data visualization. Bioinformatics. 2013; 29(8): 1103–1104. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKawashima S, Katayama T, Sato Y, et al.: KEGG API: A web service using SOAP/WSDL to access the kegg system. Genome Inform. 2003; 14: 673–674. Reference Source\n\nCytoscape.js. Reference Source\n\nVillaveces JM, Jimenez RC, Habermann B: KEGGViewer, a BioJS component to visualize KEGG pathways. Zenodo. 2014. Data Source"
}
|
[
{
"id": "3667",
"date": "25 Feb 2014",
"name": "Hedi Peterson",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nKEGGViewer is a BioJS component for easy visualization of KEGG pathways. Although the article is quite short it provides all the essential information about the BioJS component for KEGG pathway visualization and points interested users to the source code for its implementation. We do however have some minor comments about the text:The emphasis of signaling pathways is not reasoned enough in the text. KEGG has signaling pathways, but it is so much more (e.g. metabolism, information processing, diseases, etc). For the usage of the given component it makes no difference between pathway classification, this should be clarified.KEGG also has information about metabolites but this has not been mentioned in the text nor in the documentation of the component. I would assume that KEGGViewer is capable of handling metabolite data as well, but it would be nice to have it specified in the text and/or in the documentation of the component.Although KEGGViewer is an easy plugin for visualizing KEGG pathways it is not a unique way for visualizing user data and alternative options could be mentioned in the Introduction section. KEGG itself allows for user data mapping, for example, KEGGanim is a special web tool for mapping metabolite and gene expression data to the pathways. Other alternatives that could be mentioned include Reactome, which allows expression analysis from user provided data.Although the BioJS KEGGViewer component page has enough information to create working examples of the component, not all the requirements are self-explanatory (missing UI icons, display problems on certain mac chrome versions, expression range setup bar is confusing and it could be set to a default state at 0,0, the proxy setup is confusing and needs better documentation).Currently, the description of parameters and options allows only basic usage. To make the component usable for a wider range of users and to display it's full power, the authors will have to considerably update the component description with additional details and 3-4 use cases.",
"responses": []
},
{
"id": "3666",
"date": "28 Feb 2014",
"name": "Alexander Pico",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe BioJS library of components has a lot of potential. It's encouraging to see a diversity of interactive viewers already registered with BioJS. The intersection of modern JavaSript (JS) components with network biology in particular is ripe for development to bring powerful perspectives on massive biomedical datasets to researchers. I decided to critique this article introducing the BioJS KEGGViewer from three points of view to acknowledge the broad set of use cases and challenges this work takes on. While there are a number of things to improve upon (as always) and a few points requiring clarification, the project is a nice addition to the BioJS library and may provide a useful data visualization option when deployed with a complementary set of web tools for selecting pathways, managing datasets and viewing details.Generic User:The \"play\" feature is great for comparing conditions. Nicely done!Panning is tricky, I seem to have to hold cmd, click, pause, and then drag. Without the 'pause' I invoke a selection tool.There is no additional information or link-outs when you click on a node; only the gene symbol is provided.There is no interface for accessing the data values underlying the visualization. There is a disconnect between the web developer who sets up the viewer with all the underlying expression data and the end-user who views the data with only limited access and controls.Biomedical Researcher:The default expression range appears to be set at min-min, which results in all data values visualized as up-regulation. I would recommend default values centered on 0 in addition to support for user-provided parameters.Unfortunately, the parameter names and value ranges for data overlays are unnecessarily restricted to \"expression\", \"upColor\" and \"downColor\". A generic solution for data overlay that could work with any type of data (KEGGViewer shouldn't care if it's expression or not) and color gradients or discrete mapping options would be much more useful.All of these sorts of options are in fact already available in closely related tools (also free and open source, and which I happen to work on) that the authors neglected to cite: PathVisio [1] and Cytoscape [2]. These projects have both Java and JavaScript flavors. The JS version of Cytoscape was obviously used and cited in this work, but the Java version with its built-in data import, style and overlay options -- as well as KEGG import -- was missed. Speaking of KEGG, I'm dubious about the blanket statement that it is \"free of charge for academics\". It's a complicated situation that I know many colleagues are unclear about, so I think it's important to describe it thoroughly. According to their own website [3], \"Academic users who utilize KEGG for providing academic services are requested to obtain a KEGG FTP subscription for organizational use, which includes a proper license agreement.\" This leads to a licensing agent with various paid subscription options [4,5]. The KEGG API, which KEGGViewer uses, is indeed freely provided for academic use, but only for individual downloads. Bulk downloads, such as those required to do analysis of over representation or enrichment, are explicitly forbidden and require a KEGG FTP subscription [6].Software Developer:It is unfortunate that the EBI host site has resources in conflict with the KEGGViewer. This seems counter to the whole point of BioJS and should be addressed in future releases of the EBI web site, cytoscape.js and/or KEGGViewer (whichever CSS is the most intrusive or classes least specific).Beyond a bit of copy/paste JS (including a 5-level deep JS object), asking users to host a php proxy will likely turn some away. Is there any way around this? References 1. http://pathvisio.org2. http://cytoscape.org3. http://www.kegg.jp/kegg/legal.html4. http://www.bioinformatics.jp/en/keggftp.html5. http://www.pathway.jp/licensing/commercial.html6. http://www.kegg.jp/kegg/rest/",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-43
|
https://f1000research.com/articles/3-41/v1
|
11 Feb 14
|
{
"type": "Case Report",
"title": "Cerebral venous thrombosis in post-lumbar puncture intracranial hypotension: case report and review of literature",
"authors": [
"Mahesh P. Kate",
"Bejoy Thomas",
"P.N. Sylaja",
"Bejoy Thomas",
"P.N. Sylaja"
],
"abstract": "The spectrum of presentation of intracranial hypotension is clinically perplexing. We report a case of 31-year-old post-partum woman who underwent an uneventful caesarean section under spinal anesthesia. From the second day of surgery she developed postural headache, the headache lost its postural character after few days. She then developed seizures and ataxic hemiparesis. Magnetic resonance imaging showed features of severe intracranial hypotension in the brain and the spinal cord, and magnetic resonance venography showed cortical vein and partial superior sagittal sinus thrombosis. Prothrombotic (etiological) work-up showed Protein C and S deficiency. She responded to anticoagulation therapy and recovered completely. On review of literature two distinct groups could be identified obstetric and non-obstetric. The non-obstetric group included patients who underwent diagnostic lumbar puncture, intrathecal injection of medications and epidural anesthesia for non-obstetric surgeries. Poor outcome and mortality was noted in non-obstetric group, while obstetric group had an excellent recovery.",
"keywords": [
"lumbar puncture",
"cerebral venous thrombosis",
"intracranial hypotension"
],
"content": "Introduction\n\nPost-lumbar puncture (PL) headache is common, occurring in 10–30% of patients due to persistent cerebrospinal fluid (CSF) leakage after a lumbar puncture performed for anesthesia or diagnostic purposes1. Post-lumbar puncture intracranial hypotension syndrome (PL-IHS) with persistent headache and neurological deficits is rare. PL-IHS is observed in multiple settings post-diagnostic lumbar puncture2, post-spinal anesthesia/analgesia for abdominal and lower-limb surgeries3, post-epidural anesthesia/analgesia during labor for pain and caesarean section4, and post-intrathecal injection for chemotherapy and post-myelography5. Usually it has a benign course, with most patients making complete recovery with or without an epidural blood patch (EBP). Rarely complications may develop in the form of subdural hygroma6 and neurological deficits due to brainstem compression7. We present a case with PL headache in a post-partum female progressing to cerebral venous thrombosis (CVT). We reviewed the literature to identify predictors of outcome.\n\n\nCase report\n\nA 31-yr-old female with pregnancy-induced hypertension (PIH) underwent caesarean section (CS) at 35 weeks of pregnancy (due to fetal growth retardation). Spinal anesthesia was used for the procedure. On the second day post-partum, she noticed diffuse occipital headache and neck pain when she tried to get off the bed, which improved on lying down. This postural headache was persistent over next 8 days following which it changed character and became continuous. A diagnosis of PL headache was made and she was discharged on Ibuprofen 400mg thrice daily. On 12th post-partum day she had a generalized tonic clonic seizure and had persistent drowsiness. She was taken to local hospital for seizure; there she received lorazepam 4mg slow IV bolus followed by phenytoin 900mg IV infusion over 60 minutes. After stabilization of vitals she was transferred to our comprehensive stroke care center. Prior to this episode she had been detected to have PIH in two earlier pregnancies and the first pregnancy had resulted in intrauterine death. During the present pregnancy she received dalteparin 5000 IU/day subcutaneous injection from 6 weeks pregnancy from local maternity centre with suspicion of anti-phospholipid syndrome; it was continued until the 8th post-partum day. There was no past history of abortions or deep vein thrombosis.\n\nOn examination in the emergency room after 12 hours of seizure she was conscious and oriented. She had no papilledema and her visual acuity and visual field were normal. She had weakness and appendicular ataxia of the left upper limb and mild dysarthria. Her National Institute of Health Stroke Scale on admission was 3.\n\nShe was investigated with a CT scan of the head (Figure 1) after 8 hours of ictus at a local hospital; this showed a right high parietal hemorrhagic infarct. MR imaging of the brain (Figure 2a–g) and spine (Figure 3) (sequences: T1 Weighted, T2 Weighted, fluid attenuated inversion recovery (FLAIR), susceptibility weighted imaging (SWI), diffusion weighted imaging (DWI), apparent diffusion coefficient (ADC) and MR venography (MRV) was done after 14 hours of seizure ictus. This showed a right posterior high parietal lobe hematoma with mass effect and right side superficial cortical veins and partial sagittal sinus thrombosis. In addition there was evidence of intracranial hypotension with CSF leakage at the lumbar puncture site, as evidenced by corpus callosum sagging, pachymeningeal enhancement, and diffuse prominence of the cortical vein and rim of CSF seen in the epidural space from the D10 to the L3 vertebra. Her routine blood investigations, including a haemogram, liver function test and renal function test, were within normal limits. Erythrocyte sedimentation rate was elevated with 40 mm/hour. Her coagulation parameters including prothrombin time and antithromboplastin time were normal. Prothrombotic (etiological) work-up sent prior to starting of heparin therapy revealed reduced Protein C and Protein S activity of 31% (normal range 67–195%, plasma-clotting time based assay) and 26% (normal range 55–123%, plasma-clotting time based assay), respectively. Antithrombin III antigen level was normal at 209 mg/l (normal range: 170–300 mg/l, chromogenic assay). Factor V Leiden mutation (real time PCR method) was not detected. The vasculitic work up (antineutrophil antibody, ANA, double stranded DNA, dsDNA, antiphospholipid antibody, APLA, IgG and IgM and antineutrophil cytoplasmic antibody, ANCA) was negative.\n\na-Flair, b-SWI, c-DWI map, d-ADC map show right high parietal hematoma without diffusion restriction; e-MR venogram shows thrombus in the sagittal sinus thrombus (white solid arrow); f-post-contrast T1W coronal sequence shows enhancement of the pachymeninges (black solid arrow), sagging of the corpus callosum (black open arrow) and filling defect of the sagittal sinus (white solid arrow); g-post-contrast T1W sagittal sequence shows thrombus in the sagittal sinus (white solid arrow) and narrowing of angle between the vein of Galen and straight sinus (black open arrow).\n\nT2W sequence shows rim epidural CSF collection in the posterior aspect D11–L3 vertebra suggestive of CSF leak (white solid arrow).\n\nUnfractionated heparin was started (16 hours after time of onset) at 800 units/hour to maintain plasma thromboplastin time between 75–90 seconds along with bed rest and caffeine immediately after the MRI venography. She responded early and was discharged after 6 days on warfarin 4mg once daily and optimized international normalized ratio (INR) of 2.1. On discharge she did not have any headache and neurological deficit had subsided, with NIHSS of 0 and modified Rankin Scale (mRS) of 0. On follow-up after 3 months, she had no further recurrences or new symptoms.\n\n\nDiscussion\n\nWe describe a case of PL-IHS as suggested by the typical postural headache of the patient and the change of pattern of headache, which could have been the time of transition when cortical venous thrombosis developed. The brain and spine features seen on MR imaging are also supportive of this diagnosis, showing classical features of intracranial hypotension and cortical venous thrombosis. Our patient had multiple predisposing conditions, such as the early post-partum period, a history of previous intrauterine death and reduced activity of Protein C and Protein S. These predisposing conditions increased the likelihood of occurrence of thrombosis in our patient with PL-IHS. Similar findings have been also described by Wilder-Smith et al.3; of their five PL-IHS patients, three had a predisposing prothrombotic condition.\n\nThe pathogenesis of the PL-IHS-induced CVT can be explained by the Monro-Kellie-Abercrombie doctrine. This suggests that the skull is a rigid structure, and the brain volume, venous blood and CSF are in state of equilibrium, so reduction or increase of either element leads alteration in the volume of the other two. In IHS, the CSF volume and pressure are reduced significantly. Two changes occur as a result of this: first in the venous compartment, there is increase in the intracranial venous volume, and second there is descent of the brain and brainstem structures. The venous volume expansion is due to the venous stasis and dilation of the sinuses, cortical and spinal veins. This change occurs first in the meninges, both pachymeninges and leptomeninges. The pachymeninges do not have a blood-brain barrier; this leads to contrast extravasation and hence the post-contrast enhancement. Further, due to the descent of the brain there is distortion and stretching of the veins. All the above changes are further aggravated in erect posture when there is acute dilation of the veins and further stretch on the venous walls, which leads to postural headache. This along with venous stasis may lead to CVT in some patients. This hypothesis has been further bolstered by the study by Canhao et al.8 who showed reduction in the velocity of blood flow by approximately 50% in the straight sinus in patients after lumbar puncture.\n\n\nReview of literature\n\nOn reviewing the published literature for PL-IHS followed by CVT, we were able to extract 52 cases from search on PubMed on September 2013 (Data Set 1). These patients could be divided into two major groups (Table 1): an obstetric associated PL-IHS group and non-obstetric associated group, including post-diagnostic lumbar puncture spinal or epidural anesthesia for non-obstetric surgeries, lumbar intrathecal injection for chemotherapy or myelography, and insertion of lumbar drain.\n\nThe CVT symptoms developed after 4–12 days of the lumbar puncture. Approximately three quarters of the patients had postural headache at the onset and a change in the pattern of headache was observed in approximately half of the patients with the onset of CVT symptoms. This was the headache becoming continuous, becoming diffuse or losing its postural character. These patients had prothrombotic predisposition in 35% of cases or prior history of oral contraceptive pill intake as additional pathogenetic factors. The prothrombotic conditions were Protein C and S deficiency9–11, heterozygous factor V Leiden mutation12 and prothrombin mutation 20210A13. Patients in this group had uniformly good response with complete resolution of symptoms in less than 4 weeks. Neither prior epidural blood patch11,12 nor prophylactic heparin were able to prevent occurrence of CVT in post-partum PL-IHS.\n\na) Post-diagnostic lumbar puncture (n=17)\n\nThe majority of the patients (82.5%) in this group had demyelinating disorder and had received intravenous high dose corticosteroids (IVCS) within a few days, such as multiple sclerosis2, recurrent optic neuritis3 and probable Neuromyelitis optica14. None of these patients had any prothrombotic condition as observed in the Obstetric associated Group. All patients uniformly had postural headache at the onset and this was followed by a change in the pattern of headache as described above in 77.8% of patients with the development of CVT. Superior sagittal sinus was involved in 70% of the cases. One-quarter of the patients in this group were reported to have had a poor outcome2,15. The underlying disease and sagittal sinus involvement may have been responsible for the poor outcome.\n\nReports of CVT in demyelinating disorders have increased in last decade2 but the exact pathogenesis remains to be elucidated. The concomitant use of IVCS has been proposed to be causative factor for the CVT. This can partly be explained by the role of high dose steroids on CSF dynamics and vascular endothelium: steroid treatment has a paradoxical effect on CSF absorption and can either impair or facilitate its absorption. Steroids have a vasodilatory effect on the vessel; this could further aggravate the venous stasis present in the PL-IHS. We hypothesize that the combined changes in CSF absorption and increased venous stasis in patients with lower intracranial hypotension may increase the likelihood of the venous thrombosis inpatient receiving IVCS.\n\nb) Other procedures group\n\nThe average age of at presentation was 35.5±10.76 years. This group was heterogeneous, including patients that had experienced post-epidural steroid injection16,17, post-intrathecal methotrexate5, post-myelography3, post-epidural anesthesia3, post-spinal anesthesia18 and post-lumbar drain19. Underlying prothrombotic conditions were Factor V Leiden mutation3, Protein C deficiency17 and lymphocytic lymphoma5. In other cases either etiological work-up was non-revealing or was not done (Data Set 1). Poor outcome was noted in 27.2% (3/11) patients and one patient died during the illness3. Apart from the age, post-partum state and outcome this group had similar characteristics and risk factors as obstetric associated group.\n\nSince its initial description by Schou and Scherb in 1986 of PL-IHS and CVT18, there has been the ongoing debate about whether they are both mere associations and whether their occurrence is only coincidence. But over the last two decades there is formidable evidence suggesting causality8, that is, of PL-IHS causing CVT, though the exact pathogenetic mechanism remains to be conclusively demonstrated. Further, the various settings in which the phenomenon has been observed suggest multiple risk factors. Over the last 25 years of its description, the clinical characteristics have consistently demonstrated the headache pattern, starting as postural headache and then with a change in the headache character as it loses its orthostatic character; this should alert the treating clinician that the patient may have developed CVT. Though the phenomenon is very rare compared to the number of lumbar punctures being performed, the morbid outcome in one-quarter of patients reported should caution us. The propensity for PL-IHS is increased in patients with prothrombotic conditions; prophylactic heparin may prevent the occurrence of the CVT in this group of patients. This generates questions regarding the dose and duration of the heparin, type of heparin to be used, assessment measures etc, which further studies can answer. Patients with known prothrombotic conditions who undergo lumbar puncture for various procedures should be aggressively managed, including hydration and supine posture, and they may benefit from heparin prophylaxis.\n\n\nData availability\n\nFigshare: Review of literature of cerebral venous thrombosis associated with post-lumbar puncture intracranial hypotension, http://dx.doi.org/10.6084/m9.figshare.92019220\n\n\nConsent\n\nWritten informed consent for publication of clinical details and clinical images was obtained from the patient on Institutional format.",
"appendix": "Author contributions\n\n\n\nMK, manuscript preparation, literature review; BT, figure preparation, literature review; SPN, manuscript preparation, literature review.\n\n\nCompeting interests\n\n\n\nNo relevant competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nEvans RW, Armon C, Frohman EM, et al.: Assessment: prevention of post-lumbar puncture headaches: report of the therapeutics and technology assessment subcommittee of the american academy of neurology. Neurology. 2000; 55(7): 909–914. PubMed Abstract | Publisher Full Text\n\nVandenberghe N, Debouverie M, Anxionnat R, et al.: Cerebral venous thrombosis in four patients with multiple sclerosis. Eur J Neurol. 2003; 10(1): 63–66. PubMed Abstract | Publisher Full Text\n\nWilder-Smith E, Kothbauer-Margreiter I, Lammle B, et al.: Dural puncture and activated protein C resistance: risk factors for cerebral venous sinus thrombosis. J Neurol Neurosurg Psychiatry. 1997; 63(3): 351–356. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKapessidou Y, Vokaer M, Laureys M, et al.: Case report: cerebral vein thrombosis after subarachnoid analgesia for labour. Can J Anaesth. 2006; 53(10): 1015–1019. PubMed Abstract | Publisher Full Text\n\nBienfait HP, Gijtenbeek JM, van den Bent MJ, et al.: Cerebral venous and sinus thrombosis with cerebrospinal fluid circulation block after the first methotrexate administration by lumbar puncture. Neuroradiology. 2002; 44(11): 929–932. PubMed Abstract | Publisher Full Text\n\nSinha A, Petkov S, Meldrum D: Unrecognised dural puncture resulting in subdural hygroma and cortical vein thrombosis. Anaesthesia. 2010; 65(1): 70–73. PubMed Abstract | Publisher Full Text\n\nBloch J, Regli L: Brain stem and cerebellar dysfunction after lumbar spinal fluid drainage: case report. J Neurol Neurosurg Psychiatry. 2003; 74(7): 992–994. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCanhao P, Batista P, Falcao F: Lumbar puncture and dural sinus thrombosis--a causal or casual association? Cerebrovasc Dis. 2005; 19(1): 53–56. PubMed Abstract | Publisher Full Text\n\nTodorov L, Laurito CE, Schwartz DE: Postural headache in the presence of cerebral venous sinus thrombosis. Anesth Analg. 2005; 101(5): 1499–1500. PubMed Abstract | Publisher Full Text\n\nKarci A, Boyaci F, Yaka E, et al.: Cerebral venous thrombosis initially considered as a complication of spinal-epidural anaesthesia. J Int Med Res. 2005; 33(6): 711–714. PubMed Abstract | Publisher Full Text\n\nCan OS, Yilmaz AA, Gurcan E, et al.: Is post partum headache after epidural anaesthesia always innocent? Eur J Anaesthesiol. 2008; 25(8): 697–700. PubMed Abstract | Publisher Full Text\n\nKueper M, Goericke SL, Kastrup O: Cerebral venous thrombosis after epidural blood patch: coincidence or causal relation? A case report and review of the literature. Cephalalgia. 2008; 28(7): 769–773. PubMed Abstract | Publisher Full Text\n\nJungmann V, Werner R, Bergmann J, et al.: [Postpartum cerebral venous sinus thrombosis after epidural anaesthesia]. Anaesthesist. 2009; 58(3): 268–272. PubMed Abstract | Publisher Full Text\n\nCasado-Menendez I, Uria DF, Jimenez L: [Cerebral venous thrombosis as a complication following a diagnostic lumbar puncture]. Rev Neurol. 2011; 52(4): 252–253. PubMed Abstract\n\nPfeilschifter W, Neumann-Haefelin T, Hattingen E, et al.: [Cortical venous thrombosis after a diagnostic lumbar puncture]. Nervenarzt. 2009; 80(10): 1219–1221. PubMed Abstract | Publisher Full Text\n\nFerrante E, Spreafico C, Regna-Gladin C, et al.: Images from Headache. Cerebral venous thrombosis complicating lumbar puncture. Headache. 2009; 49(2): 276–277. PubMed Abstract | Publisher Full Text\n\nMilhaud D, Heroum C, Charif M, et al.: Dural puncture and corticotherapy as risks factors for cerebral venous sinus thrombosis. Eur J Neurol. 2000; 7(1): 123–124. PubMed Abstract | Publisher Full Text\n\nSchou J, Scherb M: Postoperative sagittal sinus thrombosis after spinal anesthesia. Anesth Analg. 1986; 65(5): 541–542. PubMed Abstract\n\nMiglis MG, Levine DN: Intracranial venous thrombosis after placement of a lumbar drain. Neurocriti Care. 2010; 12(1): 83–87. PubMed Abstract | Publisher Full Text\n\nReview of literature of cerebral venous thrombosis associated with post-lumbar puncture intracranial hypotension. F1000Res. Mahesh P. Kate, Bejoy Thomas, Sylaja P.N. Data Source"
}
|
[
{
"id": "3613",
"date": "12 Feb 2014",
"name": "Bruce Campbell",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAn interesting case with a well conducted review of similar cases in the literature. It is a good clinical teaching point to consider dural sinus thrombosis if there is a change in pattern of post-lumbar puncture headache.",
"responses": []
},
{
"id": "3612",
"date": "17 Feb 2014",
"name": "J David Spence",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis case report and review of the literature may be helpful to the patients of physicians who are not aware of this syndrome.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-41
|
https://f1000research.com/articles/2-242/v1
|
13 Nov 13
|
{
"type": "Research Article",
"title": "From zebrafish heart jogging genes to mouse and human orthologs: using Gene Ontology to investigate mammalian heart development.",
"authors": [
"Varsha K Khodiyar",
"Doug Howe",
"Philippa J Talmud",
"Ross Breckenridge",
"Ruth C Lovering",
"Varsha K Khodiyar",
"Doug Howe",
"Philippa J Talmud",
"Ross Breckenridge"
],
"abstract": "For the majority of organs in developing vertebrate embryos, left-right asymmetry is controlled by a ciliated region; the left-right organizer node in the mouse and human, and the Kuppfer’s vesicle in the zebrafish. In the zebrafish, laterality cues from the Kuppfer’s vesicle determine asymmetry in the developing heart, the direction of ‘heart jogging’ and the direction of ‘heart looping’. ‘Heart jogging’ is the term given to the process by which the symmetrical zebrafish heart tube is displaced relative to the dorsal midline, with a leftward ‘jog’. Heart jogging is not considered to occur in mammals, although a leftward shift of the developing mouse caudal heart does occur prior to looping, which may be analogous to zebrafish heart jogging. Previous studies have characterized 30 genes involved in zebrafish heart jogging, the majority of which have well defined orthologs in mouse and human and many of these orthologs have been associated with early mammalian heart development. We undertook manual curation of a specific set of genes associated with heart development and we describe the use of Gene Ontology term enrichment analyses to examine the cellular processes associated with heart jogging. We found that the human, mouse and zebrafish ‘heart jogging orthologs’ are involved in similar organ developmental processes across the three species, such as heart, kidney and nervous system development, as well as more specific cellular processes such as cilium development and function. The results of these analyses are consistent with a role for cilia in the determination of left-right asymmetry of many internal organs, in addition to their known role in zebrafish heart jogging. This study highlights the importance of model organisms in the study of human heart development, and emphasises both the conservation and divergence of developmental processes across vertebrates, as well as the limitations of this approach.",
"keywords": [
"Early heart development",
"heart jogging",
"Gene Ontology",
"Orthology"
],
"content": "Introduction\n\nAn understanding of heart development is important for the treatment of both congenital and acquired heart disease. The majority of heart development studies use model organisms for ethical and practical reasons. Transparent fish embryos, as well chick embryos, enable the developing heart to be studied in real time1, and the mouse continues to be a key model organism used to investigate mammalian heart development2. Although there is substantial evolutionary conservation in the development of left-right axis asymmetry, there is divergence between species3. The earliest events in mammalian heart development are of great interest, but are poorly understood relative to externally developing organs, due to practical constraints.\n\nFor the majority of developing vertebrate embryos left-right asymmetry is controlled by a ciliated region; the left-right organizer node in the mouse and human, and the Kuppfer’s vesicle in the zebrafish4,5. In the zebrafish, laterality cues from the Kuppfer’s vesicle determine asymmetry in the developing heart, and consequently the direction of heart jogging and heart looping. At 24 hours post-fertilization (hpf) the symmetrical zebrafish heart tube is displaced relative to the dorsal midline, with a leftward ‘jog’. At 36hpf the heart tube then loops to the right to create the asymmetric heart5,6. Cilia within the Kuppfer’s vesicle are known to be instrumental in establishing left-right asymmetry and consequently play a significant role in determining the direction of heart jogging7 and heart looping8. However, a failure of heart jogging does not necessarily imply that there will be a failure in heart looping, and vice versa. In addition, asymmetric cell migration has been implicated as a key factor in the process of heart jogging9–13. Several of the genes involved in zebrafish heart jogging have been identified from mutation, morpholino and functional complementation studies6,10,13–26.\n\nWe sought to determine whether the use of Gene Ontology (GO) annotation could offer mechanistic clues to early mammalian heart development. GO is a controlled vocabulary that is used to describe gene product function27. GO describes three aspects of a gene product’s biology: the biological process that the gene product is involved in, the specific molecular function of the gene product and the cellular component that the gene product is located in. GO terms are associated in a directed acyclic graph (DAG), and thus have defined relationships to each other.\n\nThe process of heart looping has been described in a variety of higher eukaryotes2,28,29, and the occurrence of dextral-looping, the early phase of heart looping, appears to be conserved from zebrafish to chicken to humans. In addition, many congenital heart abnormalities, such as dextrocardia and isomerisms are thought to be due to abnormal heart looping2,30 and ciliary dysfunction has been associated with 50% of patients with congenital heart disease and heterotaxy31. However, the process of heart jogging has only been described in zebrafish6. Biben and Harvey describe a leftward shift of the developing mouse caudal heart prior to looping, which may be analogous to heart jogging in zebrafish28, but to our knowledge this has not been investigated further, and heart jogging is not considered to occur in mammals. Consequently, when the ontology describing heart development was expanded32, limitations were included to prevent the association of the GO term ‘heart jogging’ to mammalian gene products33. However, an absence of evidence is not evidence of absence, hence it remains a possibility that heart jogging also occurs in mammalian systems.\n\nAlthough there has been substantial progress in heart development research1,3,4,29, there are clearly gaps in our understanding of early heart development, particularly in the mammal. Functional enrichment analysis of genes known to be involved in zebrafish heart jogging, and also of the human and mouse orthologs of these zebrafish heart jogging genes, identifies many conserved biological processes, functions and cellular locations across these three species. The results of these analyses support the role of cilia in symmetry breaking and the importance of cell signalling in early heart development.\n\n\nMethods\n\nA list of 30 zebrafish heart jogging genes was compiled using a variety of approaches. Twelve zebrafish proteins were identified as they were already annotated to the ‘heart jogging’ GO terms, a further 18 proteins were then identified using the ZFIN database, using a keyword search (heart jogging). The ZFIN (http://zfin.org/) browser searches figure legends of papers that are known to describe specific zebrafish genes (and proteins), but which have not yet been curated with GO terms. This search identified a further 23 zebrafish genes, however manual review of these publications led to 5 being disregarded, as the evidence for an involvement in heart jogging was not strong enough. This left 30 zebrafish proteins with strong evidence for a role in the heart jogging process (Table 1). The experimental evidence describing the association of each gene to the process of heart jogging was manually reviewed, to ensure consistent criteria were applied.\n\nThe evidence for these 30 zebrafish proteins having a role in heart jogging comes from mutant, morpholino or functional complementation studies, as described in the associated publications.\n\nThe HUGO Gene Nomenclature Committee Comparison of Orthology Predictions (HCOP) search tool (http://www.genenames.org/cgi-bin/hcop.pl) was used to identify the closest possible human and mouse ortholog for each of the 30 zebrafish genes. HCOP displays predictions from 11 homology prediction tools, including EnsemblCompara, Homologene and Inparanoid34. For all but one gene, southpaw, HCOP returned human or mouse homologs for the zebrafish genes. The lack of a close mammalian ortholog of southpaw was confirmed with a UCSC BLAT analysis against the human and mouse genomes35. BLAST analysis36 showed that the closest possible human and mouse homolog for the zebrafish southpaw gene was Nodal (33% identity). Indeed, both southpaw and nodal are specifically expressed in the left lateral plate mesoderm5,37 and knockdown of murine Nodal in this region leads to a disruption of cardiac asymmetry, as does injection of southpaw morpholinos, suggesting a functional orthology between southpaw and Nodal5,37. However a reciprocal HCOP search showed that the zebrafish genes nodal-related 1 and 2 are the closest orthologs of human NODAL. Hence we have not included a human or mouse ortholog for zebrafish southpaw (Table 1). Three pairs of zebrafish paralogs (bmpr2a/bmpr2b; foxj1a/foxj1b; nipbla/nipblb) have a single corresponding ortholog in human and mouse. Therefore, there are 26 human and 26 mouse orthologs to the 30 zebrafish genes identified as relevant to zebrafish heart jogging (Table 1).\n\nThe human ‘jogging ortholog’ genes were fully manually annotated, by an experienced GO curator38. Individual PubMed queries were run for each gene using the approved human gene symbol and filtering on ‘human’. To achieve full annotation, all of the relevant publications (a total of 232) containing unique functional data for each gene were annotated, regardless of the specific biology described in each paper. This approach enabled consistent annotation of all experimental data relating to each gene, thus ensuring an unbiased overview of any common processes associated with these genes. In addition, the GO term ‘heart looping’ was associated with a ‘jogging ortholog’ human gene if dextrocardia or situs inversus totalis phenotypes had been associated with a mutation in the gene, in order to follow the generally agreed view that leftward heart looping will have resulted in these phenotypes2.\n\nThe Mouse Genome Informatics functional enrichment tool VLAD (VisuaL Annotation Display; http://proto.informatics.jax.org/prototypes/vlad-1.0.3/) was used to look for overrepresentation of GO terms in each gene list relative to the whole genome of the organism. The annotation datasets used for the analysis were zfin (4th March 2013), goa_human (5th March 2013) and mgi (7th March 2013) for the zebrafish, human and mouse analyses respectively, and the ontology dataset used was dated 10th March 2013. The query gene lists (as UniProt IDs) were pasted into the ‘Query Set’ field, the ‘Universe Set’ field was left blank (to specify all genes in species specific annotation file) and the ‘Display Settings’ options selected were ‘pruning threshold’:3 and ‘collapsing threshold’:6. No evidence codes were excluded from the analyses. For this analysis the total number of genes (universe set size) having annotations in the biological process ontology were 14,577, 30,441 and 24,813 for zebrafish, human and mouse respectively. In line with common practice, when using functional analysis tools, enriched GO terms with 1 or 2 associated query genes were excluded from the final results table.\n\nA list of 103 mouse genes likely to play a role in early heart development was created by combining gene lists derived from three sources: The Mouse Genome Informatics Mammalian Phenotype Ontology browser http://www.informatics.jax.org/searches/MP_form.shtml39, the QuickGO browser http://www.ebi.ac.uk/QuickGO/40 and the ‘jogging ortholog’ gene list described above (see Mousegenelist.csv in Data File). The Mammalian Phenotype Ontology browser was queried for genotypes annotated with the terms ‘abnormal direction of heart looping’, ‘situs inversus totalis’, ‘dextrocardia’ and ‘mesocardia’, creating a list of 180 genotypes with an associated gene. Due to the multiple phenotypes associated with each of these genotypes only 58 genes were identified through this approach, and of these only 5 overlap with the 26 ‘jogging ortholog’ genes. Thirty-five genes were identified by filtering on the GO term ‘determination of heart left/right asymmetry’ and its child terms, the evidence code IMP (Inferred by Mutant Phenotype), and the mouse taxon. Of these only two are also present in the ‘jogging ortholog’ gene lists and 11 are present in the phenotype gene list. Twenty-six mouse ‘jogging ortholog’ genes were added to this combined gene list, and any duplicated genes were removed.\n\n\nResults\n\nThirty zebrafish genes were annotated to the GO term ‘heart jogging’ or one of its child terms based on experimental data from the literature (Table 1). Human and mouse orthologs of these genes were identified, as described in the Methods section, resulting in a list of 26 mammalian ‘jogging orthologs’.\n\nThe human ‘jogging ortholog’ genes were then fully annotated with GO terms based on published experimental data. All manual annotations to the human, mouse and zebrafish genes can be visualized with the QuickGO Gene Ontology browser http://tinyurl.com/humanortholog, http://tinyurl.com/mouseortholog and http://tinyurl.com/zebrafishgenes.\n\nThe zebrafish heart jogging gene list and the human and mouse ‘jogging ortholog’ gene lists were analysed using the VLAD enrichment tool. This identified 155 biological process GO terms that were significantly enriched in the zebrafish (see Human_data.csv in Data File), 431 in the human (see Human_data.csv in Data File) and 402 in the mouse (see Mouse_data.csv in Data File) gene lists. The enriched GO terms from all three species were grouped into five biological areas: Development, Patterning, Cellular Process, Signalling and Movement. The relative enrichment of key GO terms from each area was compared across all three species (see Biological_process_summary.csv in Data File; summarized in Table 2).\n\nThe enriched GO terms were grouped into specific ontology areas, with a selection of more specific child term (preceded with a dash) also included. The full list of grouped GO terms can be found in Table S4, which also shows the genes annotated to each term from each of the three species. k: the number of genes in each gene list annotated to the GO term; M: the number of genes in the species proteome annotated to the GO term.\n\nEnrichment of heart development terms. As expected there was a significant enrichment of developmental process terms in all three gene lists, including an enrichment of the GO term ‘heart development’. However, there was also enrichment of terms such as ‘renal system development’ and ‘nervous system development’, indicating the role of these proteins in regulating the development of a range of organ systems and tissues. These data analyses also show an enrichment of terms describing specific, but universal, cellular processes, such as signalling and regulation of transcription (Table 2). These terms represent essential aspects of development, but are grouped discretely due to their roles in many other biological processes.\n\n‘Pattern specification’, described in GO as a ‘developmental process that results in the creation of defined areas or spaces within an organism to which cells respond and eventually are instructed to differentiate’ and several of its more specific child terms (such as ‘specification of symmetry’), were also enriched in all three gene lists. Within the symmetry ontology, the GO term ‘determination of heart left/right asymmetry’ is annotated to all 30 genes in the zebrafish jogging gene list, however, it is only associated with 8 and 4 jogging ortholog genes in the human and mouse respectively. Of the 97 zebrafish genes associated with ‘determination of heart left/right asymmetry’ 31% are also present in the zebrafish jogging gene list. In contrast, only 11% of the human and 8% of the mouse genes associated with this term are also ‘jogging orthologs’. These results confirm an overlap in the functional role of the zebrafish jogging genes and the human and mouse orthologs in the determination of heart left/right symmetry. However, this relatively low level of overlap may reflect the limitations of model organism and human research in this area, rather than a lack of functional conservation of these genes.\n\nIn addition, there were some differences in the developmental terms that were enriched between species. For example, the GO terms ‘vasculature development’ and ‘sensory organ development’ are enriched in both the human and mouse ‘jogging ortholog’ gene lists (Table 2), but neither of these processes are enriched in the zebrafish jogging ortholog genes. This difference may reflect the type of experiments zebrafish are used for, rather than reflecting a difference between zebrafish and mammals in the genes required for these developmental processes.\n\nEnrichment of cilia terms. Terms in the cellular component organization or biogenesis ontology were enriched across all three gene lists (Table 2 and Biological_process_summary.csv in Data File). Specifically there was an enrichment of terms describing ‘cilium morphogenesis’ and ‘protein complex assembly’ (Figure 1). Within each of these, some more specific terms were enriched, for example the human and mouse ‘jogging ortholog’ gene lists were enriched for the term ‘axonemal dynein complex assembly’, whilst the zebrafish and human gene lists showed an enrichment of the term ‘cilium assembly’.\n\nThe GONUTs view of relationships between enriched terms from the cellular component organization or biogenesis ontology84. The grey arrows are used where a term has an ‘is a’ relationship to its parent term, the blue arrows indicate a ‘part of’ relationship. The bars below each GO term indicates which of these terms are enriched in the zebrafish ‘jogging’ gene list, and the human and mouse ‘jogging ortholog’ gene lists.\n\nTerms such as ‘regulation of cell projection organization’ were also enriched in the human and mouse ‘jogging ortholog’ gene lists. ‘Regulation’ terms have a ‘regulates’ relationship with the relevant processes; for example the term ‘positive regulation of cell projection organization’ has a ‘positively_regulates’ relationship to the term ‘cell projection organization’. In GO an important benefit of building a DAG, rather than a flat-list of controlled vocabulary terms, is that relationships can be used to make inferences from one term to another. However, the VLAD enrichment tool does not automatically create a transitive relationship between ‘regulation’ terms and the processes or functions they regulate. Consequently genes annotated to a ‘regulation’ term will not be associated with the regulated process term (unless there is an independent annotation to the process term). It is also important to recognise that it can be difficult for a curator to choose between annotating to the biological process itself, or to the term describing the regulation of that biological process, based on the published experimental data. Therefore, to get a full picture of the genes involved in a process, including the genes that regulate the process, it is necessary to combine the genes annotated to GO terms describing both the ‘process’ and the ‘regulation of the process’. For example, 10 zebrafish, 12 human and 14 mouse genes within the ‘jogging ortholog’ gene lists are annotated to either ‘cell projection organization’ or ‘regulation of cell projection organization’ (or children of these terms). This represents 33%, 40% and 47% of these zebrafish, human and mouse ‘jogging’ gene lists respectively, indicating that the process of cell projection organization is an important function for this group of genes. In addition, many of the ‘jogging’ and ‘jogging ortholog’ genes annotated to ‘cell projection organization’ terms have also been annotated to the cellular component term ‘cell projection’ (7, 11, 13, genes in zebrafish, human and mouse, respectively, see Data File). The enrichment of the biological process term ‘cell projection organization’ and cellular component term ‘cell projection’ within these gene lists is consistent with the key role of cilia located in the node/Kuppfer’s vesicle to determine heart left/right asymmetry in all three species.\n\nEnrichment of cell migration terms. Cell migration also plays a key role in the establishment of the heart cone, heart jogging and heart looping9,10 and enrichment of the GO term ‘cell migration’ is seen in the ‘jogging’ gene lists of all three species (Table 2 and Biological_process_summary.csv in Data File). Lenhart et al. (2013)9 identified FoxH1, spaw, Bmp4, Lefty2 and Has2 as essential to the asymmetric cell migration that leads to heart jogging. However, our literature review suggests that some genes may have functions in both cilia assembly, within the Kuppfer’s vesicle, and cell migration. For example, thymocytes from Foxj1 transgenic mice display defective migration41, whereas Foxj1-null mice are defective in ciliogenesis42. Similarly, in zebrafish, Fzd2 has been shown to play a role in cilium assembly22 as well as pancreatic insulin-cell migration43. Consequently, further investigations into the role of these genes in heart jogging cell migration may provide further insight into this process.\n\nIn order to investigate the contribution of individual genes in the multiple processes associated with early heart development we created human and mouse heart development gene lists and examined the associated GO biological processes terms. A list of 103 mouse genes with roles in early heart developmental processes was created by merging the three gene lists created using the Mouse Genome Informatics phenotype browser, the QuickGO browser as well as the ‘jogging ortholog’ gene list (Mousegenelist.csv in Data File).\n\nGO captures a range of biological processes that a single gene is involved in. By comparing the overlap between the GO terms associated with specific gene lists it is possible to see what cellular mechanisms are likely to be contributing to the various heart developmental processes. Using the QuickGO browser, genes in the zebrafish ‘heart jogging’ gene list, which were associated with the GO terms ‘heart looping’, ‘signal transduction’, ‘cell migration’ and ‘cell projection organization’ (and all child terms, including ‘regulation’ terms), were downloaded, as well as the genes associated with these terms that were also present in the mouse ‘early heart development’ gene list (Mousegenelist.csv in Data File).\n\nIn the zebrafish ‘heart jogging’ gene list a similar proportion of the genes have the potential to play a role in cell projection organisation (10 genes), cell migration (8 genes) and signal transduction (13 genes) (Figure 2A). In the list of 103 mouse genes that are associated with early heart development, either by phenotype, annotation or homology to the zebrafish ‘heart jogging’ gene list, 82 have been annotated to the GO term heart looping. In contrast to the zebrafish ‘jogging’ gene list, signal transduction appears to play a major role in the mouse early heart development, with 27 genes associated with both signal transduction and heart looping, whereas only 18 and 9 genes, respectively, are associated with cell migration and cell projection organization (Figure 2B). These results fit well with what is known about these gene lists. The zebrafish ‘jogging’ gene list defines a group of genes whose functions are required very early in heart development, when the role of cilia in symmetry breaking initiates the heart jogging process. Whereas, in the mouse ‘early heart development’ gene list the genes included have roles in heart looping, which is developmentally later event than heart jogging. Therefore, although the initial events associated with breaking of left-right symmetry are represented within this gene list, the genes involved in the later process of ensuring the complex looping of the heart tube, through controlled signalling and cell migration, contribute to a large proportion of this list.\n\nVenn diagrams showing the overlap between the GO terms associated with A) the zebrafish ‘heart jogging’ gene list (30 genes) and B) the mouse combined heart development gene list (103 genes).\n\nWhile annotating the 26 human ‘jogging ortholog’ genes we noticed that almost half of these genes have not been associated with a specific disease phenotype (Table 3). However, of the 26 genes examined, mutations in 14 had been associated with a disease phenotype, a fifth of which were ciliopathies. Dextrocardia or situs inversus totalis (reversal or mirroring of the major visceral organs) was associated with 6 of the human ‘jogging ortholog’ genes. Location of the heart on the right side (rather than the left) is generally agreed to be the result of left-handed, instead of right-handed looping of the heart tube in early embryogenesis2. The association of these ‘jogging ortholog’ genes with heart looping defects confirm that there is conserved functional homology between at least some of these orthologous zebrafish and human genes in the very early stages of heart development, which lead to the initial heart asymmetry. All four of ciliopathy-associated ‘jogging orthologs’ were also described as associated with situs inversus totalis, confirming the conserved role of these genes in the cilia within the symmetry determining left-right organizer.\n\nThe associated diseases are described in the listed publications.\n\n\n\n\nDiscussion\n\nWe have used GO to annotate the key genes involved in zebrafish heart jogging and their human and mouse orthologs. Heart jogging is not a process that is thought to occur in mammals. However, these genes are conserved between species and play essential roles in many developmental processes. The information available about these genes in several diverse species can be used to shed light on the roles of these genes and possible mechanisms in heart jogging and other heart developmental processes. Our analyses are in agreement with the well described essential role of cilia in early development4,5,31, with a third of the zebrafish ‘heart jogging’ genes associated with the biological process ‘cell projection organization’ (Table 2).\n\nHowever, it is also important to recognise that although there is considerable evidence for conserved mechanisms of heart development across vertebrates there are also areas of divergence44. For example, in the mouse, zebrafish and Xenopus the rotation of cilia is responsible for the early asymmetric gene expression pattern around the left-right organizer, whereas cilia do not play a role in symmetry breaking in the chicken or pig44.\n\nThe early phases of heart development are particularly difficult to study in mammals, however various approaches are enabling progress in this area2,29,45,46 and using phenotype, annotation and orthology data we have created a list of 103 genes with a putative role in early mouse heart developmental processes. Furthermore, the phenotypes associated with experimentally generated mutant mice provide further clues to the likely role of these genes in human heart development; the genes associated with situs inversus totalis phenotypes are most likely to have functional roles within the node. Conversely, genes not associated with situs inversus totalis but associated with an abnormal direction of heart looping, dextrocardia or mesocardia are likely to be involved in the response of the embryonic heart tube to the left/right asymmetry signals. This is not a completely reliable interpretation, for example mutations in the transcription factor Pitx2 lead to mice with situs inversus totalis, however, Pitx2 is expressed in the left lateral plate and its continued asymmetric expression is necessary for asymmetric morphogenesis of most visceral organs44. The mouse knockout consortia data47 will continue to help with the identification of additional early heart development genes, and informed interpretation of these phenotypes will make it possible to separate those genes likely to be associated with the node from those with functions within the heart tube.\n\nIn humans, defects in early heart development are likely to result in spontaneous abortion and therefore many genes required for early heart development will go undetected48. Consequently, human embryos with heart defects, which develop to full term, represent the less severe end of the spectrum. Although, mutations in several human genes have now been identified as causative of abnormal heart looping, such as ACVR2B, LEFTY2, GJA1 and ZIC349–52, only a few of the ‘jogging ortholog’ genes, CCDC103, CCDC40, DNAAF1, LRRC6, NPHP3 and PKD2, are associated with heart looping defects, and thus provide evidence which suggests an involvement of these genes in left-right asymmetry determination in the heart. Furthermore, mutations in some of the ‘jogging ortholog’ human genes, FOXH1 and PTPN11, are associated with heart septal defects in humans, which seems to imply that in individuals with these mutations early heart developmental processes have proceeded normally, suggesting that, contrary to their role in zebrafish, these genes may not be involved in the early stages of human heart development. However, there are numerous other reasons why there is a poor association of heart defects with the ‘jogging ortholog’ gene list. This may simply be due to the lack of detection of situs inversus totalis53, or reflect a redundancy in gene function, or it may be that the majority of mutations in these genes are simply not detected in humans because they are masked by first trimester spontaneous abortions, which are known to have a high level of heart defects48.\n\nThe impact of lethal mutations on detection of genes associated with heart development would suggest that mutations in these genes would only be detected in individuals with mutations with relatively minor impact on gene function. This idea is supported by the recent identification of multiple ‘minor’ heterozygous mutations within a functional network in three patients with transposition of the great arteries. All of these genes either participate or cooperate within the Nodal signaling pathway54 and the carriers of single mutations exhibit no heart or laterality defects. The impact of ‘minor’ mutations, such as these, may explain the contribution of ‘genetic modifiers’ to congenital heart defects with variable penetrance within a family55, or may suggest a polygenic basis for some of these diseases56. This is supported by model organism data, which provides evidence of multigenic origins for congenital heart disease56. However, model organisms are rarely used to examine the impact of genetic modifiers on heart development, as the majority of model organisms are inbred and examination of mutations leading to ‘minor’ phenotypic variations is often not viewed with the same level of interest as the more extreme heart development defects.\n\nNext Generation Sequencing (NGS) has the potential to identity many more instances of multiple mutations in genes which are functionally linked through a specific pathway. However, teasing out which gene mutations are contributing to a disease, as a genetic modifier or as the causative gene variant, and which are not involved in the disease, is likely to take considerable time. Gene Ontology, KEGG and Reactome pathways, along with protein interaction networks have the potential to inform the process of identifying genetic variants associated with heart defect risk through the identification of pathways and networks which are common to the genes associated with the risk gene variants. Consequently, interpretation of NGS data will be greatly improved with full annotation of the candidate genes involved. The identification of these risk gene variants is likely to be of considerable value to those patients seeking prenatal diagnosis. In addition, the identification of more genes associated with heart defects will also help clarify the conserved and divergent heart development pathways that exist between humans and key model organisms.\n\n\nConclusions\n\nThis study demonstrates that full annotation, using GO, of a set of genes known to be associated with early stages of heart development in zebrafish can be used to confirm functional conservation of the role of these genes in a variety of developmental processes. While this study supports the assertion of gene function based on orthology between genes, it also identifies that for some genes there is no direct evidence for their conserved involvement in specific developmental processes through evolution. Consequently, for evolutionary studies, manual annotation of the genome of individual species will be necessary to enable a bioinformatics approach to investigating the evolution of developmental processes.",
"appendix": "Author contributions\n\n\n\nVKK conceived and designed the study, undertook curation of the prioritized human genes, analysed the datasets and drafted the manuscript. DH participated in the design of the study, undertook curation of the prioritized zebrafish genes, and drafted the manuscript. PJT and RB participated in the study design and helped to draft the manuscript. RCL participated in the design of the study, analysed the datasets and drafted the manuscript. All authors read and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nThe authors declare that they have no conflict of interests. VKK is currently employed by F1000Research. Her role at the journal does not include any involvement in the pre-publication editorial checks, or with the refereeing process.\n\n\nGrant information\n\nThe Cardiovascular GO Annotation Initiative is funded by the British Heart Foundation (SP/07/007/23671 and RG/13/5/30112) and PJT is supported by the British Heart Foundation (RG08/014). The Zebrafish Model Organism Database is funded by the National Human Genome Research Institute (P41 HG002659) of the National Institutes of Health.\n\n\nAcknowledgements\n\nMany thanks to Dr. Jim Hu and Dr Mary Dolan for their help with the creation of Figure 1 and Dr Fotios Drenos for creating the Venn diagrams in Figure 2.\n\n\nReferences\n\nBakkers J: Zebrafish as a model to study cardiac development and human cardiac disease. Cardiovasc Res. 2011; 91(2): 279–288. 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Am J Hum Genet. 2009; 85(6): 883–889. PubMed Abstract | Publisher Full Text | Free Full Text\n\nReardon W, Winter RM, Rutland P, et al.: Mutations in the fibroblast growth factor receptor 2 gene cause Crouzon syndrome. Nat Genet. 1994; 8(1): 98–103. PubMed Abstract | Publisher Full Text\n\nJabs EW, Li X, Scott AF, et al.: Jackson-Weiss and Crouzon syndromes are allelic with mutations in fibroblast growth factor receptor 2. Nat Genet. 1994; 8(3): 275–279. PubMed Abstract | Publisher Full Text\n\nWang B, Yan J, Mi R, et al.: Forkhead box H1 (FOXH1) sequence variants in ventricular septal defect. Int J Cardiol. 2010; 145(1): 83–85. PubMed Abstract | Publisher Full Text\n\nKott E, Duquesnoy P, Copin B, et al.: Loss-of-function mutations in LRRC6, a gene essential for proper axonemal assembly of inner and outer dynein arms, cause primary ciliary dyskinesia. Am J Hum Genet. 2012; 91(5): 958–964. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGillis LA, McCallum J, Kaur M, et al.: NIPBL mutational analysis in 120 individuals with Cornelia de Lange syndrome and evaluation of genotype-phenotype correlations. Am J Hum Genet. 2004; 75(4): 610–623. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKrantz ID, McCallum J, DeScipio C, et al.: Cornelia de Lange syndrome is caused by mutations in NIPBL, the human homolog of Drosophila melanogaster Nipped-B. Nat Genet. 2004; 36(6): 631–635. PubMed Abstract | Publisher Full Text\n\nGuo J, Cagatay T, Zhou G, et al.: Mutations in the human naked cuticle homolog NKD1 found in colorectal cancer alter Wnt/Dvl/beta-catenin signaling. PLoS One. 2009; 4(11): e7982. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBergmann C, Fliegauf M, Bruchle NO, et al.: Loss of nephrocystin-3 function can cause embryonic lethality, Meckel-Gruber-like syndrome, situs inversus, and renal-hepatic-pancreatic dysplasia. Am J Hum Genet. 2008; 82(4): 959–970. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOlbrich H, Fliegauf M, Hoefele J, et al.: Mutations in a novel gene, NPHP3, cause adolescent nephronophthisis, tapeto-retinal degeneration and hepatic fibrosis. Nat Genet. 2003; 34(4): 455–459. PubMed Abstract | Publisher Full Text\n\nFiskerstrand T, Houge G, Sund S, et al.: Identification of a gene for renal-hepatic-pancreatic dysplasia by microarray-based homozygosity mapping. J Mol Diagn. 2010; 12(1): 125–131. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMochizuki T, Wu G, Hayashi T, et al.: PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein. Science. 1996; 272(5266): 1339–1342. PubMed Abstract | Publisher Full Text\n\nDigilio MC, Conti E, Sarkozy A, et al.: Grouping of multiple-lentigines/LEOPARD and Noonan syndromes on the PTPN11 gene. Am J Hum Genet. 2002; 71(2): 389–394. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTartaglia M, Niemeyer CM, Fragale A, et al.: Somatic mutations in PTPN11 in juvenile myelomonocytic leukemia, myelodysplastic syndromes and acute myeloid leukemia. Nat Genet. 2003; 34(2): 148–150. PubMed Abstract | Publisher Full Text\n\nTartaglia M, Mehler EL, Goldberg R, et al.: Mutations in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome. Nat Genet. 2001; 29(4): 465–468. PubMed Abstract | Publisher Full Text\n\nRenfro DP, McIntosh BK, Venkatraman A, et al.: GONUTS: the Gene Ontology Normal Usage Tracking System. Nucleic Acids Res. 2012; 40(Database issue): D1262–D1269. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "2480",
"date": "29 Nov 2013",
"name": "Vincent VanBuren",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe title is fine, but it would be better if it summarized findings rather than summarized the procedures. The abstract is an adequate summary of the article.Required revisions:Methods- Generation of the list of zebrafish jogging genes: \"The search identified a further 23 zebrafish genes, however manual review of these publications led to 5 being disregarded, as the evidence for an involvement in heart jogging was not strong enough.\" The phrase, \"was not strong enough\" does not provide sufficient detail to make construction of this list reproducible. The details should be provided. Discussion- paragraph 4: \"only a few of the 'jogging ortholog' genes, CCDC103, CCDC40, DNAAF1, LRRC6, NPHP3, and PKD2, are associated with heart looping defects, and thus provide evidence which suggests an involvement of these genesis left-right asymmetry determination in the heart.\" First, making an assertion that genes are not associated with a particular process requires that high-powered studies were performed to reach a negative conclusion. Does the literature support this? Second, while it is true that eliminating one possibility increases the probability of other explanations, it is not very solid evidence for a particular explanation. The logic of the above statement should be better supported, or the statement should be removed. Discussion- paragraph 4: \"However, there are numerous other reasons why there is a poor association of heart defects with the 'jogging ortholog' gene list.\" This should be, \"…numerous other possible reasons…\".",
"responses": [
{
"c_id": "710",
"date": "19 Feb 2014",
"name": "Ruth Lovering",
"role": "Author Response",
"response": "Dear Vincent, Thank you for your helpful comments. We also recognise that since the original search was conducted that there are now an additional 7 other zebrafish genes (Celf1, Cdc14aa, Cdc14b, Dmrt2a, Enpp2, Grem2, Ipar3) with experimental evidence supporting their role in heart jogging (Matsui, T. et al., 2012; Clément, A. et al., 2012; Lai, S.L. et al., 2012; Müller, I.I. et al., 2013) As this comment will remain associated with this manuscript we will not add a comment about these additional genes to the manuscript itself, but we will revise the manuscript as follows: Methods - Generation of the list of zebrafish jogging genes section:To make it clearer how this list of zebrafish jogging genes was generated we will replace part of this section with the following: \"A list of 30 zebrafish heart jogging genes was compiled using a variety of approaches. Twelve zebrafish proteins were identified as they were already annotated to the ‘heart jogging’ GO terms, the remaining 18 proteins were then identified using the ZFIN (http://zfin.org/) Site Search, with the search phrase 'heart jogging', and filtering using the 'Expression/Phenotypes' category. This search retrieves figures from papers that have ‘heart jogging’ in the figure legend, and thus are likely to be describing specific zebrafish genes (and proteins) involved in this process. Many of these genes had not not yet been curated with GO terms. Each of the papers identified in this way were reviewed; of the 23 zebrafish genes identified in these papers five (Bmpr1aa, Tbx1, unm_hu119, unm_hu202, unm_hu304) were eliminated, as none of these papers provided experimental evidence for the involvement of these genes in heart jogging.\" In Discussion - paragraph 4:We did not intend to make a negative conclusion here, this statement was making a positive statement that some of the 'jogging ortholog' genes, CCDC103, CCDC40, DNAAF1, LRRC6, NPHP3, and PKD2, are associated with heart looping defects. And as included in the comment below we do state that ‘there are numerous other reasons why there is a poor association of heart defects with the 'jogging ortholog' gene list’. To make this statement less controversial, we will modify it as follows:\"Mutations in several human genes have now been identified as causative of abnormal heart looping, such as ACVR2B, LEFTY2, GJA1 and ZIC3(49–52), and some of the ‘jogging ortholog’ genes (CCDC103, CCDC40, DNAAF1, LRRC6, NPHP3 and PKD2) are also associated with heart looping defects. Thus providing evidence to support an involvement of these genes in left-right asymmetry determination in the heart.\" Discussion- paragraph 4 (2nd comment): As suggested we will revise this to \"However, there are numerous other possible reasons why there is a poor association of heart defects with the 'jogging ortholog' gene list.\""
}
]
},
{
"id": "3269",
"date": "29 Jan 2014",
"name": "Jeroen Bakkers",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe title and abstract accurately describes the content of the manuscript. Method:The analysis described here is based on the selected 30 genes from literature that are associated with the GO term ‘heart jogging’. According to the description by the authors these genes are involved in zebrafish heart jogging, but a better definition would be that these genes affect heart jogging (either direct or indirect). Indeed some of the genes that were included play a role in cilia function and establishment of the left-right axis.Many more genes have been identified in zebrafish that affect left-right patterning and thus ‘heart jogging’. It remains unclear why these were not included in the search. The observation that many zebrafish left-right genes are not associated with the GO term ‘cardiac jogging’ could be due to annotation issues (e.g. not every authors uses the term ‘jogging’ for heart laterality defects in zebrafish embryos). This limitation in the design of this study should be discussed more thoroughly.In addition the authors could make some conclusions about the usefulness of the GO term ‘heart jogging’ . Although heart jogging is a process specific to zebrafish it is controlled by a conserved left-right patterning mechanism. However the GO term ‘cardiac jogging’ is associated with genes that control heart morphogenesis and left-right patterning.",
"responses": [
{
"c_id": "709",
"date": "19 Feb 2014",
"name": "Ruth Lovering",
"role": "Author Response",
"response": "Dear Jeroen,Thank you for your comments about our paper.We have updated the methods, as suggested, to: A list of 30 zebrafish genes that affect heart jogging…As already described in the methods we only included genes in this list where there was experimental evidence in ZFIN database which confirmed that these genes had an impact on heart jogging in zebrafish. Many genes are likely to be missing from this list because the heart jogging process is often not studied in zebrafish carrying mutations in relevant genes.We have added the following statement to the methods:This list does not represent all genes which play a role in heart jogging, as the process of heart jogging is not always studied in zebrafish carrying mutations in relevant genes.We have not provided a detailed discussion about the GO term ‘heart jogging’ as we do not want to describe the detail of GO transitivity any further here.RegardsRuth"
}
]
}
] | 1
|
https://f1000research.com/articles/2-242
|
https://f1000research.com/articles/2-227/v1
|
28 Oct 13
|
{
"type": "Web Tool",
"title": "ANGDelMut – a web-based tool for predicting and analyzing functional loss mechanisms of deleterious angiogenin mutations causing amyotrophic lateral sclerosis",
"authors": [
"Aditya K Padhi",
"Suhas V Vasaikar",
"Bhyravabhotla Jayaram",
"James Gomes",
"Aditya K Padhi",
"Suhas V Vasaikar",
"Bhyravabhotla Jayaram"
],
"abstract": "ANGDelMut is a web-based tool for predicting the functional consequences of missense mutations in the angiogenin (ANG) protein, which is associated with amyotrophic lateral sclerosis (ALS). Missense mutations in ANG result in loss of either ribonucleolytic activity or nuclear translocation activity or both of these functions, and in turn cause ALS. However, there are no web-based tools available to predict whether a newly identified ANG mutation will be ALS causative. More importantly, no web-implemented method is currently available to elucidate the mechanisms of loss-of-function(s) of ANG mutants. In light of this observation, we developed the ANGDelMut web-based tool, which predicts whether an ANG mutation is deleterious or benign. The user selects certain attributes from the input panel, which serves as a query to infer whether a mutant will exhibit loss of ribonucleolytic activity or nuclear translocation activity or whether the overall stability will be affected. The output states whether the mutation is deleterious or benign, and if it is deleterious, gives the mechanism(s) of loss-of-function. This web-based tool, freely available at http://bioschool.iitd.ernet.in/DelMut/, is the first of its kind to provide a platform for researchers and clinicians, to infer the functional consequences of ANG mutations and their association with ALS ahead of experimental findings.",
"keywords": [
"Amyotrophic lateral sclerosis (ALS) is a rapidly progressive",
"invariably fatal neurodegenerative disorder that causes the selective destruction of motor neurons",
"primarily the voluntary muscles. As a result of this degenerative process",
"most patients usually die from respiratory failure within 3–5 years from the onset of symptoms1",
"2. The etiology and mechanisms underlying this debilitating disease are not fully understood",
"and hence",
"there is currently no successful therapy for this disorder. Among the genetic factors",
"about 110 genes have documented association with ALS to date3",
"4. Since its first discovery by Greenway et al.5",
"the ANG gene has emerged as one of the most frequently mutated genes found in ALS patients of diverse ethnic groups. A total of 19 missense mutations in ANG have been associated with ALS6",
"7. In addition",
"several rare mutations have also been discovered in the ANG gene",
"however",
"it is not yet known whether these are instrumental in causing ALS7. The human ANG gene encodes a 14.1 kDa long monomeric ANG protein that induces neovascularization",
"maintains the physiology and health of motor neurons by inducing angiogenesis",
"stimulates neurite outgrowth and path-finding and protects motor neurons from hypoxia-induced death and hence acts as a neuroprotective factor8–12. ANG binds to its target cells and undergoes nuclear translocation due to the presence of two functional sites such as the receptor-binding site (60NKNGNPHREN68) and the nuclear localization signal (29IMRRRGL35) respectively. Another important function of ANG is the ribonucleolytic activity governed by the catalytic triad residues His13",
"Lys40 and His1148",
"9. Several reports on functional assay experiments9–12 and molecular dynamics (MD) simulations6",
"7",
"13 have shown that loss of either ribonucleolytic activity or nuclear translocation activity or both of these functions due to missense mutations in ANG cause ALS. However",
"no web-based tool is currently available that can establish the functional consequences of ANG mutations and precisely predict the loss-of-function(s) mechanisms. In light of these observations",
"we developed and hosted the ANGDelMut web-based tool",
"available at http://bioschool.iitd.ernet.in/DelMut/",
"which utilizes a MD simulation-based protocol to capture certain structural and dynamic features of simulated mutant proteins to predict mechanisms of functional loss."
],
"content": "Introduction\n\nAmyotrophic lateral sclerosis (ALS) is a rapidly progressive, invariably fatal neurodegenerative disorder that causes the selective destruction of motor neurons, primarily the voluntary muscles. As a result of this degenerative process, most patients usually die from respiratory failure within 3–5 years from the onset of symptoms1,2. The etiology and mechanisms underlying this debilitating disease are not fully understood, and hence, there is currently no successful therapy for this disorder. Among the genetic factors, about 110 genes have documented association with ALS to date3,4. Since its first discovery by Greenway et al.5, the ANG gene has emerged as one of the most frequently mutated genes found in ALS patients of diverse ethnic groups. A total of 19 missense mutations in ANG have been associated with ALS6,7. In addition, several rare mutations have also been discovered in the ANG gene; however, it is not yet known whether these are instrumental in causing ALS7. The human ANG gene encodes a 14.1 kDa long monomeric ANG protein that induces neovascularization, maintains the physiology and health of motor neurons by inducing angiogenesis, stimulates neurite outgrowth and path-finding and protects motor neurons from hypoxia-induced death and hence acts as a neuroprotective factor8–12. ANG binds to its target cells and undergoes nuclear translocation due to the presence of two functional sites such as the receptor-binding site (60NKNGNPHREN68) and the nuclear localization signal (29IMRRRGL35) respectively. Another important function of ANG is the ribonucleolytic activity governed by the catalytic triad residues His13, Lys40 and His1148,9. Several reports on functional assay experiments9–12 and molecular dynamics (MD) simulations6,7,13 have shown that loss of either ribonucleolytic activity or nuclear translocation activity or both of these functions due to missense mutations in ANG cause ALS. However, no web-based tool is currently available that can establish the functional consequences of ANG mutations and precisely predict the loss-of-function(s) mechanisms. In light of these observations, we developed and hosted the ANGDelMut web-based tool, available at http://bioschool.iitd.ernet.in/DelMut/, which utilizes a MD simulation-based protocol to capture certain structural and dynamic features of simulated mutant proteins to predict mechanisms of functional loss.\n\nThe method implemented in ANGDelMut has been previously tried and tested, and in addition, the functional loss predictions of mutants have been correlated with known experimental reports6,7,13. When a user submits a mutation, the mutant protein is prepared in silico by replacing the target residue with the desired amino acid residue. Subsequently, a standard MD simulation-based protocol is followed based on certain optimized parameters, which performs extensive MD simulations for 25 ns. The simulated trajectories are then visualized and analyzed for the presence of certain global attributes6,7,13, which in turn indicate whether a mutant will exhibit loss-of-function(s). The output of ANGDelMut suggests the nature of the mutation; whether it is ALS causative or not, and highlights the molecular mechanisms which were responsible for the loss-of-function(s) to the user (Figure 1).\n\nSteps involved to predict whether an ANG mutation will be ALS causative or not, with a brief understanding of the mechanism(s) of its loss-of-functions, implemented in the ANGDelMut web-tool. In Step 1, the user submits a missense ANG mutation to the web-tool and the mutated ANG protein is prepared. Next, in Step 2, an implicit-solvent MD simulation for 25 ns is performed after adding hydrogen atoms, and executing energy minimization, equilibration. After completion of the simulation, in Step 3 certain structural and dynamic markers/attributes from the MD simulation trajectory are analyzed, namely, the RMSD versus time, conformational switching of catalytic residue His114, presence of hydrogen bond interaction path from the site of mutation to His114 mediated through Leu115, local folding of nuclear localization signal residues 31RRR33 and change in SASA versus time. After analysis in Step 3, the PDB file extracted from the MD simulation and a text file containing the RMSD data are uploaded into the web-tool.\n\n\nMaterials and methods\n\nThe ANGDelMut web-based tool was developed in MySQL and is hosted on an APACHE http server located in the computer service centre of the Indian Institute of Technology, Delhi, India. The following are the minimum hardware and software requirements to run this tool –\n\nAt least 64 MB of available random-access memory (RAM) (256 MB recommended).\n\nInternet Explorer 7.0 or higher (IE8.0 or higher recommended), Firefox 3.x, Chrome 9, 10.\n\nJavaScript and cookies enabled, recommend ActiveX enabled for Internet Explorer.\n\nFlash player installed.\n\nThe ANGDelMut algorithm is executed in four steps, namely, mutation selection, implicit-solvent molecular dynamics simulation, analysis of simulation trajectory, and data output (Figure 1). The source code is freely and permanently available at: 10.5281/zenodo.7478.\n\nStep 1: Mutation selection. In the first step known as “Mutation selection”, the user submits a missense mutation as the input. Once the mutation is submitted, the mutated protein is prepared in silico (Figure 1). The X-ray structure of human angiogenin (PDB code: 1B1I)14 is used as a starting structure to prepare the mutant, from which the crystallographic water and cofactor, citric acid (CIT) is removed while keeping the secondary structure intact.\n\nStep 2: Implicit-solvent molecular dynamics simulation. Before performing MD simulations, the mutated protein is checked for accuracy and missing atoms, if present, are fixed. Following this, hydrogen atoms are added using the Xleap tool of AMBER 1115 and if required, the system is neutralized with counter ions (Figure 1). The SANDER module of the AMBER 11 package with the \"ff99SB\" force field is used for all MD simulations16. A standard protocol for MD simulations is then implemented that consists of an energy minimization (2500 steps of steepest descent followed by 1000 steps of conjugate gradient method), an equilibration phase involving gradual heating from 0 to 300 K in 200 ps followed by a constant temperature equilibration for 1000 ps at 300 K13. Finally, 25 ns production MD simulations are carried out with periodic boundary conditions in the isothermal–isobaric (NPT) ensemble at a temperature of 300 K with Berendsen temperature coupling and a constant pressure of 1 atm with isotropic molecule-based scaling17. The commonly used SHAKE algorithm and particle-mesh Ewald (PME) method are used to constrain bond lengths involving hydrogen atom(s) and in the calculation of long range electrostatic forces, respectively18,19. An implicit-solvent based MD simulation method which employs the Generalized Born (GB) model to describe the solvation effects implicitly, executes the 25 ns simulation in about 42 hours compared to 625 hours taken by the explicit method. For this, the GB model proposed by Onufriev, Bashford, and Case {generalized Born solvent (igb) = 5)} and the analytical linearized Poisson–Boltzmann (ALPB) approximations are used20,21. The Born radii were adopted from Bondi with modification (mbondi2). For analysing the simulated trajectories, the trajectory of each system is recorded at every 1 ps. Analysis of simulation results are carried out using PTRAJ module implemented in AMBER 11. All the simulations are performed on a 320 processor SUN Microsystems cluster at the Supercomputing Facility (http://www.scfbio-iitd.res.in) of the Indian Institute of Technology, Delhi, India. As a quality control measure, each simulation trajectory is visualized using visual molecular dynamics (VMD)22 and monitored by calculating certain parameters, such as, total energy and the root mean square deviation (RMSD) at regular intervals.\n\nStep 3: Analysis of simulation trajectory. Once the simulation is completed, each trajectory is analyzed for the presence of several structural and dynamic attributes. Broadly, these attributes are indicators of overall stability of the mutant, residue-specific conformational changes during the dynamics run, presence of a hydrogen bond interaction path, and changes in solvent-accessible surface area (SASA) values (Figure 1). The overall stability of the mutant is assessed by calculating the RMSD of the backbone atoms, sampled once every picosecond, using the PTRAJ module of AMBER 1115 (Figure 1). Conformational changes of the catalytic triad residues are visualized and monitored using VMD22. After this, the hydrogen bond interaction paths are computed using UCSF CHIMERA23 and visualized using Cytoscape24 to examine the ribonucleolytic activity. The nuclear translocation activity of the submitted ANG mutant is examined by calculating the SASA of nuclear localization signal residues 31RRR33 using VolArea (http://www2.fc.up.pt/PortoBioComp/Software/Volarea/Home.html), a VMD plug-in.\n\nStep 4: Data output. The output panel shows whether the user-submitted ANG mutation exhibited conformational switching of His114 and if it possessed a hydrogen bond interaction path mediated through Leu115, to predict the loss of ribonucleolytic activity. Next, it is determined if the mutated protein exhibited reduction in SASA and local folding of nuclear localization signal residues 31RRR33 to ascertain loss of nuclear translocation activity. Finally, the back bone RMSD data is examined to evaluate its stability (Figure 1). In addition to all of this information, a Protein Data Bank (PDB) file from the simulation trajectory and the backbone RMSD data computed from the simulated trajectory is made available to the user for downloading, visualization and analyses. Moreover, the functional loss mechanism data interpreted by the host is also suggested and the user is notified via an e-mail.\n\n\nResults and discussion\n\nThe main interface of ANGDelMut provides the user with a brief description about the tool, how mutations in ANG cause ALS and web links to certain important related articles. Further, the interface has an information panel that gives a brief overview of the methodology employed in ANGDelMut and the various stages involved in query processing and in analysing and obtaining the output data. To submit a mutation, the user selects the source amino acid residue which needs to be mutated with the desired residue. In order to understand the functional loss mechanisms of mutants, the user picks certain attributes from the input panel, such as {Conformational switching of His114, Hydrogen bond interaction path mediated through Leu115 - for loss of ribonucleolytic activity}, {Reduction of SASA, Local folding of nuclear localization signal residues 31RRR33 - for loss of nuclear translocation activity} and {RMSD - for stability}, which are analyzed from the simulation trajectory to predict whether a mutation is deleterious or benign. Once all the information is uploaded to the server, the processing pipeline is allowed to run. In addition, the host is notified by a confirmatory e-mail and with the corresponding job ID-used to track the job. After checking the accuracy of the input data, the web-tool starts processing the ANG mutant protein and performs the simulations. The simulated trajectories are then visualized, analyzed, and the results are uploaded to the server. The user can download the output file, which is a PDB file, that has certain structural and dynamic attributes, and if those attributes are detected, is then used to illustrate if a mutation is deleterious or benign. Further, the user is notified through an e-mail, once the result is available. The detailed working principle of ANGDelMut, illustrated using two ANG mutations, K17I (found in ALS patients, and known to cause loss of ribonucleolytic activity)6,7,13 and L35P (not yet identified in ALS patients but predicted to cause loss of both ribonucleolytic activity and nuclear translocation activity)6,7,13 and WT-ANG as control, according to the four steps (Figure 1) is described below.\n\nK17I and L35P mutant preparation. When the user submits an ANG mutation, for example K17I and/or L35P, the PyMOL molecular graphics system25 is used to mutate residue K17 to I17 and L35 to P35 in the crystal structure of ANG (PDB ID: 1B1I without the heteroatoms) and the modified file is then saved in PDB format (Figure 1). For WT-ANG, only the heteroatoms are removed from the structure and saved in PDB format. Before setting up the simulations, the PDB file of the mutant is inspected to check whether the mutation has affected its secondary structure. We noticed that before simulations, the K17I and L35P mutant had structurally aligned well with the WT-ANG.\n\nPerforming implicit-solvent molecular dynamics simulation and analysing the trajectory. The K17I, L35P mutants and WT-ANG prepared from the above step were subjected to implicit-solvent MD simulations for 25 ns (Figure 1). The trajectories of the K17I and L35P mutants and WT-ANG were then visualized using VMD22. First, the ribonucleolytic activity was investigated. We had earlier established that the conformational switching of catalytic residue His114 is responsible for loss of ribonucleolytic activity of certain ANG mutants, and the predictions were correlated with experimental reports6,7,13. The whole trajectories of the K17I and L35P mutants, and WT-ANG were scanned and it was observed that the conformation of catalytic residue His114 changed significantly in the K17I and L35P mutants, while the WT-ANG did not show any His114 conformational alterations (Figure 2A). Further, it was observed that although the site of mutation (Ile17 and Pro35) is distal from the catalytic residue His114, this affected the His114 conformation. In our previous reports, we have determined that in certain mutants, a conserved hydrogen bond interaction path mediated through Leu115 is responsible for conformational switching of His1146,7,13. The hydrogen bond interactions among pairs of amino acid residues were computed from the MD trajectory using UCSF Chimera23 based on a distance cut-off ≤ 3.2 Å and visualized using Cytoscape24. It was found that the K17I mutant possessed a hydrogen bond interaction path Ile17-Asp15-Ile46-His13-Leu115-Gln117-Asp116-His114 while the L35P mutant possessed a Pro35-Lys40-Gln12-His13-(Thr44 and Leu115)-Gln117-Asp116-His114 path, which is responsible for the observed conformational change of His114 (Figure 2B). The two attributes, such as conformational switching of His114 and presence of a conserved hydrogen bond interaction path mediated through Leu115, derived from the simulation data suggest that the K17I and L35P mutants exhibit loss of ribonucleolytic activity while WT-ANG retained this activity (Figure 2).\n\n(A) Snapshots of ANG proteins obtained from 25 ns simulation trajectories, showing conformational switching of catalytic residue His114 in K17I- and L35P-ANG mutants compared to WT-ANG, resulting in loss of ribonucleolytic activity. (B) Obtained conserved hydrogen bond interaction paths from the site of mutations Ile17 and Pro35 to catalytic residue His114 from simulations for K17I and L35P mutants, where node represents the amino acid residue and edge represents the bond length between them. These paths mediated through Leu115, which induces in loss of ribonucleolytic activity. (C) Computed SASA values of nuclear localization signal residues 31RRR33 from simulations for WT-ANG and K17I and L35P-ANG mutants. SASA values are represented as R31: black, R32: green and R33: red coloured lines. SASA values for WT-ANG and K17I are higher compared to L35P-ANG mutant, suggesting loss of nuclear translocation activity of L35P mutant due to reduction of SASA value. (D) Snapshots of ANG proteins obtained from 25 ns simulation trajectories, showing loose packing of 31RRR33 nuclear localization signal residues in WT-ANG and the K17I-ANG mutant suggesting no loss of nuclear translocation activity while in L35P, the 31RRR33 residues are in closely packed resulting in reduction in SASA values causing loss of nuclear translocation activity. (E) Plot showing the stability of WT-ANG and K17I-, L35P-ANG mutants during the simulations. The RMSD of the backbone atoms from the equilibrated conformation (0 ns) is presented as a function of time. The RMSD time profiles for WT-ANG, K17I and L35P are shown in black, magenta and green coloured lines respectively.\n\nThe nuclear translocation activity of K17I and L35P mutants was also studied. Earlier studies conducted by us have established that local folding and close packing of nuclear localization signal residues 31RRR33 are responsible for loss of nuclear translocation activity of ANG mutants6,7,13. As a result of this local folding, the three successive arginine residues, 31RRR33, exhibit a reduction in the SASA value. For the K17I, L35P mutants and WT-ANG, SASA of 31RRR33 residues was calculated from their respective MD trajectories. It was observed that L35P had a reduction in SASA compared to WT-ANG and the K17I mutant, as a result of local folding and close packing of nuclear localization signal residues 31RRR33 (Figure 2C and 2D). The two attributes, such as local folding of nuclear localization signal residues 31RRR33 and reduction of SASA, derived from the simulation data suggest that the L35P mutant will exhibit loss of nuclear translocation activity while WT-ANG and K17I mutant will retain this activity (Figure 2).\n\nThe overall stability of the K17I and L35P mutants and WT-ANG was studied by calculating the backbone RMSD values from the MD simulations. It was observed that the RMSD value of the K17I and L35P mutant was comparable to WT-ANG, suggesting the mutation from Lys17 to Ile17 and Leu35 to Pro35 did not affected the overall stability of the K17I and L35P mutants during the simulations (Figure 2E). It is also important to notice that due to such minor alterations in conformations of certain residues, such as the His114 and 31RRR33, the overall stability of the mutant remained unaltered.\n\nData output. The K17I mutant exhibited loss of ribonucleolytic activity due to conformational switching of catalytic residue His114 and presence of a conserved hydrogen bond interaction path mediated through Leu115 while retaining its nuclear translocation activity (Figure 2). However, the L35P mutant exhibited loss of both ribonucleolytic activity and nuclear translocation activity (Figure 2). Although the L35P mutant has not yet been identified in ALS patients6,7,13, from the simulation results using the ANGDelMut web-tool, it seems that L35P would be a deleterious mutation. The PDB files of the K17I and L35P mutants available for download and visualization show the presence of the above attributes and show the mechanisms for the loss-of-functions. The user also gets a text file which has information on the backbone RMSD value for the K17I and L35P mutants, which can be downloaded and analyzed by the user in order to infer the overall stability of the mutants. Broadly, the users get to know whether the mutation is ALS causative or not. A notification e-mail is also sent to the user informing them of the completion of job and the final output of the query.\n\n\nConclusion\n\nANGDelMut is an easy-to-use web-based tool that incorporates a methodology to accurately predict whether an ANG mutation will be ALS causative or not. A set of global attributes are usually investigated from MD simulations to predict whether a mutation will be deleterious or benign6,7,13. Therefore, it is the first tool of its kind to provide the user with details of the mechanisms of functional loss of an ANG mutant. The user can get this information relatively quickly ahead of experiments. We hope that the ANGDelMut web-tool, freely available at http://bioschool.iitd.ernet.in/DelMut/, will help clinicians and researchers to understand the pathogenesis and progression of ALS, due to an increase in the number of newly discovered ANG mutations.",
"appendix": "Author contributions\n\n\n\nA.K.P., B.J. and J.G. conceived and designed the experiments. A.K.P. carried out all the experiments. S.V.V. created the web server. A.K.P., B.J. and J.G. analyzed the results and wrote the manuscript; all the authors read and checked the manuscript for content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nFinancial support to B.J. and J.G. at the Supercomputing Facility for Bioinformatics & Computational Biology, Indian Institute of Technology Delhi from the Department of Biotechnology, Government of India (internal reference no. RP02146) is gratefully acknowledged. A.K.P. acknowledges the Council of Scientific and Industrial Research, Government of India, for a research fellowship.\n\n\nReferences\n\nFerraiuolo L, Kirby J, Grierson AJ, et al.: Molecular pathways of motor neuron injury in amyotrophic lateral sclerosis. Nat Rev Neurol. 2011; 7(11): 616–630. PubMed Abstract | Publisher Full Text\n\nHardiman O, van den Berg LH, Kiernan MC: Clinical diagnosis and management of amyotrophic lateral sclerosis. Nat Rev Neurol. 2011; 7(11): 639–649. PubMed Abstract | Publisher Full Text\n\nAbel O, Powell JF, Andersen PM, et al.: ALSoD: A user-friendly online bioinformatics tool for amyotrophic lateral sclerosis genetics. Hum Mutat. 2012; 33(9): 1345–51. PubMed Abstract | Publisher Full Text\n\nVasaikar SV, Padhi AK, Jayaram B, et al.: NeuroDNet - an open source platform for constructing and analyzing neurodegenerative disease networks. BMC Neurosci. 2013; 14: 3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGreenway MJ, Alexander MD, Ennis S, et al.: A novel candidate region for ALS on chromosome 14q11.2. Neurology. 2004; 63(10): 1936–1938. PubMed Abstract | Publisher Full Text\n\nPadhi AK, Jayaram B, Gomes J: Prediction of functional loss of human angiogenin mutants associated with ALS by molecular dynamics simulations. Sci Rep. 2013; 3: 1225. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPadhi AK, Vasaikar SV, Jayaram B, et al.: Fast prediction of deleterious angiogenin mutations causing amyotrophic lateral sclerosis. FEBS Lett. 2013; 587(12): 1762–6. PubMed Abstract | Publisher Full Text\n\nKishikawa H, Wu D, Hu GF: Targeting angiogenin in therapy of amyotropic lateral sclerosis. Expert Opin Ther Targets. 2008; 12(10): 1229–1242. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWu D, Yu W, Kishikawa H, et al.: Angiogenin loss-of-function mutations in amyotrophic lateral sclerosis. Ann Neurol. 2007; 62(6): 609–617. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSubramanian V, Crabtree B, Acharya KR: Human angiogenin is a neuroprotective factor and amyotrophic lateral sclerosis associated angiogenin variants affect neurite extension/pathfinding and survival of motor neurons. Hum Mol Genet. 2008; 17(1): 130–149. PubMed Abstract | Publisher Full Text\n\nCrabtree B, Thiyagarajan N, Prior SH, et al.: Characterization of human angiogenin variants implicated in amyotrophic lateral sclerosis. Biochemistry. 2007; 46(42): 11810–11818. PubMed Abstract | Publisher Full Text\n\nThiyagarajan N, Ferguson R, Subramanian V, et al.: Structural and molecular insights into the mechanism of action of human angiogenin-ALS variants in neurons. Nat Commun. 2012; 3: 1121. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPadhi AK, Kumar H, Vasaikar SV, et al.: Mechanisms of loss of functions of human angiogenin variants implicated in amyotrophic lateral sclerosis. PLoS One. 2012; 7(2): e32479. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLeonidas DD, Shapiro R, Allen SC, et al.: Refined crystal structures of native human angiogenin and two active site variants: implications for the unique functional properties of an enzyme involved in neovascularisation during tumour growth. J Mol Biol. 1999; 285(3): 1209–1233. PubMed Abstract | Publisher Full Text\n\nCase DA, Darden TA, Cheatham TE, et al.: AMBER 10, University of California, San Francisco. 2008. Reference Source\n\nHornak V, Abel R, Okur A, et al.: Comparison of multiple Amber force fields and development of improved protein backbone parameters. Proteins. 2006; 65(3): 712–725. PubMed Abstract | Publisher Full Text\n\nBerendsen HJC, Postma JPM, van Gunsteren WF, et al.: Molecular dynamics with coupling to an external bath. J Chem Phys. 1984; 81(8): 3684–3690. Publisher Full Text\n\nRyckaert JP, Ciccotti G, Berendsen HJC: Numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes. J Comput Phys. 1977; 23(3): 327–341. Publisher Full Text\n\nEssmann U, Perera L, Berkowitz ML, et al.: A smooth particle mesh Ewald method. J Chem Phys. 1995; 103(19): 8577–8593. Publisher Full Text\n\nOnufriev A, Bashford D, Case DA: Exploring protein native states and large-scale conformational changes with a modified generalized born model. Proteins. 2004; 55(2): 383–394. PubMed Abstract | Publisher Full Text\n\nSigalov G, Fenley A, Onufriev A: Analytical electrostatics for biomolecules: beyond the generalized Born approximation. J Chem Phys. 2006; 124(12): 124902. PubMed Abstract | Publisher Full Text\n\nHumphrey W, Dalke A, Schulten K VMD: visual molecular dynamics. J Mol Graph. 1996; 14(1): 33–8, 27–8. PubMed Abstract | Publisher Full Text\n\nPettersen EF, Goddard TD, Huang CC, et al.: UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004; 25(13): 1605–1612. PubMed Abstract | Publisher Full Text\n\nShannon P, Markiel A, Ozier O, et al.: Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003; 13(11): 2498–2504. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDeLano W: The PyMOL molecular graphics system. Accessed 2013 Sept 30. Reference Source"
}
|
[
{
"id": "2228",
"date": "07 Nov 2013",
"name": "Ammar Al-Chalabi",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this interesting paper, Aditya Padhi and colleagues describe a mechanism for predicting the effect of mutations in the ANG gene on the risk of ALS. The paper is well written, the title is appropriate, the abstract provides a good summary and there is an excellent explanation of the study design, methods and analysis.I have only two minor recommendations:First, the authors state that there is no successful therapy for ALS. This completely depends on what is meant by successful; there are many therapies and it is the only neurodegenerative disease for which a disease-modifying therapy exists - riluzole. I agree there are no curative or disease-halting therapies. Perhaps this sentence could be rephrased.Secondly, the authors state that functional assays have been used to predict whether certain mutations cause ALS. I would argue that this is not possible. They can only show a laboratory-based mechanism that is consistent with ALS causation. It would be helpful if the authors could provide the URL for the ALSoD database at first citation. (http://alsod.iop.kcl.ac.uk).",
"responses": []
},
{
"id": "2227",
"date": "25 Nov 2013",
"name": "Rafael Najmanovich",
"expertise": [],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have created a web-based tool to predict the effect of mutations on the angiogenin protein that utilizes Molecular Dynamics simulations to evaluate if the selected mutation is benign or otherwise deleterious. I have several criticisms regarding this work both in terms of: 1- The implementation of the web-tool described here and more importantly, 2- The methodology behind the web-tool. These are discussed below:Web-tool implementation and function.The web-tool seems to only be accessible online intermittently. On several occasions I could not access it. When I could, using Firefox or Safari browsers in Mac OS 10.9, the interface seemed badly designed, not allowing the visual inspection of the selected potential mutations in a 3D model of the protein. The authors could for example, use Jmol for that. 48 hours after submitting a query I am still waiting for any results. The MD protocol implemented by the authors calls for a 25 ns simulation, in our experience we observed that a 25 ns simulation takes approximately 5 days using 48 cores. I would therefore expect the MD simulations performed by the authors to take less than one day. While the language used in the manuscript is not sufficiently precise, the authors mention that \"each trajectory is analyzed for the presence of several structural and dynamic attributes\". To me this suggests that there is possibly manual human intervention in the analysis of each query as part of the results and as such, the method would be impractical unless using mechanisms such as Amazon's Mechanical Turk. I cannot comment further on any potential outputs for the submitted queries as I never obtained a result to my first submitted query. The methodology behind the web-toolDespite the authors having published 3 papers in peer-reviewed journals using the same methodology as proposed in this web-tool, the reliability of the method in my opinion remains to be proven. I will proceed here to describe the reasons why. The first publication by the authors (PLoS ONE 7(2): e32479. doi:10.1371/journal.pone.0032479) analyzes 6 mutations (K17I, S28N, P112L, L35P, K60E and V113I). In this work, the authors observe computationally several changes. Notably a conformational change of the catalytic H114 residue side chain and decrease in solvent accessible surface area of the 31RRR33 signal residues. Later (Scientific Reports, January 2013, doi:10.1038/srep01225), the authors considerably expanded their analysis to other mutations and predicted the effect of several other mutations entirely (I46V, K17E, R31K) or partially (R121H and K54E). This was followed by a third paper (FEBS Letters, Volume 587, Issue 12, 19 June 2013, Pages 1762–1766 doi: 10.1016/j.febslet.2013.04.022) where some other, rarer mutations are analyzed using the methodology. While the authors present compelling computational results in all these three papers, these results should be viewed as a hypothesis for the mechanism by which such mutations lead to loss of activity. These mechanisms remains to be proved experimentally. It is important to always keep in mind that correlation does not mean causation. As these suggested mechanisms have not been proven to cause the loss of function, their correlation with loss-of-function cannot be used to for ‘predicting and analyzing functional loss mechanisms of deleterious angiogenin mutations casing amyotrophic lateral sclerosis”.In summary, I have several reservations regarding the usability of the web-tool presented in this manuscript and I object to the use of the mechanisms suggested by the MD simulations, no matter how compelling their likelihood based on computational evidence, as if they were accepted mechanisms without having ever been experimentally validated. The fact that the authors have already published three papers in which the reviewers did not seem to point out this issue, makes this issue all the more important.",
"responses": [
{
"c_id": "629",
"date": "26 Nov 2013",
"name": "James Gomes",
"role": "Author Response",
"response": "To the best of our knowledge, the web-tool always opens from different browsers and works fine in different operating systems such as, Windows and Mac. We do not know why the reviewer was unable to access it because during this period our servers were not down. The Jmol JAVA visualizer needs a PDB file for 3D visualization. If a mutation is incorporated, the new coordinates of the C, N, H, O-atoms have to be updated in the PDB file. This is possible only after MD simulations. Therefore, the reviewer’s expectation of a visual inspection of the potential mutations in 3D model in Jmol before MD simulations is unrealistic. As a part of our output, we provide the user with the PDB file for visualization. The results of the reviewer submitted query were available within 48 hours after the start of the simulation and are still available on the web server (see here for a screenshot) . However, after manual inspection of the output file for accuracy, completeness and interpreting the results (deleterious or benign mutation), the output files were uploaded to the web-server within the next 12-14 hours and an e-mail was sent to the reviewer informing this. The user was notified of the completion of the job by e-mail (a screenshot of the email is available here). A Word file, named “Reply_JobID-D00018” was also sent as part of the email to his e-mail ID on 22nd November 2013 at 4:31 PM, Indian Standard Time, to provide further information on the interpreted results.We are not sure how the reviewer got the impression that a 25 ns-simulation could be completed in approximately five days using 48 cores. We would expect the reviewer to be aware that simulation times vary depending on the size of the protein and the processors available for computation. With the computational facility available in our institute, a 25 ns time duration implicit-solvent based MD simulation for a mutant Angiogenin protein takes about 42 hours in 16 processors out of the 320 processor SUN Microsystems clusters (please see the text in the manuscript “An implicit-solvent based MD simulation method which employs the Generalized Born (GB) model to describe the solvation effects implicitly, executes the 25 ns simulation in about 42 hours compared to 625 hours taken by the explicit method” and “All the simulations are performed on a 320 processor SUN Microsystems cluster at the Supercomputing Facility (http://www.scfbio-iitd.res.in) of the Indian Institute of Technology, Delhi, India). Once the simulations are completed, the RMSD and PDB files are uploaded to the web-server. As we use two different servers (one where the web-server is located and another, the supercomputing facility, where the queries are transferred and simulations are performed), for initial query processing and data completeness, human intervention is a mandatory step. In addition, after the completion of the simulations, we visualize and analyze the requested query to check for accuracy and to avoid data incompleteness. This was stated clearly in our manuscript. We hope this answers the reviewer’s comment on this issue. If we submit the reviewer’s e-mail ID or Job-ID into the output panel of the web-tool, even at this point, the output result is still visible. In response to the reviewer’s comment about the methodology behind the web-tool we would like to state the following. We are not sure that the reviewer has read the Scientific Reports (doi:10.1038/srep01225) and FEBS Letters (doi: 10.1016/j.febslet.2013.04.022) manuscripts in detail. In the Scientific Reports paper, the last paragraph of the Introduction clearly states that “The close match between simulation results and reported experimental data establishes the strength of this method and its application as a predictive tool to determine if an ANG mutation is deleterious or benign, and if deleterious, the probable mechanism of its loss-of-functions”, rather than only expanding the number of mutations. Also, we would like to suggest that the reviewer checks Table 1 in this article, where we have shown that our predictions matched with all the reported experimental data. In the FEBS Letters paper, we have clearly stated in the abstract that “We present here a fast molecular dynamics based method for determining the mechanisms of functional loss caused by mutations, and attributes to ascertain whether a mutation causes ALS”, rather than highlighting the rare mutations. We have employed the same method in this web-tool as well to understand the functional loss mechanisms. As our predictions match with the reported experimental data, it provides a step forward to use this methodology to predict and analyze the functional loss mechanisms for other new mutations and to predict whether they are deleterious or benign. Thus, we are well aware of the fact that “correlation does not mean causation” as stated by the reviewer. At present, no experimental technique exists that can be used to validate these mechanisms, such as conformational changes of residues at the molecular level, only a time dependence dynamics method, such as, MD simulation can give insights into atomic level details. In response to the reviewer’s final comment, we would like to emphasize that all our previously published papers have been extensively peer reviewed by the respective referees, with some of them even undergoing 3 or 4 rounds of revision. We feel that the reviewer’s comments that call into question the scientific and intellectual ability of peers across different parts of the world will only discourage authors to publish in open, peer-reviewed journals like F1000Research."
},
{
"c_id": "633",
"date": "30 Nov 2013",
"name": "Rafael Najmanovich",
"role": "Reviewer Response F1000Research Advisory Board Member",
"response": "I thank the authors for their extensive response to my original review. It seems that some of the points I made were not sufficiently clear. But before I continue, I would like to acknowledge that I did indeed receive the results in an email but only after I had submitted the original report. I waited for as long as I thought reasonable before submitting it and therefore to be complete, my statement should have read that I did not receive any results up to the time of writing my report. The results are available online at the time of writing this response. With respect to the PDB file provided, it would be useful if the authors would adhere more strictly to PDB naming nomenclature, in particular, the authors use the 3-letter code WAT for water molecules instead of HOH. It would also be useful if the authors would provide the possibility to download a Pymol .pse file of the MD trajectory where the regions of interest used in their manual analysis are differentially displayed and also include the possibility to download the raw MD trajectory. The only results that are provided online are a PDB file and an RMSD file, the latter very poorly documented. Additionally, the conclusions of their manual (Human) analysis of the results are displayed but no data are given online as to the fraction of time that systems spends out of the 25 ns simulation in any of the local conformations that lead to their proposed mechanism of loss of ribonucleolytic or nuclear translocation activity mechanisms. Additionally the authors should state online as part of the results given what threshold values are used to conclude based on the dynamics that any of these proposed mechanisms are not favoured in the mutant under study. It is clear that even in the wild type such conformations will be present at least a fraction of the time. This again alludes to my criticism of the human interventions in concluding that any of these proposed mechanisms are prominent during the simulation.Now, the first point about a Jmol visualization, perhaps I didn't express myself clearly enough, I didn't mean to suggest that the Jmol visualization would show the resulting mutation, of course that requires some form of minimization at the very least. What I would like to see in Jmol is the position of the selected mutation (with the original amino acid).Regarding the speed of MD simulations, it is not relevant as this discussion was part of my original report in trying to quantify when I could expect the results to arrive as no calculation progress report is given. For example, an email could be sent for a query still running with a link to a results page where the percentage to completion is displayed while the results are not available.The author may rest assured that I do indeed fulfill their expectation that I am aware that simulation times vary depending on the size of the protein and processors available. The 5 days for a 25 ns simulation using 48 cores represents my subjective experience. I thank the authors for clarifying that several steps require manual intervention. I still think that manual intervention in the generation of results (be it transferring files across servers, checking the output file for accuracy, completeness and interpretation of results), as noted in the original report, is neither dependable not scalable and therefore is not acceptable. What will happens in five years when the postdoc or student responsible for the manual intervention leave? Or when they go on vacation next time?Contrary to what the authors suggest, I did indeed carefully read their three previous published manuscripts before writing my report on this manuscript. The authors cite in their current response some of their conclusions in these previous reports. While the language used in the Scientific Reports manuscript is correct, stating 'probable mechanism' in the FEBS Letters paper it becomes 'determining the mechanism'. In this manuscript it turns to even more emphatic language saying 'elucidating the mechanisms'. I am glad that the authors are aware that correlation does not imply causation. Irrespective of what experimental techniques can or cannot be used to prove the veracity of the proposed mechanisms, until experimental validation confirms the mechanisms, the proposed mechanisms will remain proposed mechanisms only. I encourage the authors to be clear about this and I remain convinced that this point should have been more clearly stated by the authors in the other papers. Unfortunately, precisely because the peer reviews of these other papers are not openly accessible, I cannot judge the quality of the peer review. Nowhere did I call into question the 'scientific and intellectual abilities' of previous peer reviewers. I said that the fact that it had been missed makes emphasizing this issue here all the more important.As much as the problems with manual intervention are in my mind sufficient to object to the current manuscript, I am willing change my classification to 'accept the with reservations' as long as the manuscript is extensively rewritten, including changing the title to remove any suggestions, implicit or otherwise, that the mechanisms used to classify the mutations are in any way proven as well as to clearly state the limitations of the manual intervention steps. Furthermore, the proposed hybrid Human/Web-tool presented should be modified to give users access to a job progression report page that is frequently updated and that can be accessed at any time after submission until the results are available."
},
{
"c_id": "656",
"date": "23 Dec 2013",
"name": "James Gomes",
"role": "Author Response",
"response": "\"I would like to acknowledge that I did indeed receive the results in an email but only after I had submitted the original report. I waited for as long as I thought reasonable before submitting it and therefore to be complete, my statement should have read that I did not receive any results up to the time of writing my report.\"Our record shows that the e-mail was sent to the reviewer on 22nd November 2013 at 4:31 PM, Indian Standard Time (as shown in the attached screen shots of our earlier response) and the output result files were available in time. \"The results are available online at the time of writing this response. With respect to the PDB file provided, it would be useful if the authors would adhere more strictly to PDB naming nomenclature, in particular, the authors use the 3-letter code WAT for water molecules instead of HOH.\"Our web-tool uses the AMBER software package for MD simulations and thus the PDB file, which was retrieved from the MD trajectory, has the WAT code for water molecules instead of HOH. As this has no affect in the interpretation and visualization of the output PDB file, we did not replace WAT with HOH. \"It would also be useful if the authors would provide the possibility to download a Pymol .pse file of the MD trajectory where the regions of interest used in their manual analysis are differentially displayed and also include the possibility to download the raw MD trajectory. The only results that are provided online are a PDB file and an RMSD file, the latter very poorly documented.\"We agree with the reviewer that uploading a Pymol .pse session file can help the user in directly visualizing the regions of interest. Therefore, we have now accepted the above suggestions of the reviewer. As the raw MD trajectory files are usually very large in size, it is not possible to provide the user an option of downloading these files. For the reviewer’s comment about the RMSD data, we are now providing an excel file of RMSD for downloading. \"Additionally, the conclusions of their manual (Human) analysis of the results are displayed but no data are given online as to the fraction of time that systems spends out of the 25 ns simulation in any of the local conformations that lead to their proposed mechanism of loss of ribonucleolytic or nuclear translocation activity mechanisms. Additionally the authors should state online as part of the results given what threshold values are used to conclude based on the dynamics that any of these proposed mechanisms are not favoured in the mutant under study.\"As suggested by the reviewer, we are now incorporating the data on the time spent by the mutant out of 25 ns in the local conformations in the file sent to the user through e-mail. We are also adding the threshold values used to predict whether a mutation will exhibit any loss-of-functions. \"What I would like to see in Jmol is the position of the selected mutation (with the original amino acid).\"We are working on the Jmol visualization at present and will be adding this feature as soon as possible. \"Regarding the speed of MD simulations, it is not relevant as this discussion was part of my original report in trying to quantify when I could expect the results to arrive as no calculation progress report is given. For example, an email could be sent for a query still running with a link to a results page where the percentage to completion is displayed while the results are not available.\"We have incorporated a progress bar for the query progression. Once the user submits his/her Email-ID or Job-ID into the Output section of the web-tool, the output displays “Query in queue” or a “progress bar” showing the step number which has been completed. \"I thank the authors for clarifying that several steps require manual intervention. I still think that manual intervention in the generation of results (be it transferring files across servers, checking the output file for accuracy, completeness and interpretation of results), as noted in the original report, is neither dependable not scalable and therefore is not acceptable.\"We agree with the reviewer that a completely automated process flow is desirable. However, our institute LAN does not permit us to access the Supercomputing facility (http://www.scfbio-iitd.res.in) servers without log-in. Therefore, the two steps – uploading the jobs and downloading the results are inevitably manual. Even if the next version of the web-tool is hosted in the Supercomputing facility, we will need to manually download the result, analyse the trajectory and link it to the output page of the web-tool. \"The authors cite in their current response some of their conclusions in [their] previous reports. While the language used in the Scientific Reports manuscript is correct, stating 'probable mechanism' in the FEBS Letters paper it becomes 'determining the mechanism'. In this manuscript it turns to even more emphatic language saying 'elucidating the mechanisms'.\"As per the reviewer’s comments, we have made the necessary changes in this version of our manuscript. We have replaced the word “elucidate” with “predict” and made the required changes as suggested by the reviewer. Regarding validation of the proposed mechanisms, we would again like to emphasize that apart from a time dependence dynamics method, such as MD simulations, no other experimental technique exists at present that can be used to validate these mechanisms. However, if the reviewer reads our previous paper (Padhi et al, 2012), he can note that we have validated the proposed mechanism using proper computational control. We suggest the reviewer reads the “MD Simulations” subsection of the “Results” section - “We also validated the 50 ns period by performing a simulation of……..which was then used for subsequent simulation experiments and analyses”. \"As much as the problems with manual intervention are in my mind sufficient to object to the current manuscript, I am willing change my classification to 'accept the with reservations' as long as the manuscript is extensively rewritten, including changing the title to remove any suggestions, implicit or otherwise, that the mechanisms used to classify the mutations are in any way proven as well as to clearly state the limitations of the manual intervention steps. Furthermore, the proposed hybrid Human/Web-tool presented should be modified to give users access to a job progression report page that is frequently updated and that can be accessed at any time after submission until the results are available.\"We have addressed the reviewer’s major concerns and modified the title of our manuscript. After reading, we have modified and added text in several sections of the manuscript. We have also indicated that we require manual intervention in providing the output to the user. We hope the reviewer finds this revised version of our manuscript suitable for changing the current status to “Approval”."
}
]
}
] | 1
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https://f1000research.com/articles/2-227
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https://f1000research.com/articles/2-244/v1
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14 Nov 13
|
{
"type": "Research Article",
"title": "Mapping whole genome shotgun sequence and variant calling in mammalian species without their reference genomes",
"authors": [
"Ted Kalbfleisch",
"Michael P. Heaton",
"Michael P. Heaton"
],
"abstract": "Genomics research in mammals has produced reference genome sequences that are essential for identifying variation associated with disease. High quality reference genome sequences are now available for humans, model species, and economically important agricultural animals. Comparisons between these species have provided unique insights into mammalian gene function. However, the number of species with reference genomes is small compared to those needed for studying molecular evolutionary relationships in the tree of life. For example, among the even-toed ungulates there are approximately 300 species whose phylogenetic relationships have been calculated in the 10k trees project. Only six of these have reference genomes: cattle, swine, sheep, goat, water buffalo, and bison. Although reference sequences will eventually be developed for additional hoof stock, the resources in terms of time, money, infrastructure and expertise required to develop a quality reference genome may be unattainable for most species for at least another decade. In this work we mapped 35 Gb of next generation sequence data of a Katahdin sheep to its own species’ reference genome (Ovis aries Oar3.1) and to that of a species that diverged 15 to 30 million years ago (Bos taurus UMD3.1). In total, 56% of reads covered 76% of UMD3.1 to an average depth of 6.8 reads per site, 83 million variants were identified, of which 78 million were homozygous and likely represent interspecies nucleotide differences. Excluding genome repeat regions and sex chromosomes, approximately 3.7 million heterozygous sites were identified in this animal vs. bovine UMD3.1, representing polymorphisms occurring in sheep. Of these, 41% could be readily mapped to orthologous positions in ovine Oar3.1 with 80% corroborated as heterozygous. These variant sites, identified via interspecies mapping could be used for comparative genomics, disease association studies, and ultimately to understand mammalian gene function.",
"keywords": [
"As the price per base for next generation sequencing continues to fall",
"sequencing projects that are broad in scope become possible for research groups with modest budgets. As a result",
"research tools and approaches that once required large consortia1–3",
"may now be used by small groups of collaborators or even independent labs. Although high throughput technology has been democratized",
"formidable impediments remain that prohibit researchers whose work is not in human",
"model human",
"or agriculturally important species from realizing its benefits. Specifically",
"sequence data",
"once produced",
"is mapped to a reference genome for the species of the subject under investigation. The 10ktrees4 project describes the phylogenetic relationship of 299 even-toed ungulates. Of these",
"only cattle",
"swine",
"sheep",
"goat",
"water buffalo",
"and bison have annotated reference genomes. For the other species a reference genome has not been built",
"and will likely not be built for another decade or more."
],
"content": "Introduction\n\nAs the price per base for next generation sequencing continues to fall, sequencing projects that are broad in scope become possible for research groups with modest budgets. As a result, research tools and approaches that once required large consortia1–3, may now be used by small groups of collaborators or even independent labs. Although high throughput technology has been democratized, formidable impediments remain that prohibit researchers whose work is not in human, model human, or agriculturally important species from realizing its benefits. Specifically, sequence data, once produced, is mapped to a reference genome for the species of the subject under investigation. The 10ktrees4 project describes the phylogenetic relationship of 299 even-toed ungulates. Of these, only cattle, swine, sheep, goat, water buffalo, and bison have annotated reference genomes. For the other species a reference genome has not been built, and will likely not be built for another decade or more.\n\nThe goal of this study is to investigate whether or not an even-toed ungulate could benefit from the reference genomes of the few member species that do have them. To test this, one lane of paired-end Illumina sequence data (~35 billion bases) for a Katahdin ram was generated and mapped to its ovine reference assembly Oarv3.15 and to the bovine reference assembly UMD3.16. The variants measured for the Katahdin ram vs. UMD3.1 demonstrate the wealth of information that can be derived from an interspecies mapping. The majority of these variants are homozygous, and for the most part represent species-specific differences. Any heterozygous variant plausibly represents an intraspecies variation present in sheep. Approximately 78 million homozygous, and 3.6 million heterozygous variants were identified for this ram in non-repeat regions of the cattle genome (which excluded the X chromosome; chrX). Mapping the same dataset to Oar3.1 corroborated more than 1.2 million of the heterozygous variants (>80% of what could be checked, see Table 1). This result suggests that high throughput sequence data for any of the distantly related even-toed ungulates may be mapped to the reference genomes of related species that have annotated references for variant discovery, comparative genetics, and even, perhaps genotype-phenotype association studies.\n\nNo variants identified on the X-chromosome of either reference were included in these totals.\n\nResults are presented here that include the variants identified for the animal against the bovine genome, as well as those corroborated by mapping to the sheep genome. Additionally, the mapped data sets (binary alignment map files) for this sheep are made available for inspection by researchers interested in analyzing the findings of this study for loci most relevant to their work. The datasets are stored in the data management system developed and maintained by Intrepid Bioinformatics, and may be viewed in a version of the Integrative Genomics Viewer7 modified to use their web service application programming interface (https://sourceforge.net/projects/intrepidbioinfo/), the UCSC Genome Browser (http://genome.ucsc.edu)8, or dragged and dropped into a number of other analytical tools such as SAMtools (http://samtools.sourceforge.net)9. This data set, and its direct access is designed to benefit researchers interested in comparative genomics, disease association studies, and ultimately understanding mammalian gene function10.\n\n\nMethods\n\nPrior to their implementation, all animal procedures were reviewed and approved by the care and use committees at the United States Department of Agriculture (USDA), Agricultural Research Service (ARS) Meat Animal Research Center (USMARC) in Clay Center, Nebraska.\n\nThe DNA used for whole genome shotgun sequencing (WGS) was from a Katahdin ram that is part of a U.S. sheep reference panel (USMARC animal number 200008100). The USMARC Sheep Diversity Panel version 2.4 (MSDPv2.4) consists of 96 rams from nine breeds, a composite population, and one Navajo-Churro: Dorper, White Dorper, Dorset, Finnsheep, Katahdin, Rambouillet, Romanov, Suffolk, Texel, USMARCIII composite (1/2 Columbia, 1/4 Hampshire, and 1/4 Suffolk11), and one Navajo-Churro ram as previously described12. For sequencing, ram 200008100 was chosen simply for its breed type (Katahdin) and because it sired numerous progeny in the research flock.\n\nDNA from the reference animal was extracted by a typical phenol-chloroform-method from 3 ml of thawed whole blood previously stored at -20C13. The concentration and quality of the DNA was initially estimated spectrophotometrically by dissolving in a solution of 10 mM TrisCl, 1 mM EDTA (pH 8.0) and measuring the absorbance at 260 and 280 nm (NanoDrop, Wilmington, DE). Sample degradation and quality was also measured by electrophoresis on 1.5% agarose gel. Approximately 15 to 20 µg of DNA was sent to the sequencing facility (BGI Americas Corporation, Cambridge, Massachusets, USA). The sequencing facility subsequently determined the final sample concentration and integrity by fluorimetry (Qubit, Life Technologies, Grand Island, New York USA) and 1% agarose gel electrophoresis.\n\nApproximately 5 µg of sheep genomic DNA was fragmented by focused-ultrasonication to generate fragments less than 800 bp long (Covaris, Inc. Woburn, Massachusetts USA). The these fragments were used to make a paired-end library according to the manufacturer's instructions (TruSeq DNA Sample Preparation Kit, Illumina, Inc., San Diego, California USA). Paired-end library sequencing was performed on a HiSeq2000 machine (Illumina) with one lane of a flow cell to obtain 100 bp reads from each end of the library insert. After sequencing by synthesis, the raw reads were filtered to remove adaptor sequences, contaminating dimer sequences and low quality reads. The reads have been deposited in the NCBI Sequence Read Archive with accession SRR1013441.\n\nThe fastq files from the paired end sequence run for the Katahdin ram were downloaded from the sequencing facility’s ftp site. Once downloaded, each genome was indexed for use by the Burrows-Wheeler Aligner (BWA)14, and the BLAST Like Alignment Tool (BLAT)15. The reference assemblies for both UMD3.1 and Oar3.1 were downloaded from the NCBI genomes download site. Repeat information was acquired from UCSC via their genome browser’s download site using:\n\nwget ftp://hgdownload.cse.ucsc.edu/goldenPath/bosTau6/database/nestedRepeats.txt.gz.\n\nThe fastq files corresponding to R1 and R2 runs for the paired end library were aligned individually using BWA aln, vs UMD3.1 then merged and collated with BWA sampe. The mapping process was repeated for the Oar3.1 reference genome. The resulting sequence alignment map (SAM) files were converted to binary alignment map (BAM) files, and subsequently sorted via SAMtools9. PCR duplicates were marked in the BAM files using the Genome Analysis Toolkit (GATK) (http://www.broadinstitute.org/gatk/)16. Regions in the mapped dataset that would benefit from realignment due to small in/dels were identified using the GATK module RealignerTargetCreator, and realigned using the module IndelRealigner. The BAM file produced at each of these steps was indexed using SAMtools. The resulting indexed BAM files are made available via the Intrepid Bioinformatics genome browser described in greater detail below.\n\nThe above mapping efforts produced BAM files for the alignments to both UMD3.1, and Oar3.1, and each BAM file was analyzed for variation against their respective genomes. The GATK UnifiedGenotyper was used with the genotype mode (-gt_mode) flag set to DISCOVERY, and the likelihood model (-glm) flag was set to BOTH in order to identify both single nucleotide variants, and small insertions and deletions. The maximum number of alternate alleles (--max_alternate_alleles) flag was set to allow only 3. Other than those mentioned, default parameters were used. A BED annotation file was created from the nestedRepeats file for the UMD3.1 assembly. The variant call format (VCF) file produced from the dataset mapped to UMD3.1 was filtered to remove any variants that were detected in repeat regions of the UMD3.1 reference assembly using vcftools (http://vcftools.sourceforge.net)17. Since this was a ram, variants identified on chrX were filtered out of the resulting dataset. This filtered file was used in all subsequent analyses.\n\nOnce variants for this animal were identified vs. the cattle reference sequences, a process was created to determine how many of those variants could be corroborated in the alignment to the sheep genome. This required a translation table that listed the corresponding position in the sheep reference for the variants identified vs. the cattle. The process we created was based on the BLAST-like alignment tool, BLAT15. For each of the 3,672,099 heterozygous variants identified in non-repeat regions of the UMD3.1 alignment, 100 bases of reference sequence flanking each side of the variant position was extracted, and a fasta record was created for each variant. These fasta records were collected in groups of 10,000, and BLATed against the sheep genome. The results were output in the BLAST file format. The BLAT result for each fasta record was analyzed, and high scoring pairs (hsps) were selected that contained the variant position and at least 50% of the 201 bases in the fasta record with greater than or equal to 90% identity across the hsp. From within that hsp, it was possible to identify the chromosome and position in the ovine reference that corresponded to the variant being searched.\n\nSince the animal being studied is a ram, any record amongst these heterozygotes that had a corresponding mapping to chrX on Oar3.1 was removed. With this process, we were able to identify corresponding coordinates in the ovine genome for 1,524,297 of the heterozygotes measured in autosomal, non-repeat regions of the bovine genome. Only variants that had exactly one corresponding hsp spanning the fasta record were considered further.\n\nA VCF file was created with the ovine coordinates derived above, the ovine reference allele, and non- ovine reference base identified at each position and was subsequently passed to the UnifiedGenotyper as the –alleles value, and used in genotyping mode (arguments –glm BOTH, -gt_mode GENOTYPE_GIVEN_ALLELES and -out_mode EMIT_ALL_SITES). The results are summarized in Table 1.\n\nA representative screenshot of the Intrepid Web page (http://server1.intrepidbio.com/FeatureBrowser/customlist/record?listid=7632246067) is shown in Figure 1. By clicking on the any of the \"View in IGV\" links, a java webstart file (.jnlp) is downloaded to the users download directory. That file may be opened to webstart Intrepid’s version of IGV that has been modified to use Intrepid’s application programming interface. The modified source code is posted on our SourceForge site (http://sourceforge.net/projects/intrepidbio/files/?source=navbar). The source code is also permanently available at 10.5281/zenodo.7523. Once opened, a window will appear from which a user may click to \"Load\" the data described in the text boxes that correspond to the dataset represented by the link. This will load the dataset and the user may browse to specific locations in IGV. Otherwise, the application will function in a manner consistent with the native application. The \"View All in IGV\" link may be clicked, and as above will download the .jnlp file which when opened will allow the user to simultaneously load all tracks presented on the web page. The \"Click to load to UCSC\" link will push a URL corresponding to the BAM file up to the UCSC Genome Browser8, and will add it as a new track onto the appropriate genome. Once loaded, the UCSC browser may be used as it would otherwise. The \"Drag to other Apps\" link, on Mac OS X machines may be dragged into a terminal window or other third party applications capable of accepting them. The links for the BAM files may be used directly in SAMtools to download subsets of data for use locally, such as by typing\n\nsamtools view -h\n\nAt this point, drag the link to the terminal window, and append the text\n\nchr13:47,398,414-47,420,508\n\nproducing a command line that resembles\n\nsamtools view -h <dragged link> chr13:47,398,414-47,420,508\n\nThis command, tested using SAMtools-0.1.19, will download in SAM format the SAM header and alignment information for the reads mapping between the coordinates specified on chromosome 13. On a Windows machine, the link may be copied via right click, and pasted into a command window in the appropriate place. These links may be dragged or copied directly into standard versions of IGV available from the Broad Institute or elsewhere by selecting the \"File\" tab in IGV, and the \"Load from URL\" option within. Finally, these links may be used with the wget application to download the datasets entirely. It is suggested that wget be used with the –c option in case the download is disrupted for any reason. If the subsets of the files are downloaded via SAMtools, the resulting files will need to be converted (if downloaded in the SAM format) to BAM files and indexed via SAMtools. Downloaded VCF files may be indexed with IGVTools within IGV.\n\n\nResults\n\nSequencing results for one lane of paired-end reads for the Katahdin ram consisted of 35,917,868 filtered (i.e. clean) reads comprising 35,891,768,800 bases. The average read length and insert size was 100 and 500 bp, respectively with 95.4% of the reads meeting the Q20 quality score. These reads were mapped to the sheep and cattle reference genomes Oar3.1 and UMD3.1, respectively. In total, 56% of reads covered 76% of UMD3.1 to an average depth of 6.8 reads per site (Table 2). More than 83 million variants were identified by the interspecies mapping, of which 78 million were homozygous and likely represent interspecies nucleotide differences (Table 3). An aim of this work was to determine how many heterozygous sites in this animal could be identified via interspecies mapping.\n\naAll variants measured on chromosome X were removed from these totals.\n\nbThe variants identified vs. Oar3.1 that occurred in repeat regions were not filtered out.\n\ncNot determined.\n\ndNonRef Hets are heterozygous variants where neither detected allele corresponds to the bovine reference allele at that position.\n\nExcluding genome repeat regions and sex chromosomes, approximately 3.7 million heterozygous sites were identified in this animal vs. bovine UMD3.1, representing polymorphisms occurring in sheep. The homozygous variants are also directly informative for comparative genomics studies and may be used to create a catalogue of interspecies variation.\n\nThe reads for this sheep were also mapped to the ovine reference genome Oar3.1. The statistics for mapping, and variant discovery are shown in Table 2 and Table 3 respectively. The objectives of this exercise were twofold. 1) to get a rough estimate of the number of interspecies variants that could be measured, and 2) to determine how many of the intraspecies variants could be measured using the heterozygous variants measured against UMD3.1. The total number of variants measured vs. Oar3.1 were in excess of 16.2 million, 9.1 million of those were heterozygotes, all of which would be considered intraspecies. The total number of heterozygous variants detected vs. the UMD3.1 reference was 4.8 million. There are certainly variants identified in both sets that are artifacts, as well as some that would have been missed due to low read coverage (even though they are the same set of reads, in conserved regions, the depth of coverage was lower in the UMD3.1 mapped dataset (Table 2)). These numbers provide a rough estimate that suggests roughly 52.7% (4.8M/9.1) of the heterozygous variation can be measured via this interspecies approach.\n\nDue to the myriad mapping artifacts that will occur in an interspecies mapping, measurement of putative intraspecies variation with this approach is likely to be the most error prone. To estimate an error rate, an attempt was made to corroborate these heterozygous measurements vs. UMD3.1 using the Oar3.1 mapping result. Using the process described in the Methods, a table was created that listed the positions identified as heterozygotes vs. non-repeat, regions for UMD3.1, and the coordinate of the corresponding position in the Oar3.1 reference. The UnifiedGenotyper was used to genotype the dataset mapped to Oar3.1 at these corresponding coordinates, and the results are summarized in Table 1. Of the called heterozygous variants vs. UMD3.1, it was possible via our method to identify the corresponding position in Oar3.1 for 1,524,297 variants. Of these variants, heterozygotes could be corroborated for slightly more than 80% of them. For the nearly 20% of the variants that could not be corroborated, there are several explanations including, incorrect mapping of interspecies reads, as well as overzealous calls on the part of the UnifiedGenotyper vs. the UMD3.1 mapping, and errors in our process for identification of corresponding positions between the two assemblies. This error rate could be mitigated significantly with either more coverage, or better yet, more animals from the same species. By adding more coverage there will be a benefit to the genotype likelihood models that identify variants. By adding more animals, many of the artifacts that are the result of inappropriate mappings of reads from orthologous regions will manifest themselves as fixed heterozygotes. In fact, if one were to use this approach to perform an association study, many of the genotyping errors will be weeded out by producing high P-value associations. Regardless, this result suggests that of the 3.67 million heterozygous variants identified in the autosomal, non-repeat regions of the UMD3.1 reference, as many, or more than, 2.94 million (3.67M×0.8) of them are legitimate intraspecies variation.\n\nThis approach to variant detection using moderate coverage whole genome shotgun (WGS) sequence data in species without reference genomes shows a great deal of promise. However when studying only one animal, an estimated 20% error rate for heterozygous variant detection is sufficiently high to suggest caution in using this information in a high throughput analysis pipelines. When implementing analysis pipelines that use variation data, it is common practice to work directly with a distilled list of variants such as in a VCF file, or other list that would provide the variant as well as summary information derived from the mapped dataset. This summary information includes probability likelihoods, and information about the number of times each allele was measured. This information is very useful, but visual inspection of the alignments are a far more effective approach when working to gain an understanding of the quality of the mappings used to derive the genotype. For this reason, the BAM files used as the foundation of this analysis as well as the VCF files derived from them are provided for direct visual interrogation, and use in analytical pipelines.\n\nThe BAM and VCF files for the UMD3.1, and Oar3.1 mappings are made available for visualization directly within the IGV modified to use the Intrepid Bioinformatics application programming interface. The links to access the data are provided in Table 4. An example of one of the pages is shown in Figure 1, and the use of the information on the page is described in the Methods. The \"View All in IGV\" link will display all data mapped to the respective assemblies in IGV.\n\nFrom these web links it is possible to readily view, retrieve, and transfer subsets of these voluminous datasets between autonomous applications such as SAMtools, wget, and other third party applications in a straightforward fashion without requiring researchers to download, or reprocess them.\n\n\nDiscussion and conclusion\n\nIn this work it has been demonstrated that WGS sequence data from one ruminant species (sheep) could be mapped to a mature reference genome from another ruminants species (cattle) diverged 15 to 30 million years ago for the purpose of identifying both inter-, and intraspecies variation in highly conserved genomic regions. Although there is a high quality, annotated reference genome for the sheep, we chose this species for two reasons. First, it provided the opportunity to determine what percentage of intraspecies variation could be identified, and second, it allowed an estimation of how many of the heterozygous variants identified by cross-species mapping could be corroborated against its own genome.\n\nThe catalogue of interspecies variation derived from the homozygous variation measured vs. the bovine genome provides insight into the relationship of genome structure and function across different biological species. Although the sequence of one animal is not intended to represent the comprehensive spectrum of alleles within a species, it provides at least one example of alleles that have evolved. Using only one animal as a representative of a species, it will be impossible to determine whether a homozygous variant is between or within species. Irrespective of this limitation whether the variation is inter- or intraspecies, a researcher is given insight as to how much variation is tolerated in an otherwise conserved region. However, for the purpose of intraspecies variation detection, homozygous variants derived from a single animal should be ignored. This problem could be mitigated if more animals were used since that would dramatically increase the number of alleles represented and greatly increase the likelihood that a heterozygote would be measured. The majority of heterozygotes will represent a pair of alleles present in this animal, and therefore intra-species variants. Caution should be exercised, as some of the heterozygotes will be due to artifacts either specific to this approach, or otherwise, of the alignment process. Artifacts specific to this approach include the difficulties inherent in performing variant discovery via interspecies mapping since there is significantly more legitimate variation between the reads and the reference sequence due to interspecies variation. This variation, even within conserved regions, results in significantly lower coverage for interspecies mappings (refer again to Table 2). As for artifacts of the alignment process, if there are two very similar regions of the ovine genome (paralogs, which are orthologous to a unique region in the bovine genome), then reads corresponding to both ovine regions may map to a single region in the bovine genome, and differences between the two ovine regions would appear as heterozygotes in the mapping vs. cattle. It is possible to identify these regions by visual inspection as there would be many heterozygotes within the length scale of a read, and very few homozygous variants in the same region, but it would be difficult to reliably identify these algorithmically. If a population of animals were being analyzed these variants would distinguish themselves as being heterozygous, fixed for all animals in the population.\n\nThe approach described here suggests that researchers can pursue important comparative genomics work as well as association studies in species that may be a decade or more from reference genomes. Eventually, our current approach to whole genome analysis, high throughput sequencing followed by mapping to a reference genome will likely be supplanted by technologies that produce, as closely as possible, fully assembled whole genomes for each individual being studied. However, this new reality is still years away.\n\nFinally, a new method for readily viewing, using, and accessing mapped, high throughput datasets and variant files is described. The links allow for access to subsets of data that may be useful to researchers without requiring them to download datasets that may be 10s to 100s of gigabytes in size. Also, the drag and drop formalism presented here introduces a mechanism for user-driven, surface-to-surface interoperation between autonomous applications. This ultimately will allow non-scientific programmers to easily move data between graphical user interfaces of the informatics platforms necessary to do their work.",
"appendix": "Author contributions\n\n\n\nConceived and designed the experiments: TSK, MPH\n\nPerformed the experiments: TSK\n\nAnalyzed the data: TSK\n\nContributed reagents/materials/analysis tools: TSK, MPH\n\nWrote the paper: TSK, MPH\n\n\nCompeting interests\n\n\n\nT.K. serves as the CEO of Intrepid Bioinformatics.\n\n\nGrant information\n\nThis work was supported in part by the KY IDeA Networks of Biomedical Research Excellence (Nigel Cooper PI, University of Louisville) NIH/NIGMS 5P20GM103436-13.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThis work was conducted in part using the resources of the University of Louisville's Research Computing Group and the Cardinal Research Cluster. TK would like to acknowledge specifically the support of Harrison Simrall, Rakesh C. Adupa, and Deepak Sharma of the University of Louisville Research Computing Group.\n\n\nReferences\n\nAbecasis GR, Auton A, Brooks LD, et al.: An integrated map of genetic variation from 1,092 human genomes. Nature. 2012; 491(7422): 56–65. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBirney E, Stamatoyannopoulos JA, Dutta A, et al.: Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature. 2007; 447(7146): 799–816. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBernstein BE, Birney E, Dunham I, et al.: An integrated encyclopedia of DNA elements in the human genome. Nature. 2012; 489(7414): 57–74. PubMed Abstract | Publisher Full Text | Free Full Text\n\nArnold C, Matthews LJ, Nunn CL: 10kTrees Website: a New Online Resource for Primate Phylogeny. Evol Anthropol. 2010; 19(3): 114–8. Publisher Full Text\n\nArchibald AL, Cockett NE, Dalrymple BP, et al.: The sheep genome reference sequence: a work in progress. Anim Genet. 2010; 41(5): 449–53. PubMed Abstract | Publisher Full Text\n\nZimin AV, Delcher AL, Florea L, et al.: A whole-genome assembly of the domestic cow Bos taurus. Genome Biol. 2009; 10(4): R42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRobinson JT, Thorvaldsdottir H, Winckler W, et al.: Integrative genomics viewer. Nat Biotechnol. 2011; 29(1): 24–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKarolchik D, Hinrichs AS, Kent WJ: The UCSC Genome Browser. Curr Protoc Bioinformatics. 2012; Chapter 1: Unit1.4. PubMed Abstract | Publisher Full Text\n\nLi H, Handsaker B, Wysoker A, et al.: The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009; 25(16): 2078–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlfoldi J, Lindblad-Toh K: Comparative genomics as a tool to understand evolution and disease. Genome Res. 2013; 23(7): 1063–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLeymaster KA: Straightbred comparison of a composite population and the Suffolk breed for performance traits of sheep. J Anim Sci. 1991; 69(3): 993–9. PubMed Abstract\n\nHeaton MP, Leymaster KA, Kalbfleisch TS, et al.: Ovine reference materials and assays for prion genetic testing. BMC Vet Res. 2010; 6: 23. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHeaton MP, Keele JW, Harhay GP, et al.: Prevalence of the prion protein gene E211K variant in U.S. cattle. BMC Vet Res. 2008; 4: 25. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi H, Durbin R: Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics. 2010; 26(5): 589–95. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKent WJ: BLAT--the BLAST-like alignment tool. Genome Res. 2002; 12(4): 656–64. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcKenna A, Hanna M, Banks E, et al.: The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010; 20(9): 1297–303. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDanecek P, Auton A, Abecasis G, et al.: The variant call format and VCFtools. Bioinformatics. 2011; 27(15): 2156–8. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "2733",
"date": "16 Jan 2014",
"name": "Claire Wade",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe majority of the paper contains well conducted bioinformatic research. I would ask for a small amount of laboratory work and the editing of one section.To infer a rate of validation of the SNP discovery, it would be useful to see a small subset of identified SNP (10-20 should be sufficient) randomly chosen from the discovery set genotyped using a different method (e.g. Sequenom) in the discovery animal DNA, and ideally a small cohort of animals of the same breed.The section \"Access to BAM and VCF file data via the Intrepid Browser\" is superfluous to the major paper and could be removed and replaced with a reference link to the paper and a second link describing access instructions.",
"responses": [
{
"c_id": "700",
"date": "10 Feb 2014",
"name": "Ted Kalbfleisch",
"role": "Author Response",
"response": "Thank you for your comments and suggestions. Please find below a description of our efforts to address them. We hope that your find them satisfactory.“[I]t would be useful to see a small subset of identified SNP (10-20 should be sufficient) randomly chosen from the discovery set genotyped using a different method (e.g. Sequenom) in the discovery animal DNA, and ideally a small cohort of animals of the same breed.” We were able to acquire an Illumina Ovine 50k SNP Chip dataset for the animal in this study. We identified 5283 assays on the chip that corresponded to positions predicted in our study as polymorphic in sheep. The process for selecting variants to include on a SNP chip is rigorous. The fact that assays for these variants were included on the chip indicates that they are demonstrated to be either informative with respect to disease risk, or have high allele frequencies in sheep. When compared, and excluding those assays that produced no genotype call, 99.03% of the genotype calls were in agreement between the Ilumina platform, and our approach. As for the section in the paper “Access to BAM and VCF file data via the Intrepid Browser”, we agree that this section does not directly support the paper’s findings. This section has been moved from “Methods” to “Supplemental materials”."
}
]
}
] | 1
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https://f1000research.com/articles/2-244
|
https://f1000research.com/articles/3-39/v1
|
06 Feb 14
|
{
"type": "Case Report",
"title": "Essential timing of orthopaedic treatment in children with Ehlers-Danlos syndrome arthrochalasia type: a case report",
"authors": [
"Maarten J. van Rooij",
"Heleen Staal",
"Merel Klaassens",
"Lodewijk Van Rhijn",
"Connie T.R.M. Stumpel",
"Heleen Staal",
"Merel Klaassens",
"Lodewijk Van Rhijn",
"Connie T.R.M. Stumpel"
],
"abstract": "Objective: This case report presents the orthopaedic impact of arthrochalasia-type Ehlers-Danlos syndrome (EDS) (former type VIIA and B) in a young girl.Methods: The characteristics of EDSs are skin abnormalities and tissue fragility with orthopaedic consequences including joint hypermobility and dislocations. In EDS arthrochalasia type (former type VIIA and B) severe general hypotonia and congenital bilateral hip dislocation are distinctive symptoms. This type of EDS is less common than other types and just a few cases have been reported.Results: This report describes a case of a 3-year old girl with EDS arthrochalasia type and bilateral high hip dislocation, spontaneous shoulder dislocation and scoliosis as orthopaedic consequences.Conclusions: Orthopaedic consequences in these cases can be severe so conservative or surgical treatment will be necessary to reduce disability in later life. Timing of either conservative or surgical intervention is essential. According to the literature, most conservative treatments fail. Also, surgical treatment is rarely effective in most of the cases. Both conservative and surgical treatment should be considered carefully in the treatment of EDS arthrochalasia type, each of which has pros and cons. The best treatment depends on the prognosis and current situation of the child.",
"keywords": [
"Ehlers-Danlos syndrome",
"arthrochalasia",
"orthopaedic surgery",
"treatment"
],
"content": "Introduction\n\nEhlers-Danlos syndrome (EDS) is a well known inherited connective-tissue disorder characterized by general joint hypermobility, tissue fragility and skin abnormalities. Six subtypes are described which show clinical overlap with each other and other syndromal connective tissue disorders (e.g. Larsen syndrome)1,2. EDS arthrochalasia type (former EDS type VII-A and VII-B) can be distinguished from other types by the presence of mild dysmorphic features (in particular hypertelorism and micrognathia), congenital bilateral hip dislocation, multiple other recurrent (sub)dislocations and severe muscular hypotonia. This type of EDS is very rare with only 27 cases described to-date1.\n\nIn this case report the history of a three year old girl with EDS arthrochalasia type is described. This girl has been described previously1.\n\n\nMedical history\n\nA 6 year old Caucasian girl, known in the paediatric, clinical genetic and orthopaedic outpatient clinic since birth, was treated for severe skin problems, hyperlaxity of her joints and hypotonia. These congenital symptoms were initially thought to be a part of Larsen syndrome, because of the facial features and hypotonia. However, this would not explain the severe skin problems. At the first visit to our outpatient clinic, when the girl was four months, a bilateral hip dislocation in 90° abduction was seen. Manual closed reduction was considered a possibility. The shoulders dislocated spontaneously with maximum anteversion. X-rays showed high dislocations of the both hip joints and a thoracolumbar scoliosis (Figure 1 and Figure 2). At the age of six months EDS arthrochalasia type was suspected and confirmed by Sanger sequencing, which showed a COL1A2 mutation.\n\nInitially, the hip dislocation was not treated with a harness because of the easy bruising and multiple lacerations of the skin, and the unknown origin of the syndrome. After confirmation of the diagnosis, surgical hip reduction in the near future was believed not to be the best intervention. The reason for this was the high chance that obtaining a stable reposition would fail, because of the severe connective tissue abnormalities. Given the severe hypotonia and multiple other dislocations, the prognosis for walking was small. In addition, the patient would have to wear a plaster cast for at least 12 weeks postoperatively, which would cause major skin problems and would probably further impair her motor development. For the same reason conservative treatment in terms of only a plaster cast was deemed unachievable. This decision was supported by findings in the literature showing that conservative treatment in these cases is not successful.\n\nFollow-up of the patient showed a girl with a progressive equinovarus deformity of both feet (Figure 3–Figure 5) starting at the age of 1 year and 6 months. Due to her foot deformity, bilateral hip dislocation and hypotonia it was not possible for the girl to mobilize. Initially, the foot deformity was mild and easy to redress. After one year (at age 2 years and 6 months) it was no longer possible to redress the foot deformity, due to contractures and stiffness of the joints. A decision for surgical treatment was made in order to allow shoe wearing. A bilateral tenotomy of the Achilles tendon and a bilateral anticus transfer was performed. Extension of the flexor hallucis tendon of the right foot and a tenotomy of the flexor hallucis tendon of the left foot were also part of the treatment. Postoperative treatment consisted of a well-padded plaster cast for 6 weeks and appropriate footwear afterwards. After this treatment, a slight foot deformity remained (Figure 6), although the intention of treatment, which was to redress the deformity in her feet so that they would fit shoes, was achieved.\n\n\nDiscussion and consideration\n\nIn 1892, Tschernogobow was the first to describe joint dislocations due to ligamentous and capsular laxity4. Ehlers-Danlos syndrome is an inherited connective-tissue disorder with an autosomal dominant mode of inheritance3. General clinical symptoms are hypermobility of the joints, tissue laxity and skin abnormalities. There are six different subtypes, based on their clinical and genetic features. EDS arthrochalasia type is caused by mutations in the COL1A1 (OMIM 130060) or COL1A2 (OMIM 130060) gene, which causes production of low quality type 1 collagen fibers. This EDS type distinguishes itself from the other types of EDS by the severity of the congenital bilateral hip dislocations, recurrent subluxations, subtle dysmorphic features, a severe muscular hypotonia, a soft velvety skin that is not hyperextensible and a ‘criss-cross’ patterning of the palms and soles1,3.\n\nOf all forms of EDS, approximately 90% are the classic and hypermobile subtypes (EDS I & III) and 5–10% is the vascular subtype (IDS IV). All the other subtypes are extremely rare. Only 27 cases of EDS arthrochalasia type are described in the literature1,2.\n\nA postnatal early diagnosis of EDS arthrochalasia type is important, because an early diagnosis will benefit follow-up and treatment. Important issues compromising development, such as the severe hypermobility with dislocations and hypotonia, can be addressed appropriately. Establishing a diagnosis can be difficult in the neonatal period, because of phenotypic overlap with other skeletal dysplasias, such as Larsen syndrome1,3.\n\nThere is no consensus in literature on treatment of EDS arthrochalasia type. The little knowledge there is on treatment of the upper limbs tells us that neither orthotics nor surgical treatment are effective5.\n\nWith regard to the lower limbs, a choice between conservative treatment vs. surgical treatment is an important step in the treatment process. Conservative treatment, such as plaster casts, Pavlic bandages or orthotics, would be preferable, but they have a high chance of causing decubitus lesions, due to high pressures on a highly vulnerable skin. Therefore, these treatments are not favoured in patients with motor retardation. Also for lower limbs it seems, according to the literature, that conservative treatment is not effective. Surgical treatment is effective in only some cases in the treatment of congenital hip dislocations (Table 1)5. Only an iliac osteotomy with or without a varus and derotation femoral osteotomy shows stability in other hyperlaxity syndromes1,5.\n\n*Several conservative treatment options preceded surgery\n\nFor the treatment of equinovarus deformities of the feet, which are common in EDS arthrochalasia type patients, treatment options are also described to be rarely effective6.\n\nAlthough surgical procedures are invasive, they seem to be the only proven treatment to be effective. In this case a bilateral hip dislocation and an equinovarus deformity of both feet was seen and conservative treatment was considered not to be effective or to possibly be even harmful in this child. Therefore, a capsular and tendon release of the feet was carried out. Another treatment option for the feet could have been a subtalar release or a triple arthrodesis6,7.\n\nIt is advisable to wait till the end of childhood for correction of the scoliosis deformity. In general, muscles will have gained strength during childhood, partly because of improvement in muscle tone. This most likely will result in an improvement of the scoliosis6.\n\n\nConclusion\n\nEDS arthrochalasia type is rare. In all patients the dislocation of the joints combined with hypotonia is a serious problem, leading to severe disability. All interventions, both conservative and surgical, should be considered carefully. As with any treatment method, complications such as wound healing problems and decubitus, resulting from the poor quality of the collagen fibers, are a risk. The best treatment depends on prognosis and current situation of the child.\n\n\nConsent\n\nWritten informed consent for publication of her clinical images was obtained from the parent of the patient.",
"appendix": "Author contributions\n\n\n\nMR, HS and MK conceived the study and prepared the draft of the manuscript. LR and CS revised the article critically.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe thank the family of the patient for their cooperation.\n\n\nReferences\n\nKlaassens M, Reinstein E, Hilhorst-Hofstee Y, et al.: Ehlers-Danlos arthrochalasia type (VIIA-B) – expanding the phenotype: from prenatal life through adulthood. Clin Genet. 2011; 82(2): 121–30. PubMed Abstract | Publisher Full Text\n\nYen JL, Lin SP, Chen MR, et al.: Clinical features of Ehlers-Danlos syndrome. J Formos Med Assoc. 2006; 105(6): 475–480. PubMed Abstract | Publisher Full Text\n\nBeighton P, De Paepe A, Steinmann B, et al.: Ehlers-Danlos syndromes: revised nosology, Villefranche, 1997. Ehlers-Danlos National Foundation (USA) and Ehlers-Danlos Support Group (UK). Am J Med Genet. 1998; 77(1): 31–37. PubMed Abstract | Publisher Full Text\n\nBeighton P, Horan F: Orthopaedic aspects of the Ehlers-Danlos syndrome. J Bone Joint Surg Br. 1969; 51(3): 444–453. PubMed Abstract\n\nGiunta C, Superti-Furga A, Spranger S, et al.: Ehlers-Danlos syndrome type VII: clinical features and molecular defects. J Bone Joint Surg Am. 1999; 81(2): 225–238. PubMed Abstract\n\nHamada S, Hiroshima K, Oshita S, et al.: Ehlers-Danlos syndrome with soft-tissue contractures. J Bone Joint Surg Br. 1992; 74(6): 902–905. PubMed Abstract\n\nTrudgian J, Trotman S: Ehlers-Danlos syndrome and wound healing: injury in a collagen disorder. Br J Nurs. 2011; 120(6): S10, S12, S14 Passim. PubMed Abstract"
}
|
[
{
"id": "4333",
"date": "03 Apr 2014",
"name": "Patrick Calders",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well written case study which I 'Approve' without any revision necessary. It is clear and the fundamental information needed to understand each step is well described.",
"responses": []
},
{
"id": "4502",
"date": "16 Apr 2014",
"name": "Zbigniew Gugala",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAll available reports in the literature emphasize the extreme musculoskeletal challenges in the course and managements of patients with arthrochalasia-type (formerly type VIIA and VIIB; OMIM 130060) Ehlers-Danlos syndrome (EDS), and indicate no progress in effective therapy regardless of whether it is nonsurgical or surgical. All sorts of orthopaedic surgical interventions have been tried to improve patient gait and/or mobility, however, they all typically produce disappointing outcomes, thereby justifying surgery for only selected and specific indications in favor of physical therapy and/or bracing.The case report by van Rooij et al. describes a patient with arthrochalasia-type EDS who has the characteristic severe musculoskeletal phenotype, specifically related to profound joint laxity and severe muscular hypotonia. The authors report a case of a girl with arthrochalasia EDS exhibiting phenotypic features typical for the disease (congenital bilateral hip dislocations, multiple and recurrent other joint sub- or dislocations, severe muscle hypotonia, velvety skin, facial signs) with the diagnosis confirmed genetically with a presence of COL1A mutation. Considering the protracted bilateral hip dislocation combined with severe muscle hypotonia any intervention, surgical or nonsurgical, had a very poor chance for tangible improvement of the patient’s mobility. Furthermore, the skin fragility and abnormity posed significant risk for developing laceration, bruises, healing problems, thereby limiting all possible treatment options. Because the patient developed bilateral equinovarus foot deformity, and subsequent contractures and joint stiffness prevented a conservative treatment, the decision was made to surgically correct the foot deformity. Postsurgical cast treatment was uneventful, and ultimately a satisfactory outcome was achieved. This surgical intervention prompted the authors to discuss the challenges with any orthopaedic intervention for arthrochalasia-type EDS patients, and focus on the importance of identifying true surgical goals, while accepting high risk for complications.The manuscript comprises a valuable reminder about the formidable treatment challenges that exist for these patients—the cases fortunately very rare.There are, however, some inconsistencies and/or deficiencies in providing important and complete information about the case. The patient’s age is contradictorily specified (i.e., 3 years old in the Introduction versus 6 years old in the Medical history section), and it remains unclear if any - and if so, what - treatment was performed to address the congenital bilateral hip dislocation, and how this was tolerated by the patient. The reader would also benefit from a more thorough discussion on the benefits and risks of early versus late surgical treatment, and the specific goals of surgery, if it is indeed indicated for these patients.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-39
|
https://f1000research.com/articles/3-35/v1
|
04 Feb 14
|
{
"type": "Case Report",
"title": "Functional abdominal pain causing Scurvy, Pellagra, and Hypovitaminosis A",
"authors": [
"Edith Y. Ho",
"Christian Mathy",
"Christian Mathy"
],
"abstract": "Severe vitamin deficiency disease is rarely seen in developed countries. We present an atypical case of a young man with scurvy, pellagra, and hypovitaminosis A, caused by longstanding functional abdominal pain that severely limited his ability to eat.",
"keywords": [
"Scurvy",
"Pellagra",
"hypovitaminosis A",
"vitamin deficiency",
"malnutrition",
"functional disorder",
"abdominal pain"
],
"content": "Case presentation\n\nThis case describes a spirited 20 year-old male Caucasian college student, with no significant past medical history, who has been suffering from severe mid-epigastric and lower abdominal pain for ten years. He has seen multiple doctors for these symptoms and was diagnosed with irritable bowel syndrome. Various medical therapies were tried but they did not provide durable benefit. He described having acute onset of sharp, debilitating pain, lasting twenty minutes to several hours on a daily basis. The pain was often triggered by eating or drinking. Associated gastrointestinal symptoms included watery diarrhea. Over the last five years, the pain had become so severe, especially post-prandially, that he could only tolerate small amount of plain food, such as plain white bread and red meat, and drink only water. As a result, he had difficulty gaining weight and was frustrated with his dietary limitations. He also complained of blurry vision at night. Upon physical examination, the patient appeared thin but not cachectic. His examination was notable for dermatitis of his dorsal hands bilaterally demarcating at the wrists with hyperkeratosis over his metacarpophalangeal and proximal interphalangeal joints. Follicular hyperkeratosis with perifollicular hemorrhage and corkscrew hair were apparent on his thighs, calves, and buttocks symmetrically.\n\nThe patient underwent an extensive workup for chronic abdominal pain. His initial laboratory tests, including a complete blood count, metabolic panel, liver profile tests, and fecal fat tests were unremarkable. He underwent an abdominal MRI, CT enterography, cholescintigraphy (HIDA scan), upper endoscopy, colonoscopy, capsule endoscopy, endoscopic ultrasound and a gastric emptying study, which were all unrevealing. He was tested as positive for Helicobacter pylori infection and small intestinal bacterial overgrowth, for which he received an appropriate course of antibiotics, but his symptoms persisted. Screenings for heavy metal exposure and porphyria were negative. When vitamin levels were checked, he was found to have undetectable levels of vitamin A and vitamin C. Clinical suspicion of severe nutritional deficiency was further confirmed by his dermatologist who commented that the patient’s pattern of photodistributed, erythematous patches, well demarcated on dorsal hand with hyperkeratosis” over his finger joints was consistent with pellagra and “follicular hyperkeratosis with perifollicular hemorrhage and corkscrew hair” were consistent with vitamin A and C deficiencies. Based on this patient’s clinical presentation, laboratory values, as well as dermatopathological findings, diagnoses of pellagra (vitamin B3 or niacin deficiency), scurvy (vitamin C deficiency), and phrynoderma (vitamin A deficiency) were made.\n\n\nCourse follow-up and outcome\n\nThe patient was started on intravenous infusions of vitamin C and liquid multivitamin for niacin and vitamin A repletion. To treat his abdominal pain, the patient tried a number of pharmacological therapies, including antispasmodic agents, tricyclic antidepressants, and gabapentin, none of which provided relief. The patient was referred for psychiatry and to a pain specialist for further management. After a few months of nutritional supplementation, his vitamin levels returned to the normal range. His diarrhea and night vision also improved.\n\n\nDiscussion\n\nVitamin deficiency diseases are common in developing countries where there is limited access to a healthy diet, particularly with fruits and vegetables. In industrialized countries, nutritional deficiencies are rarely seen, but can occur in alcoholics, institutionalized elderly, or those with restrictive dietary intake related to eating disorders, psychiatric conditions, food allergies, or organic gastrointestinal disorders1–5. This case highlights three specific vitamin deficiencies: scurvy, pellagra, and hypovitaminosis A.\n\nScurvy is a clinical syndrome that results from ascorbic acid or vitamin C deficiency, largely due to impaired collagen synthesis resulting in defective connective tissue. Symptoms can occur as early as three months after insufficient intake. Symptoms include ecchymosis, petechiae, coiled hairs, hyperkeratosis, bleeding gums, arthralgia, Sjogren’s syndrome, and poor wound healing. Scurvy generally occurs when the plasma concentration of ascorbic acid is < 0.2 mg/dL (11 µmol/L). Humans require exogenous intake of ascorbic acid for maintenance, particularly from fruit and vegetables. The recommended dietary allowance for ascorbic acid is 75 mg per day for most women, 90 mg per day for men, and 120 mg per day for pregnant/lactating women or the elderly6,7.\n\nNiacin, or vitamin B3, deficiency can cause pellagra, a clinical syndrome classically referred to as the four D’s: diarrhea, dermatitis, dementia, and death. The most common finding is a symmetric hyperpigmented rash in exposed areas of skin, similar to a sunburn4. The recommended dietary allowance for niacin is 14 NEs (Niacin Equivalents) daily for adult females, 16 NEs for adult males, 17 NEs for pregnant women, and 18 NEs for lactating mothers. One NE is equivalent to 1 mg of niacin, which is equal to 60 mg of dietary tryptophan7,8.\n\nVitamin A is essential to cellular differentiation and integrity of the eye. Therefore, deficiency may lead to eye dryness, fragility, and night blindness. It is also associated with impaired bone growth, hyperkeratosis, and poor immune function. The recommended daily allowance for vitamin A is 3000 IU (international units) (900 µg retinol) daily for adult males and 2300 IU (700 µg retinol) for females7.\n\nOne important lesson to be gleaned from this case report is the potentially serious complications of a functional disorder. In this case, the patient suffered from severe chronic abdominal pain resulting in dietary restrictions that ultimately led him to profound malnutrition. If left untreated, this could progress to severe morbidity and even death in extreme cases. Therefore, functional gastrointestinal disorders, although seemingly benign conditions, can lead to serious long-term complications and should not be overlooked.\n\n\nConsent\n\nThe patient provided informed consent for publication of his clinical details.",
"appendix": "Author contributions\n\n\n\nEH: acquisition of data, interpretation of data, drafting of manuscript, assistance in clinical care.\n\nCM: interpretation of data, guidance of clinical management plan, revision of manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nAcknowledgements\n\nThe authors would like to acknowledge Timothy Berger, MD, from the Division of Dermatology at the University of California San Francisco for his expertise in clinical care. We would also like to thank the patient for giving us the opportunity to share his story with the medical community and add to our understanding of these conditions.\n\n\nReferences\n\nReuler JB, Broudy VC, Cooney TG: Adult scurvy. JAMA. 1985; 253(6): 805–807. PubMed Abstract | Publisher Full Text\n\nMertens MT, Gertner E: Rheumatic manifestations of scurvy: a report of three recent cases in a major urban center and a review. Semin Arthritis Rheum. 2011; 41(2): 286–290. PubMed Abstract | Publisher Full Text\n\nSmith A, Di Primio G, Humphrey-Murto S: Scurvy in the developed world. CMAJ. 2011; 183(11): E752–755. PubMed Abstract | Publisher Full Text | Free Full Text\n\nProusky JE: Pellagra may be a rare secondary complication of anorexia nervosa: a systematic review of the literature. Altern Med Rev. 2003; 8(2): 180–185. PubMed Abstract\n\nSommer A, Vyas KS: A global clinical view on vitamin A and carotenoids. Am J Clin Nutr. 2012; 96(5): 1204S–1206S. PubMed Abstract | Publisher Full Text\n\nFood and Nutrition Board - Institute of Medicine. Dietary reference intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids. Washington DC: National Academy Press; 2000. Reference Source\n\nOtten JJ, Hellwig J, Meyers LD (Eds): Dietary Reference Intakes: The Essential Guide to Nutrient Requirements. Washington DC: The National Academies Press; 2006. Reference Source\n\nFood and Nutrition Board-Institute of Medicine. Dietary reference intakes for Thiamin, riboflavin, niacin, vitamin B6, folate, vitamin B12, pantothenic acid, biotin, and choline. Washington DC: National Academy Press; 1998. Reference Source"
}
|
[
{
"id": "3507",
"date": "05 Feb 2014",
"name": "Bruce Bistrian",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting case report that demonstrated the appearance of undetectable vitamin A and C levels in the setting of dermatologic evidence of niacin deficiency (pellagra) and vitamin C (scurvy) that developed in an individual with severe gastrointestinal symptoms but without weight loss presumed secondary to a functional disorder.\n\nThat severe dietary deficiencies could develop over the period of years from a disturbed diet is likely, and the laboratory evidence for deficiency is not refutable for vitamins A and C. However it would be interesting to know whether there was biochemical evidence for niacin deficiency and whether the presumed pellagrous skin lesions responded to vitamin supplementation, since the routine B vitamin fortification of grain products in developed countries would make this somewhat less likely. Although the authors state that red meat was tolerated to some extent, it is not likely that it would be a substantial component of the diet, since meat is a good source of essential nutrients. The publication of this report is important to remind us that nutritional deficiencies can develop under such conditions.",
"responses": []
},
{
"id": "4159",
"date": "28 Mar 2014",
"name": "Sarah Sheibani",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a very well-written and informative case report that highlights a potential complication that can occur in an apparently benign disorder. Functional abdominal pain is one of the most common disorders evaluated by gastroenterologists. To my knowledge, this is the first report to suggest that vitamin deficiencies should be checked in patients with functional abdominal pain that present with malnutrition and diarrhea along with unexplained clinical findings, such as a rash.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-35
|
https://f1000research.com/articles/3-34/v1
|
31 Jan 14
|
{
"type": "Research Article",
"title": "Genetic and environmental determinants of insect herbivore community structure in a Betula pendula population",
"authors": [
"Tarja Silfver",
"Matti Rousi",
"Elina Oksanen",
"Heikki Roininen",
"Matti Rousi",
"Elina Oksanen",
"Heikki Roininen"
],
"abstract": "A number of recent studies have shown that intraspecific genetic variation of plants may have a profound effect on the herbivorous communities which depend on them. However less is known about the relative importance of intraspecific variation compared to other ecological factors, for example environmental variation or the effects of herbivore damage. We randomly selected 22 Betula pendula genotypes from a local population (< 0.9 ha), cloned them and planted cloned seedlings on two study sites separated at a regional scale (distance between sites about 30 km) to examine an insect community of 23-27 species on these genotypes. B. pendula genotypes did not differ in their species richness, but the total mean abundance and the structure of the insect herbivore community was significantly affected by the genotype, which could account for up to 27% of the total variation in community structure. B. pendula genotype accounted for two to four times more variation in the arthropod community structure than did environmental (block) variation on a local scale, while on a regional scale, genotypic and environmental (site) variation accounted for 4-14% of the arthropod community structure. The genetic effects were modified by environmental variation on both a local and regional scale over one study year, and locally, the largest part of the variation (38%) could be explained by the genotype × environment (block) interactions. Suppression of insect herbivores during one growing season led to changed arthropod community structure in the following growing season, but this effect was minimal and could explain only 4% of the total variation in insect community structure. Our results suggest that both genetic and environmental factors are important determinants of the community structure of herbivorous insects. Together these mechanisms appear to maintain the high diversity of insects in B. pendula forest ecosystems.",
"keywords": [
"Genetic variation within one species can affect the structure and dynamics of associated communities and entire ecosystems1",
"2. This may be considerable",
"especially for keystone species",
"such as forest trees",
"which serve as food and habitat for numerous primary consumers. A vast number of studies have already shown that arthropod communities respond to genetic differences among individual plants within interspecific hybridizing complexes (e.g. Eucalyptus3",
"Salix4",
"Populus5",
"Quercus6) or specific genotypes within species (e.g. Oenothera biennis7",
"Eucalyptus globulus8",
"Solidago altissima9",
"Populus angustifolia10). However",
"it has recently been argued that the role of plant genetic variation in structuring arthropod communities has been considerably inflated due to the common methodological flaw that genotypes are collected from diverse and often distant environments",
"which maximizes genetic variation",
"whilst experiments are performed in a single common garden where environmental variation is minimized11",
"12. Indeed",
"when this mismatch in scale was avoided in the experimental design",
"spatial processes relegated host plant genotype to a secondary role in structuring insect communities of Quercus robur L.13. Whether this applies to all systems is",
"however",
"not yet known."
],
"content": "Introduction\n\nGenetic variation within one species can affect the structure and dynamics of associated communities and entire ecosystems1,2. This may be considerable, especially for keystone species, such as forest trees, which serve as food and habitat for numerous primary consumers. A vast number of studies have already shown that arthropod communities respond to genetic differences among individual plants within interspecific hybridizing complexes (e.g. Eucalyptus3, Salix4, Populus5, Quercus6) or specific genotypes within species (e.g. Oenothera biennis7, Eucalyptus globulus8, Solidago altissima9, Populus angustifolia10). However, it has recently been argued that the role of plant genetic variation in structuring arthropod communities has been considerably inflated due to the common methodological flaw that genotypes are collected from diverse and often distant environments, which maximizes genetic variation, whilst experiments are performed in a single common garden where environmental variation is minimized11,12. Indeed, when this mismatch in scale was avoided in the experimental design, spatial processes relegated host plant genotype to a secondary role in structuring insect communities of Quercus robur L.13. Whether this applies to all systems is, however, not yet known.\n\nGenes encounter a range of environments in nature and it has long been recognized that genetic determination of plant susceptibility to a herbivorous insect depends on environmental context14. However, most studies that have examined the role of genotype × environment interactions in the abundance and distribution of herbivorous species, have used only one or a few closely related herbivore species (e.g.15–18), and much fewer studies have examined genotype × environment interactions in a community context7,13,19,20. It is well recognized that we know too little of the relative importance of intraspecific genetic variation compared to other ecological factors that also influence multi-trophic communities and ecosystem processes11. Thus, the examination of genotype × environment interactions in a community context may be essential for improving our knowledge in the developing field of community genetics.\n\nSilver birch (Betula pendula Roth) is an ideal tree species in which to examine the mechanisms of plant-herbivore interactions and the community-level consequences of trait variation, because the species shows remarkable genetic variation in its resistance to herbivores21–24. In addition, the genetic variation of secondary metabolites26, nutrient concentrations27, and phenological traits28,29 of B. pendula are known to be substantial, and all these traits are known to affect herbivores and higher trophic level interactions2,30,31. Most of the studies that have been conducted using B. pendula have used genotypes that were originally randomly selected from a local B. pendula population, i.e. from a naturally regenerated forest stand < 0.9 ha. None of these earlier studies have, however, investigated the within-population genotypic variation in B. pendula insect herbivore species richness and community composition. We cloned 22 B. pendula genotypes, planted them in two common gardens separated at a regional scale (distance between sites about 30 km), and studied the relative importance of genetic variation in community patterns, comparing both local and regional environmental variation. In addition, we examined how strongly herbivores themselves can modify arthropod communities associated with B. pendula by suppressing herbivores from half of the saplings over one growing season in one common garden and surveying their arthropod communities the following season.\n\n\nMaterials and methods\n\nThe 22 different genotypes of B. pendula were cloned during spring 1998 from randomly selected B. pendula trees taken from a naturally regenerated B. pendula - B. pubescens Ehr forest in Punkaharju, southeastern Finland (61°48′ N, 29°18′ E), to study genetic variation in phenology, growth, reproduction and resistance-related traits among individual birch trees25. Sampling was stratified random sampling: six spots where forest lift could be transferred were first selected around the forest, and 2–5 trees within the reach of forest lift in each spot were then randomly (by throwing a coin) selected for our study purposes. B. pendula is predominantly a sexual species, but genotypes can be cloned for study purposes or for plantations using standard tissue-culture methods32. Cloned B. pendula saplings were planted at the growing sites (i.e. common gardens, each approximately 0.25 ha) in June 1999 to find out the degree to which the genotype and environment affect birch traits and to test how genotypes differ in their response to the environment26. The Kuikanniitty study site (61°47′ N, 29°21′ E) is an abandoned cultivated field and the Parikkala study site (61°36′ N, 29°36′ E) is Myrtillus type forest33. Soil type was defined as fine sandy till for both sites26. The distance between these sites was around 30 km and they were situated at approximately the same altitude (Kuikanniitty 79 m and Parikkala 93 m above sea level). Thus, the mean summer (June–August) temperatures were very similar at these sites: in 2002 mean temperatures were 17.6°C and 17.9°C and in 2003 they were 15.9°C and 15.6°C in Kuikanniitty and Parikkala, respectively. Both study sites were divided into six blocks, each of which included four saplings from each genotype. To prevent edge effects, the experimental saplings were surrounded by one row of extra saplings. From each block, one of the four saplings of a total of 22 genotypes was randomly selected for the present study in order to have six replicates per genotype.\n\nIn addition, we collected additional data from Kuikanniitty in 2003 to investigate the effect of previous insect herbivory on insect community structure and abundance, and surveyed one extra sapling from each block and genotype. These extra saplings were protected from insect herbivory in the previous growing season by regular sprayings with synthetic pyrethrin23, which has no direct or side effects on the growth or chemistry of birch seedlings34.\n\nThe insect herbivore community of each sapling was assessed by surveying the abundance of 23 (in Parikkala 2002) or 27 (in Kuikanniitty 2002–2003, and Parikkala 2003) insect taxa from diverse orders (Lepidoptera, Hymenoptera, Coleoptera, Diptera, Hemiptera; Table 1). These taxa were generally the most abundant taxa in both sites. However, species that were rare in both sites were included in the surveys as well. Species identifications were undertaken following Saalas35 species identification guide, using several web pages (http://www.funet.fi/pub/sci/bio/life/insecta/index.html; http://www.leafmines.co.uk/index.htm; http://www.bladmineerders.nl/; http://www.nrm.se/) with the assistance of specialists. Euceraphis betulae eggs were counted from the side of twelve (2002) or eight (2003) topmost buds in April before budburst. However, in 2002 E. betulae eggs were counted from two saplings per genotype per block (sum of the eggs on the sides of 24 buds was used in the analysis), because regular sprayings with synthetic pyrethrin on the other sapling was started only after egg counts in both sites. Trichiosoma sp. pupae were counted in April/May when the timing of budburst of the same saplings was observed (Possen, submitted manuscript). The abundance of Eriocrania sp. was determined at the end of June, Deporaus betulae at the beginning of July and Heteropteran 1 (sap sucker) in August. Croesus septentrionalis larval colonies and the number of larvae in each colony were recorded along with Eriocrania and D. betulae measurements in both years. The abundance of all other insects were determined indirectly by counting damaged leaves at the beginning of September in both years, since the damage caused by most of the surveyed taxa remained identifiable for a long time after the initial damage.\n\na E. tetraquetrana counts represent the damage during the whole lifetime of the saplings (see Materials and methods). Note also that years are not directly comparable because of the changed sampling protocol between years.\n\nIn general, the insect abundance in 2002 was determined by surveying the whole sapling. The mean height of these saplings at the end of 2002 was 253 ± 4.3 cm (mean ± SE) in Kuikanniitty and 227 ± 3.8 cm in Parikkala. Because B. pendula genotypes differ in their height and diameter growth23 and large saplings may harbor more insects than smaller saplings, we determined the whole sapling “surface area” and used it as a covariate hereafter called “size index” in statistical analysis. Surface area was determined by photographing each sapling sideways from their southern side against a white background, converting the picture to a black and white silhouette picture in Adobe Photoshop 7.0 and determining the number of black pixels (i.e. leaf and branch area) within the picture. The number of pixels was converted to m2 using the number of pixels of a known area as a reference. The amount of pixels significantly (p < 0.001) explained over 73% of the sapling volume [Y = (3.14 * {base diameter/2}2 * height)/3] in both sites. The abundance of Phyllonorycter cavella, Phyllonorycter sp. 1, Parornix betulae and Parornix sp. was not examined on the whole sapling, but was determined as the damage (i.e. number of mines per each species) found within a period of 30 seconds. The period of time (30 sec) was chosen so that even the smallest saplings had leaves uncounted when the time was up.\n\nSince the method of assessing herbivore abundance/resistance by time counts has been successfully used in the past35,36 we decided to use time counts to determine the abundance of almost all taxa (except E. betulae, Trichiosoma sp. and C. septentrionalis) in 2003. The same person undertook all surveys. The abundance of easily visible damage (large mines and rolls) of Eriocrania sp., D. betulae and Heteropteran 1 were determined as the number of damaged areas found within a period of 30 seconds. Epinotia tetraquetrana “knobs” in the branches of the saplings were counted within a period of 20 seconds in 2003 starting at the top of the tree. Since the “knobs” in the branches remain visible for years and we did not separate different year’s growth while surveying, the values represent the accumulation of E. tetraquetrana damage during the last few years. Therefore the same values were used in both years’ insect community analyses. The abundance of all other 20 taxa in 2003 was determined within a single time count of each sapling at the beginning of September. To examine a similar proportion of each sapling, they were divided into three size categories according to their height and number of leaves. Small saplings (average height 2.8 and 3.2 m in Parikkala and Kuikanniitty, respectively) were surveyed for 30 seconds, average sized saplings (3.5 and 3.9 m in Parikkala and Kuikanniitty, respectively) for 60 seconds and large saplings (4.5 m in Kuikanniitty, large saplings were not found in Parikkala) for 120 seconds. Surveying time was used as a covariate called size index in statistical analysis.\n\nAll multivariate analyses were performed with Primer 6 (Primer-E Ltd, United Kingdom). The full data matrix consists of the abundance of 23–27 (23 in Parikkala 2002) insect species in 264 saplings (22 genotypes, 6 blocks, 2 sites) that were surveyed in two consecutive years. All surveyed insect species were included in the statistical analysis when sites were tested separately, but those four species that were not surveyed in Parikkala 2002 were excluded also from Kuikanniitty 2002 data when sites were compared. Arthropod community composition data was analyzed using non-parametric multivariate analysis of variance (PERMANOVA), which is well suited to non-normal ecological data such as ours38,39. Years were analyzed separately in all statistical tests, because of the changed sampling protocol between years (surveying the whole tree in 2002, using time counts in 2003). All data was fourth root transformed prior to analysis to reduce differences between common and rare species. The semimetric Bay-Curtis distance, which generally seems to provide the most meaningful measure of dissimilarity in ecological community structure39, was used to calculate distances between each pair of observations. The resulting distance matrix was used to obtain p-values using a random subset of 4999 permutations in PERMANOVA. The permutation method was permutation of residuals under a reduced model. The statistical model was designed to test the effect of genotype, site, block (nested within site) and the interaction of genotype × site using sapling size index (sapling surface area in 2002 and surveying time in 2003, see above) as a covariate. Site was treated as a fixed factor and block and genotype as random factors in the model. In addition to these analyses, we separately tested the effect of genotype and block on insect assemblages in each site and year to calculate the proportion of variance explained by B. pendula genotype and local environment (i.e. replicated block). Additional data collected from those saplings that were protected from insect herbivory in the previous growing season in Kuikanniitty 2003, were combined with the Kuikanniitty 2003 non-treated sapling data prior to analyzing the effects of insect removal, block and genotype, and their two-way interactions with the insect assemblages with PERMANOVA. Sapling size index was used as a covariate.\n\nTo visualize the multivariate patterns among observations, non-metric multidimensional scaling (nMDS) was performed on the Bay-Curtis distances. The distance among centroids for groups of samples was determined prior to nMDS to increase clarity, e.g. when the whole data was visualized we had 88 genotype-site-year points (22 genotypes in 2 sites over 2 years) instead of 528 genotype-block-site-year points. To visualize the effect of genotype in individual site and year, we separately determined the distance among genotype centroids in each site and year and produced one nMDS plot from each of these “environments”. Additional Kuikanniitty 2003 data combined with Kuikanniitty 2003 raw data was used to visualize the effect of insect removal on insect assemblages using nMDS on the genotype centroids of those saplings that were either protected from herbivory or grown under natural herbivory.\n\nSpecies richness (number of species/sapling) and total mean abundance (number of herbivores/sapling) was statistically tested by analysis of covariance using SPSS 20.0.0.1 (IBM SPSS Statistics) General Linear Models (GLM) procedure. Those four species that were not surveyed in Parikkala 2002 were excluded also from Kuikanniitty species richness and total mean abundance calculations to better enable site comparisons. Genotype and block (nested within site) were treated as random factors and site as a fixed factor in the statistical model while sapling size index was used as a covariate. Additional Kuikanniitty 2003 data combined with Kuikanniitty 2003 basic data was used to analyze the effects of insect removal, block and genotype, and their interactions with the species richness and total mean abundance. Genotype and block were treated as random factors and insect removal as a fixed factor while sapling size index was used as a covariate. Total mean abundance was log(x+1)-transformed to equalize the error variances across groups in both analyses.\n\n\nResults\n\nStudy years and sites were distinctly grouped apart into two-dimensional ordination space, when the genotype centroids of different years and sites were analyzed using nMDS (Figure 1). The MANOVA Table in turn, shows that sites had statistically significantly different insect species community composition in both years (Table 2). Sites were also clearly different in their total mean abundance (Table 3) and species richness (p < 0.008 for the site effect in species richness): the forest site of Parikkala had a 49–78% higher total mean abundance, but 18–25% lower species richness than the abandoned field site of Kuikanniitty in 2002–2003, respectively. These findings indicate that each year and site had significantly different herbivorous insect assemblages, thus creating different biotic environments.\n\nStress is 0.14, which indicates a good representation of the data in two-dimensional ordination plot. Each point is a centroid of six replicates. Numbers in the centre of the markers are genotype identification numbers.\n\nSapling size index, which is a measure of height and number of leaves (see material and methods), was used as a covariate.\n\nSapling size index, which is a measure of height and number of leaves (see material and methods), was used as a covariate.\n\nB. pendula genotypes were significantly different in their insect species community composition in both study years (Table 2). In 2002, regional scale environmental (site) variation explained more of the total variation in species composition than the genotype (13.9 and 8.0%, respectively), while in 2003 the genotype explained more of the total variation than the site (12.1 and 3.8%, respectively). Significant genotype × site interaction, which explained 8.2% of the total variation, was found only in 2003. When the sites were tested separately in both years we found that the effect of genotype was significant in Kuikanniitty 2002 and both study sites in 2003 (Table 4, Figure 2). B. pendula genotype could account for 15.8–27.0% of the total variation in community structure, while local scale environmental (block) variation explained 5.9–7.6% of the total variation in community structure (Table 4, Figure 2).\n\nSapling size index, which is a measure of height and number of leaves (see material and methods), was used as a covariate.\n\nNon-metric MDS plot of insect assemblages of 23 (Parikkala 2002) or 27 species colonizing B. pendula genotypes in a) Kuikanniitty 2002, b) Parikkala 2002, c) Kuikanniitty 2003 and d) Parikkala 2003. Each point is a centroid of six replicates. Numbers in the centre of the markers are genotype identification numbers. White circles denote genotypes in Kuikanniitty, black circles denote genotypes in Parikkala. Stress values >0.2 indicate that this data may be better visualized with more dimensions (stress for three-dimensional solutions varied between 0.14 to 0.16).\n\nB. pendula genotypes also significantly differed in their total mean abundance of herbivores (mean number of herbivores/sapling): the total mean abundance of the most susceptible genotype was 5.4- and 3.2-fold compared to the total mean abundance of the most resistant genotype in Kuikanniitty and Parikkala 2002, respectively (Table 3, Figure 3). In 2003, only the genotype × site interaction was statistically significant, which indicates that the genotype effect strongly depended on the study site. Indeed, when we tested the study sites separately, genotype effect was significant only in Parikkala (ANCOVA: Parikkala F21,104=2.29, p=0.003; Kuikanniitty F21,103=1.48, p=0.103). The species richness (number of insect species/sapling) was not significantly affected by the B. pendula genotype or genotype × site interactions in either year (p>0.134).\n\nWhite bars: Kuikanniitty, black bars: Parikkala.\n\nLocal scale genotype × environment interaction (i.e. the interaction of genotype × replicated block) was studied in Kuikanniitty 2003. Insect species community composition was significantly affected by both genotype and genotype × block interaction (Table 5). Genotype variation explained 10.6% and genotype × block variation 38.0% of the total variation in insect community composition, indicating that genotype effect is also strongly affected by local scale environmental variation. Total mean abundance or species richness was not affected by genotype or genotype × block interaction (p>0.097).\n\nPrevious year herbivory changed the insect community composition of B. pendula saplings (Table 5). The genotype centroids of those saplings that were either subjected to natural herbivory or protected from it were located on the opposite sides of the two-dimensional nMDS ordination plot, although overlapping is evident (Figure 4). Previous year herbivory did, however, explain only 4.4% of the total variation in insect community composition. Total mean abundance was affected by the previous year’s herbivory as well, but species richness was not (ANCOVA: effects of insect removal on total mean abundance F1,5.28=34.6, p=0.002 and species richness p>0.829).\n\nSapling size index, which is a measure of height and number of leaves (see material and methods), was used as a covariate.\n\nEach point is a centroid of six replicates. Numbers in the centre of the markers are genotype identification numbers.\n\nThe associations between insect species across genotypes in different sites and years seemed to be based on random associations, since we found only one correlation that was significant after sequential Bonferroni correction40. An unidentified gallery mine (Lepidoptera 4) and E. fuliginosella were correlated across genotypes in Kuikanniitty 2002 (Pearson’s correlation; in 2002, r = 0.88, n = 22, p < 0.0001; in 2003, r = 0.545, n = 22, p = 0.009), but not in Parikkala (p>0.199).\n\n\nDiscussion\n\nOur results provide evidence that genetic variation within a natural B. pendula population can modify the structure of the arthropod community even though all genotypes supported similar insect species richness. Genetic variation in phenotypic plasticity, however, seemed to be the major factor affecting the abundance and structure of the insect herbivores associated with this tree species, because genotype effect was often dependent on the environmental variation at both regional (Table 2 and Table 3) and local scales (Table 5). Those B. pendula genotypes that were used in our study should give unbiased estimates of the true variance that is present in B. pendula populations, since we chose them randomly from one naturally regenerated population stand (< 0.9 ha) in eastern Finland, where this Eurasian deciduous tree species is particularly abundant42. By contrast, we might have exaggerated the role of regional environmental variation and genotype × environment (site) interactions by planting our genotypes on two rather different areas (open forest and abandoned field, areas that are typically rapidly colonized by B. pendula) at a much larger scale (70,000 ha). Therefore, it is not surprising that the importance of the genetic variation in structuring insect herbivore communities of B. pendula decreased from 15.8–27.0% (of variation explained) to 8.0–12.1% with increasing spatial scale in our study. Other studies have also found that while the effect of a genotype can be clear on local scales (within common gardens), it may be partially swamped by environmental variation on larger scales7,43.\n\nIt has been argued that, because host plant genotypes have often been collected from large geographic areas and studied within the confines of a single common garden, the role of the host plant genotype in arthropod community patterns has been largely overestimated12. Indeed, Tack et al.13 showed that spatial processes dominated genetic effects when genotypes of Q. robur were collected at the same local (500 ha) or regional (1 million ha) scale as that where experiments were conducted, and thus, in real landscapes, spatial impacts might relegate host plant genotype to a minor role. Our results, however, suggest otherwise, because genotype explained about three times more of the total variation in insect herbivore community structure than local environment (block) in both sites (Table 4), and the scale of our common garden(s) was approximately the same as the scale of that where genotypes were collected (< 0.9 ha). In addition, on a regional scale, genetic and environmental effects explained similar proportions of the total variation in arthropod community structure (Table 2), even though we might have inflated the role of the environment in our study. This discrepancy in our results might perhaps be attributed to the difference in the distribution of these wind-pollinated tree species: the populations of Q. robur are strongly fragmented and grow at the northern margin of the species’ European distribution in southern Finland (where Tack et al.13 conducted their experiments), while B. pendula has a wider and more continuous distribution over the whole of Finland, apart from Lapland. Q. robur populations exhibit higher geographic differentiation estimates, Fst 0.032 for B. pendula and 0.066 for Q. robur44,45, which means that the gene flow among B. pendula populations is two times higher than among Q. robur populations, and thus local B. pendula populations might express a larger amount of genetic variation than populations of Q. robur.\n\nWe found that insect herbivore communities can be affected by both local and regional genotype × environment interactions, at least in some years. But why do B. pendula genotypes support different insect communities in different environments? It is possible that resistance traits of the genotypes are changed due to differences in abiotic environment and insect communities respond to these changes. This is supported by the fact that earlier studies have found regional genotype × environment interactions in the secondary metabolites of the same study saplings26. Yet, we do not know whether genotype × environment interactions in B. pendula resistance traits exist at a local scale and recent studies suggest that secondary metabolites are not the most important anti-herbivore defence of plants31. On the other hand, spatial processes might affect local insect communities and create genotype × environment interactions. For example, in our experiment where genotypes of each block are arranged randomly, the effects of a particular genotype could be partially masked by the effects of their conspecifics in some blocks if nearby genotypes are very dissimilar, i.e. there is associational resistance (see a review by Agrawal et al.46) at the level of a genotype. Both of these processes may be affecting different insect species differently. We found only one species pair that was correlated across genotypes in one of our study sites, which, together with earlier findings47,48, indicates that generalized defenses against multiple insect species are not likely in B. pendula (see Leimu and Koricheva49). Additionally, it may also be that local insect communities differ in their response regardless of spatial processes and without any change in the traits of B. pendula.\n\nThe size of B. pendula trees is positively associated with their fitness, i.e. seed production29. It has been shown that herbivores can reduce the growth of B. pendula by up to 46% (Mikola et al. unpublished results, see also Prittinen et al.22, Silfver et al.23) and increase seedling mortality considerably50. Thus, by imposing selection in various genetically variable resistance traits of B. pendula25,26,51, herbivores may have high potential to drive the community evolution in B. pendula. Indeed, we found that only one season of protection from herbivory changed arthropod community variables (mean abundance and community composition) in five-year old field-grown B. pendula saplings. Total mean abundance, for example, was lower in saplings that were protected from herbivory in the previous growing season, which indicates that they may have had more resources to defend themselves against insects when herbivores were present again. Yet, the magnitude of these effects was smaller than the effects of local environmental (block) variation, and could explain only about 4% of the total variation in arthropod community structure. It is important to note, however, that in nature B. pendula seedlings typically establish in open patches, where high numbers of individuals compete heavily before self-thinning eliminates some of the seedlings. Surviving for these first years and consequently reaching maturity is crucial for an individual’s fitness in this long-lived tree species. Earlier studies that have used open-pollinated progeny of the same genotypes, have shown that in such dense stands, even moderate levels of insect herbivory can change the genetic structure of B. pendula populations in the first year of establishment52. This is reminiscent of recent studies, which have demonstrated that natural selection can favour different genotypes in the absence of herbivores rather than in their presence, and different genotypes in response to different herbivore species within only few generations of annual or biannual plants53,54 (see also Hare55).\n\nTo conclude, we have shown that the structure of insect herbivore communities can be significantly affected by intraspecific genetic variation when there is no mismatch in scale. However, genetic effects were modified by environmental variation on both a local and regional scale in one study year. Furthermore, insect herbivore damage in one growing season changed the community patterns of the following season, yet those effects were minimal compared to genetic and environmental factors. Our results suggest that both genetic and environmental factors are important determinants of the community structure of herbivorous insects. Together these mechanisms appear to maintain the high diversity of insects in B. pendula forest ecosystems.\n\n\nData availability\n\nfigshare: Community structure of insect herbivores on different genotypes of silver birch (Betula pendula), http://dx.doi.org/10.6084/m9.figshare.91533256",
"appendix": "Author contributions\n\n\n\nMR and EO conceived the study. MR contributed to the experimental design. HR contributed to the preparation of the manuscript and provided expertise in species identifications and statistics. TS carried out the research and prepared the first draft of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was funded by the Academy of Finland (project 78743, granted to EO), and funding granted to TS by the Graduate School in Forest Sciences, Finnish Concordia Fund and Kone Foundation (grant 2-712) is also acknowledged.\n\n\nAcknowledgements\n\nWe are grateful to the Punkaharju Research Unit of the Finnish Forest Research Institute for facilities and help. Matti Viitasaari, Tommi Nyman, and Jarmo Holopainen are thanked for help in species identification. 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HortScience. 1981; 16: 453. Reference Source\n\nJalonen J, Vanha-Majamaa I, Tonteri T: Optimal sample and plot size for inventory of field and ground layer vegetation in a mature Myrtillus-type boreal spruce forest. Ann Bot Fennici. 1998; 35: 191–196. Reference Source\n\nSilfver T, Autelo M, Paaso U, et al.: Use of an insecticide in field-scale plant-herbivore studies: no side effects of synthetic pyrethrin on Betula pendula growth or chemistry. Ann Bot Fennici. 2013; 50(5): 337–346. Publisher Full Text\n\nSaalas U: Suomen Metsähyönteiset Sekä Muut Metsälle Vahingolliset Ja Hyödylliset Eläimet. Helsinki, Suomen Tiedeakatemia. 1949. Reference Source\n\nFloate KD, Kearsley MJC, Whitham TG: Elevated herbivory in plant hybrid zones: Chrysomela confluens, Populus and phenological sinks. Ecology. 1993; 74(7): 2056–2065. Publisher Full Text\n\nMartinsen GD, Driebe EM, Whitham TG: Indirect interactions mediated by changing plant chemistry: Beaver browsing benefits beetles. Ecology. 1998; 79(1): 192–200. Publisher Full Text\n\nAnderson MJ: A new method for non-parametric multivariate analysis of variance. Austral Ecol. 2001; 26(1): 32–46. Publisher Full Text\n\nMcArdle BH, Anderson MJ: Fitting multivariate models to community data: A comment on distance-based redundance analysis. Ecology. 2001; 82(1): 290–297. Publisher Full Text\n\nFaith DP, Minchin PR, Belbin L: Compositional dissimilarity as a robust measure of ecological distance. Vegetatio. 1987; 69(1–3): 57–68. Publisher Full Text\n\nRice WR: Analyzing tables of statistical tests. Evolution. 1989; 43(1): 223–225. Publisher Full Text\n\nHynynen J, Niemistö P, Viherä-Aarnio A, et al.: Silviculture of birch (Betula pendula Roth and Betula pubescens Ehrh.) in northern Europe. Forestry. 2010; 83(1): 103–119. Publisher Full Text\n\nBangert R, Lonsdorf E, Wimp G, et al.: Genetic structure of a foundation species: scaling community phenotypes from the individual to the region. Heredity. 2008; 100(2): 121–131. PubMed Abstract | Publisher Full Text\n\nRusanen M, Vakkari P, Blom A: Genetic structure of Acer platanoides and Betula pendula in northern Europe. Can J Forest Res. 2003; 33(6): 1110–1115. Publisher Full Text\n\nVakkari P, Blom A, Rusanen M, et al.: Genetic variability of fragmented stands of pedunculate oak (Quercus robur) in Finland. Genetica. 2006; 127(1–3): 231–241. PubMed Abstract | Publisher Full Text\n\nAgrawal AA, Lau JA, Hambäck PA: Community heterogeneity and the evolution of interactions between plants and insect herbivores. Q Rev Biol. 2006; 81(4): 349–376. PubMed Abstract | Publisher Full Text\n\nRousi M, Tahvanainen J, Henttonen H, et al.: Clonal variation in susceptibility of white birches (Betula spp.) to mammalian and insect herbivores. Forest Science. 1997; 43(3): 396–402. Reference Source\n\nTikkanen O, Rousi M, Ylioja T, et al.: No negative correlation between growth and resistance to multiple herbivory in a deciduous tree, Betula pendula. Forest Ecol Manag. 2003; 177(1–3): 587–592. Publisher Full Text\n\nLeimu R, Koricheva J: A meta-analysis of genetic correlations between plant resistances to multiple enemies. Am Nat. 2006; 168(1): E15–E37. PubMed Abstract | Publisher Full Text\n\nPrittinen K, Pusenius J, Koivunoro K, et al.: Mortality in seedling populations of silver birch: Genotypic variation and herbivore effects. Funct Ecol. 2003; 17(5): 658–663. Publisher Full Text\n\nValkama E, Koricheva J, Salminen J, et al.: Leaf surface traits: Overlooked determinants of birch resistance to herbivores and foliar micro-fungi? Trees. 2005; 19(2): 191–197. Publisher Full Text\n\nPrittinen K, Pusenius J, Tahvanainen J, et al.: Herbivory modifies the genetic structure of birch populations. Oikos. 2006; 114(3): 465–470. Publisher Full Text\n\nAgrawal AA, Hastings AP, Johnson MT, et al.: Insect herbivores drive real-time ecological and evolutionary change in plant populations. Science. 2012; 338(6103): 113–116. PubMed Abstract | Publisher Full Text\n\nZüst T, Heichinger C, Grossniklaus U, et al.: Natural enemies drive geographic variation in plant defenses. Science. 2012; 338(6103): 116–119. PubMed Abstract | Publisher Full Text\n\nHare JD: How insect herbivores drive the evolution of plants. Science. 2012; 338(6103): 50–51. PubMed Abstract | Publisher Full Text\n\nTarja S, Matti R, Elina O, et al.: Community structure of insect herbivores on different genotypes of silver birch (Betula pendula). Figshare. 2014. Data Source"
}
|
[
{
"id": "3457",
"date": "07 Feb 2014",
"name": "Patrick Tobin",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this study, the authors selected and cloned 22 silver birch genotypes and planted them at two common garden sites to examine the effects of both genetic and environmental factors on the community structure of insect herbivores. By selecting genotypes from a spatially limited area (< 9 ha), and by performing the experiments at two spatially separated common garden sites, the authors were able to examine more precisely the main and interacting effects of genetic and environmental variation on the herbivore community. The authors also excluded herbivores on half of the host trees at each common garden site through application of an insecticide, which allowed them to examine the effect on the herbivore community in the following year. One limitation of the study is the use of only two common garden sites, which does limit the broader implications of the study. However, despite these limitations, the data still provide sound preliminary information on the importance of both genetic and environmental variation on the structure of the herbivore community. Although I understand and appreciate the challenges of documenting the herbivore community and the labor involved to do so, more comments are needed to address the lack of a more temporally robust sampling regime. Some species were sampled at a specific time given their respective seasonality, but most were sampled at the end of the growing season in the fall. In doing so, I suspect that authors would have missed spring and summer feeders, such as the winter moth for example, whose damage could have been difficult to ascertain and differentiate from other herbivores when herbivore damage was examined in the fall. This is not a fatal flaw, especially since the authors incorporated herbivore exclusions, which presumably would have also excluded spring and summer feeding herbivores. However, since one goal of the study was to examine the role of the herbivore community in affecting the community in the following year, it would be helpful to discuss the potential limitations of the fall sampling regime in documenting insects that feed earlier, and how the herbivore exclusion component was also (presumably) a mechanism to deal with sampling limitations. On a related note, it wasn't clear to me if potential temporal autocorrelation in the numbers and diversity of the herbivore community from one year to the next was appropriately addressed in the analysis; if so, I would suggest adding a statement in the materials and methods how this was addressed or if not, why it did not need to be addressed. Overall, this was a nicely designed experiment, the manuscript was very well written, and the data were well presented. This study adds to our knowledge of the role of genetic and environmental variation on the structure of the herbivore community on silver birch, and sets the stage for a number of interesting follow-up questions.",
"responses": [
{
"c_id": "737",
"date": "21 Mar 2014",
"name": "Tarja Silfver",
"role": "Author Response",
"response": "Our sampling regime may appear to be concentrated on fall, but actually many of those species that were examined in fall might have made their damage many months earlier in early/late summer; we sampled species that were distinguishable for a long time after the damage. It is true that due to practical reasons (and difficulties in identification) we sampled only part of the hundreds of insect species that feed on silver birch and many free feeders were missed in this study. Years, however, were analyzed separately in our study and thus, potential temporal autocorrelation is not addressed in this study."
}
]
},
{
"id": "3852",
"date": "24 Feb 2014",
"name": "Judith Myers",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting study on the role of tree genotypes on herbivorous insect attack of willow trees, Betula pendula, using a common garden approach in Finland. Twenty-two trees were randomly selected and cloned, although no details are given as to how the trees were cloned. Each tree is considered to be a different genotype. Six blocks with 4 saplings of each genotype were used from which one sapling was randomly selected to give 6 replicates per genotype for the study of insect attack. Insect sampling was done largely based on the damage done over the summer. I am surprised that it is possible to distinguish insects based on their damage as I would have guessed that the damage from leaf miners would have been similar, and damage from sucking insects difficult to find at all. I have the following questions and comments:What is meant by structure of the insect community? This term is used in the abstract and throughout the paper but it is not defined. The term local environmental variation is mentioned, and it would be clearer if this was referred to as variation among blocks. Insects were removed from some saplings but details are lacking on how this was done, how frequently it was done, and how effective it was. “Additional Kuikanniitty 2003 data combined with Kuikanniitty 2003 raw data was used” I don’t understand what this means. How would these results be interpreted by one who wanted to select for herbivore resistance among tree genotypes? Is the amount and consistency of the among genotype resistance sufficient to evolve over time? How are the results influenced by variation in insect abundance from year to year?",
"responses": [
{
"c_id": "738",
"date": "21 Mar 2014",
"name": "Tarja Silfver",
"role": "Author Response",
"response": "\"What is meant by structure of the insect community? This term is used in the abstract and throughout the paper but it is not defined.
\"The structure of the insect community means the composition of the insect community. \"The term local environmental variation is mentioned, and it would be clearer if this was referred to as variation among blocks.
\" This is a matter of opinion; we have kept it as it is, because we feel that discussion is easier to follow if term local environmental variation is used. \"Insects were removed from some saplings but details are lacking on how this was done, how frequently it was done, and how effective it was.
\"Insect removal was conducted by synthetic pyrethrin (Decis) sprayings in about every other week using portable garden sprayer. Wind drift of the insecticide was controlled with a portable shower cubicle. Effectiveness of the sprayings was not systematically determined, but it was evident that sprayed seedlings had very little visible leaf damage compared to unsprayed ones. \"“Additional Kuikanniitty 2003 data combined with Kuikanniitty 2003 raw data was used” I don’t understand what this means.
\"Additional Kuikanniitty 2003 data means the data collected from the saplings that were sprayed with insecticide in 2002 and Kuikanniitty raw data 2003 means the data collected from nonsprayed saplings, i.e. it is the same data, which is in “2003_data” raw data file. \"How would these results be interpreted by one who wanted to select for herbivore resistance among tree genotypes? Is the amount and consistency of the among genotype resistance sufficient to evolve over time?
\" I would not use these results to select for general herbivore resistance among birch genotypes, because all birch feeding insect were not included in our study. Thus, the total mean abundance of herbivores among our birch genotypes may not be strongly connected to the amount of leaf damage, which is often used as a measure of insect herbivore resistance. Our earlier studies with the same saplings, however, have shown that silver birch genotypes differ in their insect resistance (defined as a lower amount of leaf damage) consistently across years and study sites (Silfver et al., 2009). \"How are the results influenced by variation in insect abundance from year to year?\"In general, community structure should not be influenced by yearly variation in insect abundance if insect abundance changes similarly in all species. However, if the relative changes in the abundance of counted insect species are large, it can influence the results. We did not test for the year x genotype effects, because of the changed sampling protocol between years."
}
]
}
] | 1
|
https://f1000research.com/articles/3-34
|
https://f1000research.com/articles/1-22/v1
|
03 Oct 12
|
{
"type": "Case Report",
"title": "A rare variation of hydranencephaly: case report",
"authors": [
"Buddhika TB Wijerathne",
"Geetha K Rathnayake",
"Sisira K Ranaraja",
"Geetha K Rathnayake",
"Sisira K Ranaraja"
],
"abstract": "Hydranencephaly is a rare severe abnormality characterized by replacement of cerebral hemisphere with fluid covered by leptomeninges such that absent cerebral cortex. We present the Ultrasonographic diagnosis of a case of fetal Hydranencephaly at 38 weeks of gestation. Sonography revealed the absence of cerebral cortex, thalami and basal ganglia with disrupted falx and preserved posterior fossa structures. This was the first reported case of Hydranencephaly with absence thalami and basal ganglia along with midbrain. Confirmation of diagnosis was made with postnatal computed tomography. Even though it was diagnosed in the late third trimester, it allowed prompt and finest obstetric management which reduced the risk for both fetus and mother. Furthermore it allowed arrangement of optimal conditions for birth at a unit with accessible specialized pediatric facilities.",
"keywords": [
"Please note that the refereeing status of this version was changed from “indexed” to “[v1",
"ref status: approved 1",
"approved with reservations 1]”."
],
"content": "Introduction\n\nHydranencephaly is a rare abnormality in which the cerebral hemispheres are almost absent and have been replaced with fluid. It was first described by cruveilher (1892) as “Anencephalie hydrocephalique” or “Hydroanencephalie”1. Crome and Sylvester reviewed the disease and defined it as a congenital condition2. It is a very rare isolated abnormality occurring less than 1 per 10,000 births world wide3. It is said to be present in 0.2% of infant autopsies and approximately 1% of babies are diagnosed clinically as hydrocephalus4. It's important to distinguish early between Hydranencephaly and extreme hydrocephalus, because the latter carries a considerably superior prognosis5. We report a rare case of Hydranencephaly which was diagnosed in late third trimester followed by successful obstetric management.\n\n\nCase presentation\n\nA 28-year-old woman, gravida 2 para 1 was presented to our tertiary care institution at 38 weeks' gestation because of lower abdominal pain. She and her partner were Sinhalese and unrelated. There was no family history of genetic or congenital anomalies. Her first pregnancy was uncomplicated normal vaginal delivery with normal healthy male child. She had not attended the local antenatal clinic regularly where she was recognized as a noncompliant patient. She had not undergone routine dating scan, anomaly scan or the growth scan of the fetus. She was a nonsmoker and there was no history suggestive of congenital infections or exposure to toxins. The clinical obstetric examination was unremarkable. The cardiotocography of the fetus was normal.\n\nObstetric ultrasound scan showed fluid filled cranial cavity with absent cerebral cortex, thalami and basal ganglia. The third ventricle dilated and remnants of midbrain structures were seen (Figure 1). The cerebellum and other posterior fossa structures were preserved. The falx cerebri was disrupted (Figure 2). There was no polyhydramnios. Umbilical artery Doppler studies were normal. Sonographicaly it was suggestive of Hydranencephaly. Her blood group was B positive and Infectious disease Antibody test showed negative titers for Rubella, HIV, Hepatitis B and Toxoplasmosis. We planed caesarian section to avoid obstetric complications due to possible cephalopelvic disproportion and minimize the risk for both mother and fetus. Specialized Pediatric team was pre informed. Parents were counseled regarding poor prognosis of the newborn's condition. At 39 week of gestation emergency caesarian section performed due to fetal distress. The newborn was a 2,980g girl with normal physical appearance and normal sized skull (head circumference = 33.5 cm) which was brilliantly transilluminated.\n\nTransabdominal transverse section of the fetal head at 38 weeks of gestation shows dilated third ventricle (V) with absent thalami and basal ganglia and cerebral cortex.\n\nTransabdominal transverse section of the fetal head shows disrupted falx (F).\n\nNewborn was transferred to neonatal Intensive care unit because of the respiratory distress. Four days after delivery Computed tomography (CT) of the newborn's head was performed without intravenous contrast. On CT there was no cerebral cortex, thalami or basal ganglia identified. The third ventricle dilated and remnants of midbrain structures were seen (Figure 3). The cerebellum and other posterior fossa structures were preserved with disrupted falx cerebri (Figure 4a, 4b).\n\nDilated third ventricle (V) with absent thalami and basal ganglia and cerebral cortex.\n\n(a) Intact Cerebellum (C) and other posterior fossa structures. (b) Shows Disrupted Falx cerebri (F).\n\nThe CT scan confirmed the diagnosis Hydranencephaly. Baby died after two weeks of birth due to cardiac arrest. The parents declined a postmortem examination.\n\n\nDiscussion\n\nHydranencephaly is an encephaloclastic abnormality characterized by replacement of cerebral hemisphere with the cerebrospinal fluid and necrotic debris surrounded by leptomeninges such that no cerebral cortex is present but there may be partial preservation of portion of the occipital lobe6. The midbrain, thalamus, basal ganglia, choroid plexuses, cerebellum and brain stem are usually preserved and contained within normal skull. The Falx cerebri is usually present but may be partially or completely absent. The septum pellucidum may be absent.\n\nThe aetiopathogenesis of Hydranencephaly is heterogeneous, but several theories have been postulated. It has been suggested that bilateral occlusion of supraclenoid segment of the internal carotid artery or middle cerebral7 arteries before the 24 weeks of gestation cause ischemia, edema, autolysis and disappearance of cerebral hemispheres respectively8. Nevertheless some reports suggest occlusion of arteries due to temporary spasm or compression rather that direct occlusion9. Intrauterine infections also cause necrotizing vasculitis or local destruction of brain. Infections like congenital toxoplasmosis or viral infections (Adenovirus, cytomegalovirus, Enterovirus, Epstein-Barr virus, herpes simplex virus, Parvovirus, and respiratory syncytial viruses) have been implicated in numerous cases3. Fetal hypoxia due to exposure to toxin like Carbon monoxide or butane gas during antenatal period may result diffuse hypoxic ischemic brain necrosis lead to Hydranencephaly10. Thromboplastic material from a deceased co-twin in monochorionic twin pregnancies reported to be implicated11. Hydranencephaly has been described in rare syndromes12.\n\nUltrasonographic findings include large cystic mass filling entire intracranial cavity with absence of cerebral cortex. The head size may be normal or large13. Appearance of thalami and brainstem protruding in to cystic cavity is characteristic, together with a midline echo from the remnant of falx, the tentorium cerebella and cerebellum14. The third ventricle and choroid plexuses are often visible. Absence of septum pellucidum may give rise to what appears to be a single ventricle in midline15. The major differential diagnosis includes extreme hydrocephaly, alobar holoprosencephaly and porencephaly. In these conditions the above mentioned structures will still be surrounded by rim of cortex. In extreme hydrocephalus, the thin cortical mantle may be hard to identify sonographically and Magnotic resonance imaging (MRI) or rarely intrauterine CT scan might aid diagnosis13. The ultrasound is the best diagnostic tool during the prenatal period and postnatal period it's MRI16–18 or CT. It is important to distinguish Hydranencephaly from other differential diagnosis because they carry better prognosis. The most of the cases detected second half of pregnancy12 and there have been reported cases of Sonographic evaluation of fetal Hydranencephaly in the first trimeste19.\n\nHydranencephaly has an irretrievably poor prognosis, with merely remaining brainstem functions. Some die at birth, but most infant die within the first year of life20 and if survived they are profoundly retarded. The recurrence risk is negligible14. Because of the poor prognosis, termination of pregnancy is recommended once definitive diagnosis has been established13. If macrocrania is found in late pregnancy cephalocentesis may be indicated (aid delivery)13. Counsel parent regarding poor prognosis and management options are very important.\n\nUltrasonographic findings, in the case we have described, made a diagnosis of Hydranencephaly particularly likely and this was confirmed by postnatal CT of the fetal head. This is the first published case of Hydranencephaly with absent thalami and basal ganglia along with midbrain. Sonographic evaluation is sufficient for the prenatal diagnosis of Hydranencephaly in most cases, and MRI or intrauterine CT cannot be considered a first-line diagnostic tool.\n\n\nConclusions\n\nEarly diagnosis is important as an early treatment options avoids obstetrics complications and it may be very useful for giving appropriate advice to parents during the pregnancy and also for preparing the optimal conditions of birth at a unit with available specialized pediatric facilities.\n\n\nConsent\n\nWritten informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editors.",
"appendix": "Author contributions\n\n\n\nSKR and BTBW reviewed the manuscript. BTBW and GKR were involved in drafting the manuscript and reviewing the literature. BTBW was a major contributor in revising the manuscript and getting informed consent from patient. GKR, SKR and BTBW were involved in reviewing the literature. All authors were responsible for the diagnosis, treatment and follow-up of the patient. All authors read and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nWe thank Mrs. Janaki Munasinghe for correcting grammar and spellings of the manuscript.\n\n\nReferences\n\nRaybaud C: Destructive lesion of the brain. Neuroradiology. 1983; 25(4): 265–291. PubMed Abstract | Publisher Full Text\n\nCrome L, Sylvester PE: Hydranencephaly (hydrencephaly). Arch Dis Child. 1958; 33(169): 235–245. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKurtz AB, Johnson PT: Diagnosis please. Case 7: Hydranencephaly. Radiology. 1999; 210(2): 419–22. PubMed Abstract\n\nHalsey JH: Hydranencephaly. In Handbook of Clinical Neurology. Volume 50: Malformations. Edited by PJ Vinken, GW Bruyn and JL Klawans. Amsterdam: Elsevier 1987; 337–353.\n\nMalheiros JA, Trivelato FP, Oliveira MM, et al.: Endoscopic choroid plexus cauterization versus ventriculoperitoneal shunt for hydranencephaly and near hydranencephaly: a prospective study. Neurosurgery. 2010; 66(3): 459–64. PubMed Abstract | Publisher Full Text\n\nJeffrey MC: Hydranencephaly: Transillumination May Not Illuminate Diagnosis. Neoreviews. 2012; 13(4): e233–e240. Publisher Full Text\n\nMyers RE: Brain pathology following fetal vascular occlusion: an experimental study. Invest Ophthalmol. 1969; 8(1): 41–50. PubMed Abstract\n\nGovaert P: Prenatal stroke. Semin Fetal Neonatal Med. 2009; 14(5): 250–266. PubMed Abstract | Publisher Full Text\n\nLindenberg R, Swanson PD: “Infantile Hydranencephaly”--a report of five cases of infarction of both cerebral hemispheres in infancy. Brain. 1967; 90(4): 839–850. PubMed Abstract | Publisher Full Text\n\nHoyme HE, Higginbottom MC, Jones KL: Vascular etiology of disruptive structural defects in monozygotic twins. Pediatrics. 1981; 67(2): 288–291. PubMed Abstract\n\nLarroche JC, Droulle P, Dalezoide AL, et al.: Brain damage in monozygous twins. Biol Neonate. 1990; 57(5): 261–78. PubMed Abstract | Publisher Full Text\n\nAlasdair GW Hunter: Brain. in Human malformations and related anomalies. 2nd Edition. Edited by Roger E Stevenson, Judith G Hall. New York: Oxford University Press, 2006; 639–645. Reference Source\n\nKeith CD, Hylton BM, David OC, et al.: Ultrasound in obstetrics and gynecology. 2nd Edition. Michigan: Churchill Livingstone; 2001; 326–327.\n\nDavid KJ, Philip JS, Carl PW, et al.: High Risk Pregnancy: Management Options. 3rd Edition. Pennsylvania: Elsevier; 2006; 386–387. Publisher Full Text\n\nGordon IRS, Ross FGM: Diagnostic radiology in pediatrics. Illustrated edition. Michigan: Butterworths; 1977; 305–306.\n\nBarkovich AJ, Kevin RM, Elena G, et al.: Diagnostic Imaging: Pediatric Neuroradiology. 1st edition: AMIRSYS; 2007; 1-1-152. Publisher Full Text\n\nWagner AL, Rohrer D: Imaging in hydranencephaly.2008; Accessed September 15, 2012. Reference Source\n\nJones J: Hydranencephaly. 2010; Accessed September 15, 2012.\n\nLin YS, Chang FM, Liu CH: Antenatal detection of hydranencephaly at 12 weeks, menstrual age. J Clin Ultrasound. 1992; 20(1): 62–4. PubMed Abstract | Publisher Full Text\n\nHadi HA, Mashini IS, Devoe LD, et al.: Ultrasonographic prenatal diagnosis of hydranencephaly. A case report. J Reprod Med. 1986; 31(4): 254–6. PubMed Abstract"
}
|
[
{
"id": "304",
"date": "12 Nov 2012",
"name": "Jeffrey Chinsky",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis case report presents an interesting case of apparent hydranencephalic changes, in addition to more extensive developmental brain anomalies (lack of thalamus and basal ganglia). However, including the points made below would make the manuscript much more useful as an educational resource:The manuscript would be enhanced if the author’s did more than just itemize a list of associated etiologies for hydranancephaly. The authors should provide a paragraph explaining how any of these etiologies could specifically produce the anomalies observed (cerebral arterial stroke or spasm of which specific vessels) and perhaps contrast it with any syndromic or associated genomic alterations listings of similar brain findings (or at least provide evidence that they researched this aspect).I’m not sure all would accept this as a variant of the same processes that cause classic hydranencephaly since it appears to be so much more extensive anomalous formation of the final fetal brain. The authors may have to do a little more research and discussion to support their contention based on how others who have written about hydranencphaly and define the condition.There are several grammatical errors (e.g. in the Abstract “such that absent cortex” is an incomplete phrase to end the sentence) which should be addressed, and I suggest the article is proof read again to ensure it reads how a published manuscript should do.",
"responses": [
{
"c_id": "366",
"date": "31 Jan 2013",
"name": "Buddhika Wijerathne",
"role": "Author Response",
"response": "We would like to thank the reviewer, Dr. Chinsky for the valuable time spent reviewing our manuscript and the important comments he has made. We have attempted to address and incorporate most of the concerns that were raised in version 2 of our article."
}
]
},
{
"id": "305",
"date": "14 Nov 2012",
"name": "Athanasios Petridis",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis case report is interesting. Except of some grammatical mistakes, I do have one suggestion.The authors should also discuss the social aspects and the social stress which accompanies families with hydranencephalic children. It is important to induce abortion when possible in such cases since the prognosis is very poor. Early diagnosis is the most important aspect in such cases. The paper of mine, Petridis et al. (2011) deals with the social aspects of hydranencephaly and should be cited by the author.",
"responses": [
{
"c_id": "365",
"date": "31 Jan 2013",
"name": "Buddhika Wijerathne",
"role": "Author Response",
"response": "We would like to thank Dr Petridis for the valuable time spent reviewing our manuscript and the important comments he has made. We have attempted to address and incorporate most of the concerns that were raised in version 2 of our article."
}
]
}
] | 1
|
https://f1000research.com/articles/1-22
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https://f1000research.com/articles/3-32/v1
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30 Jan 14
|
{
"type": "Case Report",
"title": "Papular granuloma annulare of palms and soles: case report of a rare presentation.",
"authors": [
"Sidharth Sonthalia",
"Rahul Arora",
"Rashmi Sarkar",
"Uday Khopkar",
"Rahul Arora",
"Rashmi Sarkar",
"Uday Khopkar"
],
"abstract": "We report the case of a 44-year-old Indian male patient who presented with mildly tender isolated papular lesions confined to the palms of the hands and soles of the feet. The histopathology was characteristic of granuloma annulare. There was an excellent response with 4-week treatment with a potent topical steroid ointment and no recurrence was reported at the follow-up one year later. This report is interesting because of the rare presentation of a common disease.",
"keywords": [
"Granuloma annulare (GA) is a relatively common benign inflammatory disorder characterized clinically by dermal papules and annular plaques. First described by T. Colcott Fox1",
"it occurs in all age groups and the precise etiology remains unknown. A number of clinical variants have been described in the literature but lesions localized to palms and soles are rare1",
"2. Correlation with histopathological findings",
"including hyperkeratosis",
"presence of foci of necrobiosis (fragmented collagen)",
"interstial lymphohistiocytic infiltrate in a palisading fashion",
"and increased mucin deposition",
"are important for diagnosis of such lesions. We describe a rare case of the papular variant of granuloma annulare with isolated involvement of the palms and soles."
],
"content": "Introduction\n\nGranuloma annulare (GA) is a relatively common benign inflammatory disorder characterized clinically by dermal papules and annular plaques. First described by T. Colcott Fox1, it occurs in all age groups and the precise etiology remains unknown. A number of clinical variants have been described in the literature but lesions localized to palms and soles are rare1,2. Correlation with histopathological findings, including hyperkeratosis, presence of foci of necrobiosis (fragmented collagen), interstial lymphohistiocytic infiltrate in a palisading fashion, and increased mucin deposition, are important for diagnosis of such lesions. We describe a rare case of the papular variant of granuloma annulare with isolated involvement of the palms and soles.\n\n\nCase report\n\nA 44-year-old Indian man presented with multiple, slightly painful, raised lesions over the palms and soles that had been present for 3 months. The patient reported having sustained an insect-bite a few weeks prior to the eruption. The patient was a farmer by occupation. His past medical history was insignificant with no history or symptoms suggestive of hypertension, diabetes mellitus, chronic obstructive pulmonary disease or any nutritional deficiencies. He was a non-smoker and reported no relevant family history.\n\nClinical findings: Cutaneous examination revealed multiple mildly tender dusky red-colored papules over both palms and the medial aspects of both soles (Figure 1a and b) with truncal sparing.\n\nMultiple tender dusky red-colored papules and plaques over (a) both palms, and (b) medial aspect of both soles.\n\nDiagnostic tests: Hematological and biochemical investigations including a complete hemogram, plasma glucose levels (fasting and post prandial), liver and renal function tests were within normal limits except for a raised Erythrocyte Sedimentation Rate. Serum angiotensin converting enzyme (ACE) levels and serum calcium and phosphate levels we re also normal. Serology for HIV and VDRL tests were negative. Chest radiograph was unremarkable and Mantoux test was negative. Histological examination was done from a 3 mm sized skin biopsy specimen taken from the palm and stained with hematoxylin and eosin (H & E). It revealed hyperkeratosis, acanthosis, superficial as well as deep perivascular mixed infiltrate of lymphocytes, histiocytes and neutrophils in the dermis, over a background of incomplete collagen degeneration, with interspersed mucin (Figure 2). Based on the clinical and histopathological features, a diagnosis of the papular variant of granuloma annulare localized to the palms and soles was made.\n\nHistopathology from the palmar lesion revealed hyperkeratosis, acanthosis, superficial and deep perivacular lymphohistiocytic infiltrate (highlighted by red horizontol arrow) with few neutrophils scattered in the reticular dermis, in a background of incomplete collagen degeneration and interspersed mucin (highlighted by two straight black arrows in the left lower aspect). (HE staining, 100×).\n\nThe patient was counselled regarding the benign nature of the disease and started on a twice daily application of a potent topical corticosteroid (clobetasol propionate 0.05%) ointment. This completely resolved the papules within 4 weeks (except for mild post-inflammatory desquamation) with no recurrence reported at the follow up visit one year later (Figure 3).\n\nTreatment response with 4 weeks of treatment with potent topical corticosteroid ointment – (a) palmar lesions before treatment, (b) palmar lesions after treatment with almost complete clearance of lesions and mild desquamation, (c) plantar lesions before treatment, and (d) cleared soles with only mild erythema and focal desquamation.\n\n\nDiscussion\n\nGA, a common benign skin condition of unknown etiology has many clinical variants including localized, generalized, papular, umblicated, subcutaneous, perforating, follicular, pustular, and actinic granuloma. The localized variety is the most common and presents with skin-colored to violaceous papules in an annular configuration, most commonly over the dorsal side of the feet, ankles, lower limbs and wrists. In the generalized variety (encountered in only 15% cases), lesions are more widespread, consisting of papules as well as plaques, and involve the trunk, neck, extremities, face, scalp, palms and soles3. Therefore, while palms and soles are frequently involved in the less common generalized variant, they are usually spared in the localized papular variant, which typically affects the dorsa of hands and feet. The subcutaneous variety of GA is characterized by firm asymptomatic nodules in deep subcutaneous tissues, usually over the anterotibial plateau, ankles, dorsa of the feet, buttocks and scalp4. The rare perforating variety presents with superficial umblicated papules with keratotic crusts, usually present over the dorsa of hands and fingers, trunk or extremities5. When GA involves the palms and soles, it may present with tender erythematous annular plaques (usually as a part of generalized variant), subcutaneous nodules or perforating lesions6. In our case, the patient had localized papular GA involving the palms and soles, a rare presentation, with only few cases reported in literature6–11. The largest case series was a clinicopathological description of seven cases with palmar lesions of GA by Gutte et al.6. They reported a palisading and interstitial pattern on histopathology, which is typical of GA lesions elsewhere on the body. Perineural granuloma, perieccrine granuloma and elastophagocytosis with mucin deposition were additional findings in some cases6.\n\nA number of disorders can clinically and histologically mimic GA, including interstitial granulomatous dermatitis with arthritis, palisading neutrophilic and granulomatous dermatitis, rheumatoid nodule and granulomas of Churg Strauss disease3. Erythema elevatum diutinum, acral Sweet’s syndrome, dermatofibroma and drug-induced granulomatous eruptions are other conditions which should be considered in a patient presenting with painful acral papules6,12.\n\nGA is considered to be an immune-mediated reaction, more specifically, a delayed type IV hypersensitivity response, but the exact etiology remains unknown. Various precipitating factors have been proposed, including trauma, insect bites, viral infections, sun exposure, and medication1. Our patient reported an insect bite that had occurred a few weeks prior to the eruption.\n\nTreatments that have been proposed for GA include topical or intralesional steroids, cryotherapy, electrocoagulation, laser destruction, phototherapy, topical imiquimod, and systemic agents such as antimalarial drugs, corticosteroids, isotretinoin, dapsone, cyclosporine niacinamide, vitamin E, pentoxyfylline and infliximab13. In our patient, treatment with a potent topical corticosteroid (clobetasol propionate 0.05%) ointment resulted in complete resolution in 4 weeks. The patient did not develop lesions elsewhere, nor did he report any recurrences at the one-year follow up. Hence, the patient did not require any systemic drug treatment for his GA to date.\n\n\nConsent\n\nWritten informed consent for publication of the clinical details, and clinical images, was obtained from the patient.",
"appendix": "Author contributions\n\n\n\nSS: Study concept and design, SS and RA: Analysis of data and drafting manuscript, RS and UK: Critical revision of the manuscript for important intellectual content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nFox TC: Granuloma Annulare. Proc R Soc Med. 1910; 3(Dermatol Sect): 104. PubMed Abstract | Free Full Text\n\nStewart LR, George S, Hamacher KL, et al.: Granuloma annulare of the palms. Dermatol Online J. 2011; 17(5): 7. PubMed Abstract\n\nDahl MV: Granuloma Annulare. In: Eisen AZ Wolff K Austen KF Goldsmith LA Katz SI editors. Fitzpatrick’s Dermatology in General Medicine. 6th Ed. USA: Mc Graw-Hill; 2003; 980–4. Publisher Full Text\n\nFelner EI, Steinberg JB, Weinberg AG: Subcutaneous granuloma annulare: a review of 47 cases. Pediatrics. 1997; 100(6): 965–7. PubMed Abstract | Publisher Full Text\n\nPenas PF, Jones-Caballero M, Fraga J, et al.: Perforating granuloma annulare. Int J Dermatol. 1997; 36(5): 340–8. PubMed Abstract | Publisher Full Text\n\nGutte R, Kothari D, Khopkar U: Granuloma annulare on the palms: A clinicopathological study of seven cases. Indian J Dermatol Venereol Leprol. 2012; 78(4): 468–74. PubMed Abstract | Publisher Full Text\n\nHsu S, Lehner AC, Chang JR: Granuloma annulare localized to the palms. J Am Acad Dermatol. 1999; 41(2 pt 2): 287–8. PubMed Abstract | Publisher Full Text\n\nImamura S, Ohnishi R, Kawasaki Y, et al.: Long-standing solitary granuloma annulare on the palm of a patient with dermatomyositis. J Dermatol. 2008; 35(5): 304–5. PubMed Abstract | Publisher Full Text\n\nSpencer B, Butler D: Painful palmar granuloma annulare responsive to dapsone. J Am Acad Dermatol. 2007; 56(2 suppl 2): AB48. Publisher Full Text\n\nHaushalter K, Hughey L, Foster W, et al.: Painful acral nodules as a manifestation of interstitial granuloma annulare. J Am Acad Dermatol. 2007; 56(2 suppl 2): AB57. Publisher Full Text\n\nBrey NV, Malone J, Callen J: Acute-onset, painful acral granuloma annulare: a report of 4 cases and a discussion of the clinical and histologic spectrum of the disease. Arch Dermatol. 2006; 142: 49–54. PubMed Abstract | Publisher Full Text\n\nBarzegar M, Davatchi CC, Akhyani M, et al.: An atypical presentation of erythema elevatum diutinum involving palms and soles. Int J Dermatol. 2009; 48(1): 73–5. PubMed Abstract | Publisher Full Text\n\nPeggy R: Diagnosis and management of granuloma annulare. Am Fam Physician. 2006; 74(10): 1729–34. PubMed Abstract"
}
|
[
{
"id": "3434",
"date": "30 Jan 2014",
"name": "Maxwell Fung",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have submitted an interesting, relatively novel, and clinically relevant case of acral papular granuloma annulare (GA).I cannot independently verify the diagnosis of GA based on the photomicrograph provided. An image obtained at higher magnification may be required to clearly demonstrate the granulomatous inflammation and necrobiosis.In their Discussion, the authors describe the clinical and histologic differential diagnosis for acral GA. It may be helpful to emphasize some relevant clinical aspects, including the association with pain (beyond tenderness) and arthalgia mimicking rheumatologic disease, and a possible correlation with systemic malignancy (Barksdale et al., 1994). Idiopathic palmoplantar hidradenitis might be reasonably considered in the clinical differential diagnosis; in this regard, since neutrophils were present in the reticular dermis in this case, it may be helpful to document that eccrine glands were uninvolved to eliminate this possibility from consideration.As for any case report describing treatment of a disorder whose natural history is to wax and wane, it is uncertain whether the relationship between treatment and clinical improvement is causation versus mere correlation.",
"responses": []
},
{
"id": "3485",
"date": "04 Feb 2014",
"name": "Mohamed Badawy Hassan Tawfik Abdel-Naser",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well written and interesting case report. There are few remarks. Abstract:The phrase “palms and soles” is better than “palms of the hands and soles of the feet”.Cutaneous findings: One of the violaceous palmar papules shows a central umblication! Is it a perforating lesion? Perhaps you can add a little comment on it.Diagnostic tests:It was a good to exclude STDs, namely syphilis by serology as tender violaceous papules of palms and soles are important clinical feature of its secondary stage. The histopathology photo is of insufficient quality and the described findings of GA are difficult to confirm.Treatment and follow up:As the authors mentioned, the patient is a farmer. Was it better to apply the potent steroid once at night and under occlusion for better compliance and efficacy?",
"responses": []
}
] | 1
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https://f1000research.com/articles/3-32
|
https://f1000research.com/articles/3-31/v1
|
29 Jan 14
|
{
"type": "Case Report",
"title": "Pancreatico-pericardial fistula as a complication of chronic pancreatitis",
"authors": [
"Camille Anne Sommer",
"C. Mel Wilcox",
"Camille Anne Sommer"
],
"abstract": "Pancreatico-pericardial fistula is an extremely rare complication of chronic pancreatitis. We present a case of a 58-year-old man who presented with syncope. Transthoracic echocardiogram revealed a pericardial effusion with tamponade physiology. Pericardiocentesis and pericardial fluid analysis demonstrated a lipase level of 2321 U/L. Subsequently, an endoscopic retrograde cholangiopancreatography (ERCP) was performed, confirming the presence of a pancreatico-pericardial fistula (PPF) from the distal body of the pancreas. A pancreatic duct stent was placed across the duct disruption on two separate occasions; however, despite stent placement, the patient continued to re-accumulate pericardial fluid and deteriorated. While rare, PPFs may complicate chronic pancreatitis, may not respond to pancreatic duct stenting and may portend a poor prognosis.",
"keywords": [
"Pancreatic fistulas are a well-recognized complication of pancreatitis. They result from leakage of pancreatic enzymes from a disrupted pancreatic duct associated with acute or chronic pancreatitis",
"partial pancreatectomy",
"or injury to the pancreatic duct during surgery. Other causes include pancreatic biopsy and blunt abdominal trauma. Fluid collections develop from persistent leakage of pancreatic enzymes with subsequent erosion through the wall of the bowel and into a nearby hollow viscus (colon",
"duodenum",
"stomach",
"or esophagus)",
"where they communicate with peritoneal or pleural cavities causing effusions in the pleural space",
"mediastinum",
"lesser sac",
"retroperitoneum",
"or perihepatic space. We report a rare case of pancreatico-pericardial fistula (PPF) presenting as syncope in a patient with chronic alcoholic pancreatitis."
],
"content": "Introduction\n\nPancreatic fistulas are a well-recognized complication of pancreatitis. They result from leakage of pancreatic enzymes from a disrupted pancreatic duct associated with acute or chronic pancreatitis, partial pancreatectomy, or injury to the pancreatic duct during surgery. Other causes include pancreatic biopsy and blunt abdominal trauma. Fluid collections develop from persistent leakage of pancreatic enzymes with subsequent erosion through the wall of the bowel and into a nearby hollow viscus (colon, duodenum, stomach, or esophagus), where they communicate with peritoneal or pleural cavities causing effusions in the pleural space, mediastinum, lesser sac, retroperitoneum, or perihepatic space. We report a rare case of pancreatico-pericardial fistula (PPF) presenting as syncope in a patient with chronic alcoholic pancreatitis.\n\n\nCase report\n\nA 58 year-old black male presented with syncope related to orthostatic hypotension. He had sustained a laceration and multiple facial fractures involving the zygomatic arch and right orbit. He reported feeling weak with dyspnea on minimal exertion for several days prior to this syncopal event. He denied any abdominal pain, nausea, vomiting or chest pain. He had a history of delirium tremens post alcohol cessation and previous episodes of acute pancreatitis over the last two years. Prior surgical history was notable for an exploratory laparotomy in 1985 after sustaining a gunshot wound to the abdomen. He was on no medications on admission. He had an ongoing history of alcohol dependence (3 glasses of whisky daily) and was drinking earlier on the day of his presentation (ethanol level 178 mg/dl). He denied any family history of pancreatic disease.\n\nOn admission, his vital signs were normal without tachycardia or hypotension. Facial trauma was evident. On auscultation he had rales at both lung bases. There were no abnormalities noted on his cardiovascular exam. He had moderate ascites with an obvious fluid wave but no hepatosplenomegaly. Pertinent laboratory findings included: hemoglobin 7.7 g/dL (normal range 14–18 g/dl) and hematocrit 23.5% (normal range 40–54%). The remainder of his blood count was normal. His total bilirubin was 1.3 mg/dl (normal range 0.3–1.2 mg/dl), aspartate aminotransferase (AST) 130 U/L (normal range 15–46 U/L) and alanine aminotransferase (ALT) was 28 U/L (normal range 11–66 U/L) with albumin of 2.2 g/dl (normal range 3.5–5 g/dl). Alkaline phosphatase (ALP) was within normal limits. Electrolytes on admission were also normal. Prothrombin time (PT) and INR were 22.9 seconds (normal <12.5s) and 2.08 (normal <1.11), respectively. His serum amylase concentration the day after admission was 386 U/L (normal range 30–110 U/L). He was admitted to the Medicine Intensive Care Unit for treatment of syncope, pancreatitis, and anemia.\n\nComputed tomography (CT) of his head revealed multiple fractures involving the right posterior lateral wall of the right maxillary sinus, zygomatic arch and lateral wall of the right orbit, without intracranial hemorrhage. The admission chest X-ray demonstrated cardiomegaly without evidence of pneumonia or pulmonary edema. Overnight, the patient became confused and tachypneic and his oxygen saturation fell to 85%. A transthoracic echocardiogram was subsequently performed on the day after admission to evaluate for dilated cardiomyopathy or pericardial effusion. A large pericardial effusion was seen with systolic right atrial collapse, diastolic right ventricular collapse and left atrial collapse. Pericardiocentesis was performed with removal of 4.2 liters of straw-colored fluid and resolution of the tamponade. Fluid analysis showed: red blood cell count 573/mm3, white blood cell count 13/mm3, amylase 633 μ/L, and lipase 2321 μ/L. Despite pericardiocentesis, a repeat chest X-ray on the day after admission showed worsening cardiomegaly and a repeat echocardiogram showed re-accumulation of the fluid and an additional 1800 cc was drained over the next two days; a pericardial drain was subsequently placed.\n\nAn abdominal ultrasound three days after admission showed mild ascites and a cirrhotic liver without portal vein thrombosis. The patient was also found to have hepatitis C by blood testing. A CT of the chest, abdomen and pelvis revealed a persistent large low-attenuating pericardial effusion with the pericardial drain entering the left paramedian subcostal region with the tip terminating at the level of the ascending aorta (Figure 1); large bilateral, left greater than right, pleural effusions were also present. Changes of chronic pancreatitis were present with irregular and dilated ducts with numerous calcifications (Figure 2). There was no evidence radiographically of acute pancreatitis. The pancreatic parenchyma was homogenously enhancing. There was a moderate amount of low-attenuating free intraperitoneal fluid but no discrete peripancreatic fluid collection or stranding.\n\nLarge pericardial and pleural effusions. The tip of the drain is shown in the pericardial space.\n\nCalcifications within the pancreas are shown by arrows.\n\nThe patient continued to have a re-accumulating pericardial effusion with over 12 L removed over the ensuing several days. The cardiothoracic surgery service was consulted regarding a pericardial window, but the patient was felt to be a poor surgical candidate and too high-risk for the procedure given his co-morbidities. A technetium-labeled macro-aggregated albumin shunt study was performed five days after admission through the pericardial drain to evaluate for communication and there was no scintigraphic evidence for radiotracer communication from the pericardium into the abdomen or pelvis.\n\nDespite a pericardioplasty, the patient continued to have persistent pericardial drainage. Endoscopic retrograde cholangiopancreatogram (ERCP) was performed for suspected PPF. A scout image showed numerous calcifications throughout the pancreas that were consistent with chronic pancreatitis. After biliary sphincterotomy, the pancreatic duct was selectively cannulated and revealed numerous small stones and a diffusely dilated pancreatic duct to the tail. A leak was observed in the distal pancreatic body just proximal to the pancreatic neck (Figure 3). The fistula was seen coursing cranially surrounding a circular structure, consistent with the pericardium (Figure 4). As the ERCP contrast agent was continually injected under fluoroscopy, cardiac contractions were observed. A 7-French × 15 cm pancreatic duct stent was placed to the tail, bridging the leak.\n\nPancreatic fistula is shown by the arrows.\n\nNevertheless, the patient continued to have 3 L of output from the pericardial drain daily. A repeat CT did not show any focal peripancreatic fluid collection or tract and no visible collection or tract was seen between the pancreas and diaphragm for percutaneous drain placement to be possible. A second ERCP was subsequently performed with exchange of the stent. The fistulous tract was again noted from the neck, tracking cranially, and a 7 Fr pancreatic stent was again placed bridging the leak.\n\nThe patient continued to decline despite maximal medical therapy. While hospitalized, over 50 L of fluid was removed from the pericardial drain. He did not respond to pericardial drainage nor to treatment of pansensitive Escherichia coli that was cultured from the pericardial fluid. He was also treated with antibiotics for concurrent pneumonia and a urinary tract infection. Eventually, the patient opted for a change in his goals of care to comfort care and was transferred to hospice and expired.\n\n\nDiscussion\n\nDisruption of the pancreatic duct leads to leakage of pancreatic juice and formation of an acute fluid collection or pseudocyst. Communication to other structures can occur either indirectly, originating from a pseudocyst, or directly in the form of fistulas. Internal pancreatic fistulas result from erosion of pancreatic fluid into adjacent or distant organs whereas external fistulas drain into a defect in the skin. If the communication occurs anteriorly into the peritoneal cavity, pancreatic ascites may occur.\n\nIn most reported cases of internal pancreatic fistulas, thoracopancreatic communication occurs into the pleural space causing a pleural effusion and patients usually present with dyspnea1. Although there have been reports of mediastinal extension of pseudocysts2–5, to our knowledge only four cases of PPFs have been reported in the medical literature6–9.\n\nThe presenting symptoms can be variable depending on the location and size of the communication. Thus, patients may present with dyspnea, chest pain, palpitations, or dysphagia; sometimes with hemoptysis, acute respiratory compromise, or cardiogenic shock. Our patient presented with syncope and reported no chest or abdominal symptoms. It was only when his pericardial effusion was analysed and found to have elevated amylase and lipase concentrations that a pancreatic origin was suspected.\n\nA number of techniques may be used to establish the diagnosis of pancreatic fistula. Cross-sectional abdominal imaging by computed tomography may often be suggestive, especially if a pancreatic pseudocyst or peripancreatic fluid is observed. More recently, magnetic resonance cholangiopancreatography has been used to delineate the pancreatic duct and even to identify the fistulous tract. A variety of radiologic imaging studies were performed on our patient, including abdominal ultrasound, transthoracic echocardiogram, CT, and a technetium-labeled macroaggregated albumin shunt study, none of which were able to detect or confirm the presence of the pancreatico-pericardial fistula. ERCP is the diagnostic test of choice and offers the potential for therapy for fistulas as stenting the disrupted duct is a viable and effective therapeutic option10–15.\n\nThe treatment of pancreatic fistulas is based primarily on case reports and small observational studies16. Optimizing nutritional status and limiting oral intake may be effective with small leaks. Octreotide, a potent inhibitor of pancreatic secretion, has also been used in some small studies with efficacy16. The therapy most commonly recommended, however, is endoscopic stenting by ERCP17,18. One randomized study in patients with pancreatic duct disruption associated with necrotizing pancreatitis, however, found no significant difference between conservative and endoscopic treatment17. In the study by Kozarek et al.19, of 18 patients with pancreatic duct leaks (14 of which had associated fluid collections) who were treated with transpapillary pancreatic duct drains or stents, 7 patients required surgery for ongoing pancreatic pain or residual/recurrent fluid collections. The complications were the result of exacerbated symptoms of pancreatitis and stent occlusion leading to recurrent pancreatitis or recurrent duct blowout with pseudocyst. Covered self-expanding metal stents have been successfully used in the main pancreatic duct to manage duct disruptions and leaks20. A retrospective study of those with pancreaticopleural fistula who underwent ERCP and stenting as the primary treatment found successful treatment in only one of eight cases, mostly due to abnormal or difficult anatomy21. Location of duct disruption has been the only suggested predictor of who would best benefit from stenting for pancreatic duct leak1. Despite identifying the ductal leak and stenting twice, the leak in our patient did not resolve. If the leak persists after 2–4 weeks of conservative and endoscopic therapy, surgery may be required depending on the site of the leak. In one patient, surgical detachment of the fistula and Roux-en-Y decompression of the pancreatic duct resulted in a cure of the condition6.\n\nIn the medical literature, there is a report that described the case of a 16 year-old male who was found to have tropical calcific pancreatitis and underwent a lateral pancreatico-jejunostomy with Roux-en-Y loop performed after initial pericardiocentesis and was reported to be doing well six months later7. Another report described an adult patient who underwent an elective Roux-en-Y pancreatico-jejunostomy without complication and remained symptom-free two years after surgery8. Unfortunately, our patient had untreated cirrhosis due to hepatitis C and alcohol misuse, and had continued to consume alcohol up until the day of admission, and was deemed a poor surgical candidate. Another more recent case describes a PPF diagnosed by ERCP in a patient with acute alcoholic pancreatitis with successful stent placement in a disrupted pancreatic duct. Prior to diagnosis, the patient had been treated with intrapericardial triamcinolone and octreotide, but the pericardial effusion reccurred9.\n\nPancreatico-pericardial fistula is a very rare complication of pancreatitis and rapid and accurate diagnosis of this life-threatening complication is crucial. As our case demonstrates, endoscopic therapy may not be effective.\n\n\nConsent\n\nAs the patient died and it was not possible to locate the patient’s family, the authors obtained written permission for publication of clinical details and clinical images from Prof. Klaus Monkemuller, Chief of Endoscopy at the University of Alabama at Birmingham Hospital, where the patient was managed. Prof. Monkemuller has confirmed the absence of patient identifiers ensuring protection of any patient related information.",
"appendix": "Author contributions\n\n\n\nCamille Anne Sommer, M.D., and C. Mel Wilcox, M.D. M.S.P.H., wrote the manuscript, reviewed the figures and finalized the draft.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nIacono C, Procacci C, Frigo F, et al.: Thoracic complications of pancreatitis. Pancreas. 1989; 4(2): 228–236. PubMed Abstract | Publisher Full Text\n\nAjmera AV, Judge TA: Mediastinal extension of pancreatic pseudocyst--a case with review of topic and management guidelines. Am J Ther. 2012; 19(5): e152–e156. PubMed Abstract | Publisher Full Text\n\nImai Y, Taniguchi M, Tagawa K, et al.: A case of chronic pancreatitis complicated by massive pericardial and right pleural effusion. Gastroenterol Jpn. 1993; 28(5): 734–738. PubMed Abstract | Publisher Full Text\n\nTan MH, Kirk G, Archibold P, et al.: Cardiac compromise due to a pancreatic mediastinal pseudocyst. Eur J Gastroenterol Hepatol. 2002; 14(11): 1279–82. PubMed Abstract | Publisher Full Text\n\nKomtong S, Chanatrirattanapan R, Kongkam P, et al.: Mediastinal pseudocyst with pericardial effusion and dysphagia treated by endoscopic drainage. JOP. 2006; 7(4): 405–10. PubMed Abstract | Publisher Full Text\n\nDavidson ED, Horney JT, Salter PP 3rd: Internal pancreatic fistula to the pericardium and pleura. Surgery. 1979; 85(4): 478–80. PubMed Abstract\n\nBalasubramanian P, Jeyamani R, Govil S, et al.: Pancreatico-pericardial fistula: a rare complication of chronic pancreatitis. Indian J Gastroenterol. 2004; 23(1): 31–32. PubMed Abstract\n\nChing SS, Rao MM, Ali A, et al.: Chronic pancreatitis complicated by pancreaticopericardial fistula. Surgical Practice. 2007; 11(3): 130–133. Publisher Full Text\n\nLamparter S, Sundermann H: Swinging heart in acute pancreatitis. Am J Med Sci. 2013; 346(2): 160–161. PubMed Abstract | Publisher Full Text\n\nSchoonjans R, Vandewoude K, Cesmeli E, et al.: Recurrent pericardial effusion as a result of chronic pancreatitis. Successful treatment with somatostatin. J Clin Gastroenterol. 1996; 23(3): 224–7. PubMed Abstract\n\nChebli JM, Gaburri PD, de Souza AF, et al.: Internal pancreatic fistulas: proposal of a management algorithm based on a case series analysis. J Clin Gastroenterol. 2004; 38(9): 795–800. PubMed Abstract | Publisher Full Text\n\nSaeed ZA, Ramirez FC, Hepps KS: Endoscopic stent placement for internal and external pancreatic fistulas. Gastroenterology. 1993; 105(4): 1213–7. PubMed Abstract\n\nGarcia-Ricart F, Croizet O, El Riwini M, et al.: Endoscopic management of a persistent pancreatopleural fistula. Gastrointest Endosc. 1997; 46(4): 359–61. PubMed Abstract | Publisher Full Text\n\nPai CG, Suvarna D, Bhat G: Endoscopic treatment as first-line therapy for pancreatic ascites and pleural effusion. J Gastroenterol Hepatol. 2009; 24(7): 1198–202. PubMed Abstract | Publisher Full Text\n\nTanaka T, Kuroki T, Kitasato A, et al.: Endoscopic transpapillary pancreatic stenting for internal pancreatic fistula with the disruption of the pancreatic ductal system. Pancreatology. 2013; 13(6): 621–4. PubMed Abstract | Publisher Full Text\n\nBlatnik JA, Hardacre JM: Management of pancreatic fistulas. Surg Clin North Am. 2013; 93(3): 611–7. PubMed Abstract | Publisher Full Text\n\nBakker OJ, van Baal MC, van Santvoort HC, et al.: Endoscopic transpapillary stenting or conservative treatment for pancreatic fistulas in necrotizing pancreatitis: multicenter series and literature review. Ann Surg. 2011; 253(5): 961–7. PubMed Abstract | Publisher Full Text\n\nde-Madaria E, Abad-González A, Aparicio JR, et al.: The Spanish Pancreatic Club’s recommendations for the diagnosis and treatment of chronic pancreatitis: Part 2 (treatment). Pancreatology. 2013; 13(1): 18–28. PubMed Abstract | Publisher Full Text\n\nKozarek RA, Ball TJ, Patterson DJ, et al.: Endoscopic transpapillary therapy for disrupted pancreatic duct and peripancreatic fluid collections. Gastroenterology. 1991; 100(5 Pt 1): 1362–70. PubMed Abstract\n\nAkbar A, Baron TH: Covered self-expanding metal stent use in the pancreatic duct: a case series. Endoscopy. 2012; 44(9): 869–73. PubMed Abstract | Publisher Full Text\n\nAli T, Srinivasan N, Le V, et al.: Pancreaticopleural fistula. Pancreas. 2009; 38(1): e26–e31. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "3429",
"date": "03 Feb 2014",
"name": "Kevin McGrath",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis case report serves to remind us of a rare but potentially devastating complication of chronic pancreatitis, and one that unfortunately does not respond well to endoscopic management. Given the comorbities, this patient was not appropriate for surgical management. The authors also discuss the role of octreotide in the management of pancreatic fistulae. It would be interesting to know whether octreotide was used in conjunction with pancreatic duct stenting in this case. Given the drainage volume, I suspect it would have been of little benefit if used.",
"responses": []
},
{
"id": "3463",
"date": "03 Feb 2014",
"name": "Matthew DiMagno",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well written case report about a patient presenting with syncope due to a pancreatic-pericardial fistula, a rare complication of chronic pancreatitis that has been reported for only a handful of patients in the literature. Multiple noninvasive diagnostic tests failed to detect the fistula. ERCP proved diagnostic, showing a leak of contrast from the distal pancreatic body coursing to and surrounding a circular structure in the chest. There are no controlled data or guidelines for management of pancreatic-pleural fistula but general treatment options include watchful waiting for spontaneous resolution, medical therapy (octreotide, nasojejunal feeding, total parenteral nutrition), or drainage internally or externally by endoscopic, percutaneous, or surgical approaches. The patient was too symptomatic from the pericardial effusion to offer observation alone and he was not fit for surgery due to his comorbid illnesses. Endoscopic stent placement is a very reasonable option and potentially a first-line therapy based on the excellent outcomes reported by case series for management of pancreatic-pleural fistula and in the single of 4 cases of pancreatic-pericardial fistula offered this treatment modality. It should be noted that those having a fistula between the pericardium and a (peri)pancreatic pseudocyst, which was absent in this case, may be offered endoscopic cystenterostomy. Unfortunately, this patient failed to respond to endoscopic stenting across the body of the pancreatic duct, based on having an unremitting, large pericardial drain output of approximately 3 Liters per day of pancreatic juice. Why this particular patient did not respond is unclear. It should be emphasized that the patient underwent stent change, essentially excluding a clogged or malpositioned stent as a cause for failed therapy. Moreover, we speculate that fistula pathophysiology and perhaps the response to transpapillary or bridging pancreatic duct stenting differs between pancreatic-pleural and pancreatic-pericardial types of fistula and that the extreme, high output fistula in this particular patient would doom any treatment modality that did not include surgery.",
"responses": []
}
] | 1
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https://f1000research.com/articles/3-31
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https://f1000research.com/articles/3-30/v1
|
29 Jan 14
|
{
"type": "Research Article",
"title": "Post-fire nutrient availability in the sub-tropical forest ecosystem of the Koubru Hills, Manipur",
"authors": [
"Alice Sitlhou",
"Thingbaijam Binoy Singh",
"Thingbaijam Binoy Singh"
],
"abstract": "Forest fires are known to significantly alter soil nutrient availability. The study investigated the post-fire nutrient availability in the sub-tropical forest ecosystem of Koubru Hills, Manipur (North-East India). The forested ecosystem was disturbed by forest wildfire in January 2010.To study the fire effect, collection of burned and unburned soil samples from three soil depths (0-5, 5-10 and 10-15 cm) was started approximately three months after the fire event and continued at monthly intervals for a period of one year. The collected soil samples were analyzed for soil organic carbon (SOC), NH4+-N, Bray-extractable phosphorus, available potassium and micronutrients such as Fe, Cu, Mn and Zn. Results of the ANOVA test showed significant reduction in the SOC, NH4+-N and available K contents of the surface (0-5 cm) and sub-surface (5–10 and 10-15 cm) soil layers of the burned area. However, significant increases in the Bray-extractable phosphorus were observed in all the three soil depths studied. With respect to the effect of forest fire on soil micronutrient availability, results are contradictory. The available Fe depleted in all the three soil depths, whilst the available Cu declined in the 0-5 cm but increased in the 10-15 cm soil layer. The available Cu content of the 5-10 cm soil layer did not vary between the burned and unburned areas. While no significant effect of fire was observed on the available Mn contents of the 0-5 and 10-15 cm soil layers, there was a significant post-fire decrease in the Mn availability of the 5-10 cm soil layer of the burned area compared with the unburned area. Forest fire also resulted in a significant loss of the available Zn from the 0-5 and 5-10 cm soil layers of the burned area but in the 10-15 cm soil layer, no significant variation was observed between the burned and unburned areas.",
"keywords": [
"nutrient availability",
"soil",
"sub-tropical",
"ecosystem",
"fire"
],
"content": "Introduction\n\nForest fires form part of ecosystem disturbances and those provoked by human activities are a paramount environmental problem and a serious threat to biodiversity1. Pickett and White2 defined ecosystem disturbances as “any relatively discrete event(s) in time and space that disrupts the structure of populations, communities, and ecosystems and causes(s) change in resource availability or the physical environment”. Fire as natural or anthropogenic disturbance can cause short- or long-term changes in ecosystem dynamics3,4.\n\nThe effect of forest fire on forest soil is very complicated and less studied in comparison to its aboveground effect5. Fire effects on soil can vary greatly among different ecosystems depending on the severity (a combination of fire line intensity and duration) according to Keeley6 and frequency of fires. While low severity-fires do not substantially affect soils, high severity-fires can affect a wide range of soil properties, one of which is nutrient availability7. The concentrations, forms, and depth distribution of soil nutrients can be dramatically altered after fire disturbance8. The productivity of burned soils can be impacted, in some cases, in an irreversible way resulting from fire-induced changes on soil properties9. Post-fire nutrient losses can be accelerated through volatilization, leaching and erosion7.\n\nMost studies on fire-associated alterations in soil nutrients have been focused on evaluating changes in available macro-nutrients10–12. Very few studies have evaluated post-fire effects on soil micronutrients. This paper presents information regarding the post-fire availability of nutrients which include both macro- and micro-nutrients of the burned and unburned areas in the sub-tropical forest ecosystem of Koubru Hills, Manipur (NE India). The role of fire in this region is poorly understood, therefore, the aim of this study was to investigate changes in some of the soil nutrients following forest wildfire using conventional soil analytical tools. Results from the present study would provide insight into potential fire effects on soil nutrients at this site and also give some direction for sorely needed future research.\n\n\nMaterials and methods\n\nThis research was conducted between March 2010 and April 2011 in the sub-tropical forest ecosystem of the Koubru Hills, Manipur (NE India). The site under study lies at 24°55′N latitude and 93°48′E longitude. Average elevation of the site is approximately 1294 m above mean sea level. Mean annual precipitation is about 3.99 mm with the wet season between June and August. Average annual relative humidity is 75.99%. Average annual air temperature is 21.14°C. The data are based on long term records from the Indian Council of Agricultural Research (ICAR) located nearby the study site. The soil is sandy loam in texture. Vegetation at the site is dominated by Cinnamomum zeylanicum and some unidentified species belonging to the genus Litsaea.\n\nIn mid January 2010, a forest wildfire occurred in the study site which had been undisturbed by fire for more than 100 years. The fire removed almost all the understory vegetation and soil surface litter.\n\nSoil sampling at monthly intervals started in April 2010 and ended in March 2011. Sampling was carried out approximately three months after the occurrence of the forest wildfire. Two representative plots (with an area of 6000 m2 per plot), one each in the burned and the adjacent unburned areas, were taken. Each plot was equally divided into fifteen sub-plots (with an area of 20×20 m2 per sub-plot). From amongst the fifteen sub-plots, ten sub-plots were randomly selected following the random number table method of Fisher and Yates13. Soils were sampled from three depths (0–5, 5–10 and 10–15 cm) in each of the randomly selected ten sub-plots of both burned and adjacent unburned areas using 5 cm × 5 cm soil cores. Soils collected from the similar depths in each of the ten sub-plots of the burned area were pooled into one composite sample (for instance, soils collected from the 0–5 cm soil depth of the burned area were pooled into one composite sample representing the 0–5 cm soil depth of the burned area). The same was repeated for soils sampled from three depths in each of the ten sub-plots of the unburned area. Thus, each composite soil sample representing a specific depth of either the burned or unburned area was composed of ten pooled sub-samples collected from the randomly selected ten sub-plots. The collected soil samples were air-dried, crushed and passed through a 2 mm mesh soil sieve (Shiva Scientific Instruments, India) for nutrient analysis.\n\nSoil organic carbon (SOC) contents were analyzed using H2SO4–K2Cr2O7 oxidation method14 with five replications. Ammonical nitrogen was determined by the alkaline permanganate method using an Alpkem autoanalyser (Kjeltec system 1026 Distilling unit, Sweden) with three replications. Bray-extractable phosphorus (2.85 g soil in 0.03 N NH4F plus 0.025 N HCl) was analyzed using spectrophotometer 169 (Systronics, India) with three replications. Available potassium was extracted by 0.1 M ammonium acetate solution adjusted to pH 7 at a 1:5 soil solution ratio and determined by flame photometer 128 (Systronics, India) with three replications. The plant-available fractions of micronutrients (Fe, Cu, Mn and Zn) were determined by using 0.05 M DTPA (Diethylene Triamine Pentaacetic Acid) plus 0.01 M CaCl2 plus 0.1 M Triethanolamine, adjusted to pH 7.30 at a 1:2 soil solution ratio15. The concentrations of the elements were assessed using atomic absorption spectrophotometry (Aanalyst 200, PerkinElmer, USA).\n\nA two way ANOVA test was performed to test the effects of forest fire (burned and unburned) and sampling month (time) on the soil nutrients studied at three different soil depths (0–5, 5–10 and 10–15 cm). A one way ANOVA test was performed to detect the differences in soil nutrient concentrations between the burned and unburned areas at a specific sampling month. P≤0.05 was considered to be significant. The data analysis was carried out using Microsoft Excel 2007.\n\n\nResults\n\nThe SOC contents of the surface layer (0–5 cm) ranged from 2.10 to 2.18% in the unburned area while in the burned area they ranged from 1.94 to 2.02% (Table 1). In the sub-surface layers (5–10 and 10–15 cm) the SOC contents of the unburned area were observed to vary from 1.97 to 2.04% and from 1.76 to 1.89%, respectively (Table 1). In contrast, the SOC contents of the sub-surface layers in the burned area ranged from 1.62 to 1.74% and from 1.49 to 1.58%, respectively (Table 1).\n\nA two way ANOVA test showed that fire reduced the SOC of the surface layer (0–5 cm) varying significantly (P=0.00005) between the burned and unburned areas (Unburned > Burned, Figure 1A, Table 2) while it did not vary significantly over the sampling months (P=0.480). The SOC of the 5–10 cm soil layer was also significantly affected (P<0.001) by fire (Unburned > Burned, Figure 1B, Table 2) while there were no significant variation (P=0.090) among the sampling months. The SOC also varied significantly (P<0.001) between the burned and unburned areas in the 10–15 cm soil layer (Unburned > Burned, Figure 1C, Table 2) while it did not vary significantly (P=0.324) among the sampling months.\n\nSoil organic carbon % at 0–5 (A), 5–10 (B) and 10–15 cm (C) soil layers. Significant differences (P<0.001) between unburned and burned areas on specific sampling months are indicated by different letters above histogram bars. Data show mean ± SE (n=5).\n\nFire effect (F) and sampling month effect (E); *, ** and *** statistically significant at P<0.05, 0.01, 0.001 respectively.\n\nSoil NH4+–N contents in the surface layer (0–5 cm) ranged from 543.47 to 592.10 kg/ha in the unburned area while in the burned area, they ranged from 349.12 to 389.57 kg/ha (Table 1). In the sub-surface layers (5–10 and 10–15 cm) of the unburned area, soil NH4+–N contents ranged from 475.86 to 519.99 kg/ha and from 396.27 to 443.47 kg/ha, respectively while in the burned area, they ranged from 294.15 to 325.38 kg/ha and from 265.25 to 292.25 kg/ha, respectively (Table 1).\n\nA two way ANOVA test indicated that fire caused a significant reduction (P<0.001) of soil NH4+–N in the surface (0–5 cm) layer (Unburned > Burned, Figure 2A, Table 2) while sampling date did not significantly (P=0.141) affect the soil NH4+–N. NH4+–N of the 5–10 cm soil layer was also significantly lower (P<0.001) in the burned area compared to the adjacent unburned area (Unburned > Burned, Figure 2B, Table 2) while it showed no significant variation (P=0.073) over the sampling months. Fire also affected the NH4+–N content of the 10–15 cm soil layer (Unburned > Burned, Figure 2C, Table 2) varying significantly (P<0.001) between the burned and unburned areas while no significant effect (P=0.322) of sampling month was observed.\n\nNH4+–N (kg/ha) at 0–5 (A), 5–10 (B) and 10–15 cm (C) soil layers. Significant differences (P<0.001) between unburned and burned areas on specific sampling months are indicated by different letters above histogram bars. Data show mean ± SE (n=3).\n\nThe available P of the 0–5, 5–10 and 10–15 cm soil layers in the unburned area varied from 21.36 to 25.73 kg/ha, 18.77 to 21.42 kg/ha and from 19.10 to 21.82 kg/ha, respectively (Table 1). In contrast, the available P of the burned area in the three different soil layers ranged from 24.98 to 26.69 kg/ha, 23.72 to 26.24 kg/ha and from 23.99 to 26.30 kg/ha, respectively (Table 1).\n\nA two way ANOVA test showed that fire resulted in a significant rise (P=0.022) of the available P in the upper 5 cm of the burned area (Unburned < Burned, Figure 3A, Table 2) while it did not vary significantly (P=0.131) over the sampling months. In the 5–10 cm soil layer, fire also significantly increased (P<0.001) the available P, the concentration being higher in the burned area than the adjacent unburned area (Unburned < Burned, Figure 3B, Table 2) while sampling month had no significant effect (P=0.231) on the available P. The available P of the 10–15 cm soil layer also showed significant variation (P<0.001) between the burned and unburned areas (Unburned < Burned, Figure 3C, Table 2) while no significant variation (P=0.850) was observed over the sampling months.\n\nAvailable phosphorus (kg/ha) at 0–5 (A), 5–10 (B) and 10–15 cm (C) soil layers. Significant differences (P<0.05) between unburned and burned areas on specific sampling months are indicated by different letters above histogram bars. Data show mean ± SE (n=3).\n\nThe available potassium contents in the surface soil layer (0–5 cm) of the unburned and burned areas ranged from 697.07 to 852.76 kg/ha and from 484.62 to 623.62 kg/ha, respectively (Table 1). In the sub-surface soil layers (5–10 and 10–15 cm) of the unburned area the available potassium contents respectively ranged from 482.84 to 579.19 kg/ha and from 369.77 to 457.27 kg/ha while they ranged from 306.07 to 368.40 kg/ha in the 5–10 and from 241.78 to 288.29 kg/ha in the 10–15 cm sub-surface soil layers of the burned area (Table 1).\n\nA two way ANOVA test showed a significant fire induced loss (P<0.001) of the available potassium from the 0–5 cm soil layer of the burned area (Unburned > Burned, Figure 4A, Table 2) and there was also significant variation among the sampling months (P<0.0006). Fire also caused a significant reduction (P<0.001) of the available potassium in the 5–10 cm soil layer (Unburned > Burned, Figure 4B, Table 2) and it varied significantly among the sampling months (P=0.010). A significant difference (P<0.001) in the potassium availability between the burned and unburned areas was also observed in the 10–15 cm soil layer (Unburned > Burned, Figure 4C, Table 2) while there was no significant variation (P=0.096) among the sampling months.\n\nAvailable potassium (kg/ha) at 0–5 (A), 5–10 (B) and 10–15 cm (C) soil layers. Significant differences (P≤0.05) between unburned and burned areas on specific sampling months are indicated by different letters above histogram bars. Data show mean ± SE (n=3).\n\nThe DTPA-extractable Fe of the unburned area ranged from 87.22 to 98.37 mg/kg in the 0–5, 77.44 to 83.36 mg/kg in the 5–10 and 68.87 to 78.41 mg/kg in the 10–15 cm soil layers while in the burned area, it ranged from 73.54 to 84.42 mg/kg, 64.52 to 72.52 mg/kg and from 58.88 to 67.93 mg/kg in the 0–5, 5–10 and 10–15 cm soil layers, respectively (Table 1). Thus, the unburned area had a higher Fe availability compared to the burned area in all the three soil layers.\n\nA two way ANOVA test indicated a significant effect (P=0.001) of fire on the DTPA-extractable Fe which showed a decrease in the surface 0–5 cm soil layer of the burned area (Unburned > Burned, Figure 5A, Table 2) while no significant effect (P=0.393) of sampling month was observed. In the subsurface layer (5–10 cm), the DTPA-extractable Fe showed a significant variation (P<0.001) between the burned and unburned areas (Unburned > Burned, Figure 5B, Table 2) while no significant difference (P=0.490) was found among the sampling months. The 10–15 cm soil layer also showed a significant (P=0.001) fire-induced loss of the DTPA-extractable Fe which was found to be lower in the burned area than the adjacent unburned area (Unburned > Burned, Figure 5C, Table 2) while there was no significant difference (P=0.121) among the sampling months as confirmed by the two way ANOVA test.\n\nAvailable iron (mg/kg) at 0–5 (A), 5–10 (B) and 10–15 cm (C) soil layers. Significant differences (P<0.05) between unburned and burned areas on specific sampling months are indicated by different letters above histogram bars. Data show mean ± SE (n=4).\n\nThe DTPA-extractable Cu contents of the 0–5 cm soil layer ranged from 0.51 to 0.79 mg/kg in the unburned area while in the burned area, they ranged from 0.20 to 0.40 mg/kg (Table 1). The DTPA-extractable Cu contents of the sub-surface layers (5–10 and 10–15 cm) respectively ranged from 0.08 to 0.39 mg/kg and from 0.06 to 0.16 mg/kg in the unburned area while in the burned area they ranged from 0.12 to 0.3 mg/kg and from 0.23 to 0.39 mg/kg, respectively (Table 1).\n\nAs confirmed by the two way ANOVA test, fire caused a significant decline (P<0.001) of the DTPA-extractable Cu contents in the 0–5 cm soil layer (Unburned > Burned, Figure 6A, Table 2) but there was a significant post-fire increase (P=0.001) in the 10–15 cm soil layer. The 5–10 cm soil layer showed no significant variation (P=0.820) between the burned and unburned areas with respect to the post-fire Cu availability. No significant variation was observed among the sampling months in the DTPA-extractable Cu contents of the 0–5 (P=0.470), 5–10 (P=0.984) and 10–15 cm (P=0.668) soil layers.\n\nAvailable copper (mg/kg) at 0–5 (A), 5–10 (B) and 10–15 cm (C) soil layers. Significant differences (P<0.05) between unburned and burned areas on specific sampling months are indicated by different letters above histogram bars. Data show mean ± SE (n=4).\n\nIn the unburned area, the DTPA-extractable Mn of the 0–5, 5–10 and 10–15 cm soil layers varied from 61.28 to 79.58 mg/kg, 45.11 to 61.51 mg/kg and from 35.00 to 48.3 mg/kg, respectively (Table 1). In contrast, the DTPA-extractable Mn of the three different soil layers respectively ranged from 60.44 to 74.16 mg/kg, 39.39 to 48.36 mg/kg and from 34.38 to 41.12 mg/kg (Table 1).\n\nA two way ANOVA test indicated that fire had no significant effect on the DTPA-extractable Mn contents of the 0–5 (P=0.318) and 10–15 cm (P=0.085) soil layers. However, a significant post-fire decrease (P=0.002) in the Mn availability was noticed in the 5–10 cm soil layer (Unburned > Burned, Figure 7B, Table 2). The DTPA-extractable Mn also varied significantly among the sampling months in the 0–5 (P=0.006), 5–10 (P=0.004) and 10–15 cm (P=0.011) soil layers.\n\nAvailable manganese (mg/kg) at 0–5 (A), 5–10 (B) and 10–15 cm (C) soil layers. Significant differences (P<0.05) between unburned and burned areas on specific sampling months are indicated by different letters above histogram bars. Data show mean ± SE (n=4).\n\nIn the surface layer (0–5 cm), the DTPA-extractable Zn of the unburned area ranged from 2.18 to 4.17 mg/kg while in the burned area, it ranged from 1.41 to 2.22 mg/kg (Table 1). The DTPA-extractable Zn contents of the sub-surface layers (5–10 and 10–15 cm) in the unburned area respectively ranged from 1.59 to 2.46 mg/kg and from 1.17 to 2.17 mg/kg while in the burned area, they ranged from 1.21 to 2.12 mg/kg and from 1.05 to 2.01 mg/kg, respectively (Table 1).\n\nThe results of the two way ANOVA test showed that fire reduced the DTPA-extractable Zn contents of the 0–5 cm soil layer varying significantly (P=0.004) between the burned and unburned areas (Unburned > Burned, Figure 8A, Table 2) while it did not show significant variation (P=0.08) among the sampling months. A significant decline (P=0.020) was also observed in the 5–10 cm soil layer (Unburned > Burned, Figure 8B, Table 2). The DTPA-extractable Zn of the 5–10 cm soil layer also varied significantly (P<0.001) among the sampling months. The 10–15 cm soil layer showed no significant variation (P=0.220) in terms of the Zn availability between the burned and unburned areas. However, it showed significant variation (P<0.001) among the sampling months.\n\nAvailable zinc (mg/kg) at 0–5 (A), 5–10 (B) and 10–15 cm (C) soil layers. Significant differences (P<0.05) between unburned and burned areas on specific sampling months are indicated by different letters above histogram bars. Data show mean ± SE (n=4).\n\n\nDiscussion\n\nSOC contents decreased in all the three soil layers of the burned area as confirmed by the two way ANOVA test and the decrease persisted throughout the sampling period. The persistence of this condition throughout the study reflects the high severity of the forest fire and the fact that we found a significant decrease of SOC in the 5–10 and 10–15 cm soil layers is further evidence of the high severity of the forest fire. The fire-induced reduction in SOC in this study is consistent with the results from studies conducted elsewhere16–20. However, contrasting results have also been obtained by other researchers21–23 who reported increased SOC after fire disturbance. The decrease in SOC content of the burned area may be attributed to the volatilization of organic carbon and conversion of organic matter to ash24,25. In a laboratory experiment, Terefe et al.26 observed a significant reduction in SOC content from A horizons after heating at 200 and 300°C with the complete loss of SOC at 500°C.\n\nAs confirmed by the two way ANOVA test, forest wildfire resulted in a significant loss of NH4+–N and available K from the burned area. Most studies17,27–29 show an initial increase in inorganic forms of nitrogen such as NH4+–N after fire followed by a decline with time. In the present study, however, we found a decrease in NH4+–N concentration of the burned area and the decrease persisted until the study ended. This finding agreed with the results obtained by Shaoqing et al.30 and Longo et al.19. The burned area also had lower available potassium than the adjacent unburned area. Yinghua et al.31 reported short-term increase in available potassium after fire which then declined and was lower than the unburned area at later sampling times. The decrease in NH4+–N and available potassium of the burned area may be attributed to the individual or combined effect of several factors such as volatilization, slow mineralization rate, increased uptake by newly sprouted vegetation32, dry ravel erosion33, surface run-off and leaching. In addition to the above mentioned factors, the loss of NH4+–N from the burned area may also be due to the nitrification of NH4+ to NO3– as heat-sensitive nitrifying bacteria recolonized the area34,35. Among all nutrients vital for plant growth, N is the most sensitive to volatilization. Half of the N in organic matter can be volatilized at 500°C35,36. The volatilization temperature of K is 760°C. Because the forest fire consumed the entire understory vegetation and the litter layer, it is possible that the temperature was sufficiently high during the fire to volatilize significant quantities of NH4+–N and available potassium. The rate of nutrient mineralization may have been slowed down probably due to fire-induced reduction in soil decomposer organisms which include microbes and invertebrates. High severity-fires sterilize the soil or delay the recovery of soil microbes and consequently reduce their activity37,38. The litter loss and microclimatic changes resulting from fire can indirectly decrease the total mass and species numbers of soil and litter dwelling invertebrates39,40. Choromanska and DeLuca41 reported that N mineralization decreased after fire and did not recover after nine months of the study period.\n\nSoil that has been protected from erosional processes for decades by vegetation or litter cover is particularly vulnerable when the cover is removed42. When the vegetation and litter layer is destroyed by fire, the soil is susceptible to dry ravel43,44, which can be defined as the movement of particles by rolling, sliding and bouncing down slope under the influence of gravity45–47 and to water erosion during the first major rain event48,49. Following fire, surface run-off and erosion can be increased by several orders of magnitude50,51, mainly during the first months when the soil is bare, if intense rainfall events are frequent52–54.\n\nIn this study, the available P was higher in the burned area than the adjacent unburned area in all the three soil layers and the increase was significant as proved by the two way ANOVA test. This finding is in agreement with the findings of several other workers who have reported increased post-fire available P levels55–59. Though post-fire increase in the available P level is a common trend in most research findings, decreases have also been noted19,60. The significant post-fire increase in the available P may be attributed to a number of factors such as fire-induced conversion of organic forms of soil phosphorus to inorganic forms, lack of orthophosphate leaching and lack of plants that utilize this nutrient in the study site. P loss through volatilization generally does not take place61. However, burning converts the soil organic P to orthophosphate62,63, the sole form of soil P available to biota. The time in which the positive effect on P bioavailability runs short is highly variable depending on numerous factors. Romanya et al.64 found increased post-fire soil concentrations of available P (NaHCO3-extractable) and labile P (NH4F HCl-extractable) even at seven months after clear felling and slash burning, in a Eucalyptus forest. In a Picea dominated forest, Macadam65 found that the available P in the upper 30 cm mineral soil had increased by up to 50%, nine months after slash burning, and this increase persisted, although somewhat diminished, twenty months after the fire event. In this research, sampling at monthly intervals started approximately three months after the fire event and continued for a period of one year, so it may have been within the fire-associated P enrichment period.\n\nThe available Fe levels depleted in all the three soil layers (0–5, 5–10 and 10–15 cm) of the burned area and the depletion was significant as confirmed by the two way ANOVA test. Some recent studies have also reported a decline in the Fe availability of burned soils18,29,66–68. The fire induced depletion of the available Fe in the burned area could be associated with losses (by convection in smoke columns, transport by wind, erosion and leaching) and/or conversion to insoluble oxidized forms in soils as have been reported for several other nutrients25,69.\n\nContradictory results were obtained in terms of fire-induced effects on the soil available Cu contents of the three soil layers studied. While the available Cu content of the 0–5 cm soil layer declined significantly, it showed no noticeable variation between the burned and unburned areas in the 5–10 cm soil layer. The 10–15 cm soil layer showed an increase in the Cu availability after the fire event, the available Cu being higher in the burned area than the adjacent unburned area. Other authors68,70 have also observed fire-induced increases in the soil available Cu. The fire induced-loss of the available Cu from the 0–5 cm soil layer of the burned area may be due to the singular or combination effects of several factors such as erosion, leaching, transport by wind, etc. Solute losses increased during the first rain events following fire according to one study71. Thus, it is possible that during rains some amounts of the available Cu in the ash resulting from fire leached into the deeper soil layers causing higher Cu availability in the 10–15 cm soil layer.\n\nAs confirmed by the two way ANOVA test, fire had no significant effect on the available Mn contents of the 0–5 and 10–15 cm soil layers, although Gomez-Rey et al.29 reported higher post-fire Mn availability in the 0–5 cm soil layer, attributed to Mn supplied by ashes from the burned vegetation. In contrast to the lack of difference in terms of the Mn availability between the burned and unburned areas in the 0–5 and 10–15 cm soil layers, the 5–10 cm soil layer showed a significant post-fire decrease in the available Mn compared with the unburned areas. Research findings from several studies have been mixed in terms of post-fire Mn availability. Brye72 observed a significant decline in the soil available Mn (extracted with Mehlich 3) as a consequence of 12 years of annual burning on a tall-grass prairie. However, other authors66,68,73 have reported a significant increase in the soil available Mn after fire.\n\nWith respect to the post-fire Zn availability, we found that the available Zn decreased in the 0–5 and 5–10 cm soil layers showing significant variations between the burned and unburned areas as indicated by the two way ANOVA tests. No fire effect was seen in the 10–15 cm soil layer as regards the available Zn which did not differ between the burned and unburned areas. The post-fire decline in the available Zn of the 0–5 and 5–10 cm soil layers is consistent with the observation of Marafa and Chau74 who have also reported a post-fire reduction in the amount of Zn. Other researchers have obtained contradictory results. For example, Garcia-Marco and Gonzalez Prieto66 observed an initial rise in the available Zn after fire disturbance followed by a decline one year after the fire. However, Close et al.68 and Stankov Jovanovic et al.69 have reported enhanced Zn availability in burned soils due to the release of Zn by ashes from burned vegetation. The available Zn depletion in the 0–5 and 5–10 cm soil layers of the burned area may be attributed to leaching and erosional losses as this element could be quickly leached75 or lost in eroded sediments29.\n\n\nConclusion\n\nThe SOC was significantly reduced following fire in all the three soil layers (0–5, 5–10 and 10–15 cm) and the significant reduction of the SOC even in the sub-surface layers (5–10 and 10–15 cm) is an indication of the high severity of the forest wildfire. The forest wildfire also had a significant effect on the available macronutrient concentrations, decreasing NH4+–N and available K and increasing that of available P in all the three soil layers. Contradictory results were obtained with respect to the soil micronutrient availability. While the available Fe declined in all the three soil layers, the available Cu declined only in the 0–5 cm soil layer. However, in the 10–15 cm soil layer, the available Cu showed an increase, the concentration being higher in the burned area compared to the unburned area. The available Cu contents of the 5–10 cm soil layer did not vary significantly between the burned and unburned areas. The Mn availability declined in the 5–10 cm soil layer but no fire-induced effect was found on the available Mn contents of the 0–5 and 10–15 cm soil layers. Fire significantly reduced the available Zn contents of the 0–5 and 5–10 cm soil layers. The available Zn content of the 10–15 cm soil layer did not show significant variation between the burned and unburned areas. Thus, the forest wildfire resulted in a significant loss of most soil nutrients at this site. Taking into account the post-fire decline of most soil nutrients in one or all of the three soil layers studied, it can be concluded that forest wildfire had detrimental impacts on soil nutrients at this site and the ecologically most significant effect was the loss of N and K from the burned soil.\n\n\nData availability\n\nFigshare. Post forest fire soil nutrient levels in burned and unburned areas of the Koubru Hills, Manipur, http://dx.doi.org/10.6084/m9.figshare.90370676",
"appendix": "Author contributions\n\n\n\nTBS conceived the study, suggested expertise in soil science and contributed to the preparation of the manuscript. AS carried out the research, interpreted the data and prepared the first draft of the manuscript. Both authors critically revised the draft manuscript and have agreed to the final content of the version to be published.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe research was funded by the University Grants Commission (UGC), New Delhi under the Rajiv Gandhi National Fellowship (RGNF) awarded to Alice Sitlhou vide Grant No. F. 14-2 (SC)/2009(SA-III).\n\n\nAcknowledgements\n\nTakhellambam Bikramsen Singh is greatly acknowledged for his timely help during the selection of the study site. The authors are indebted to the village elders, Seitinhen Vaiphei and Dirinamei Dirangbou, for unrestricted access to the study site and their friendly attitude. Dr.Athokpam Herojit Singh deserves special mention for fruitful discussions of the soil analytical methods. The authors would also like to thank the technical staff at the Department of Soil Science, Central Agricultural University, for their great help and patience during the long days of soil analysis in the laboratory. Thanks also go to Hechong Sitlhou and Samson Sitlhou for their support and encouragement of this research. Ningthoujam Ranbir Singh, Khumbongmayum Rakesh Singh and Donga Mizo are also highly appreciated for their assistance in soil sampling during field work. The authors would also like to gratefully acknowledge Kshetrimayum Radheshyam Singh for his advice on statistical analysis.\n\n\nSupplementary materials\n\nThe significant P values are followed by the letters (ab) on the right.\n\nThe significant P values are followed by the letters (ab) on the right.\n\nThe significant P values are followed by the letters (ab) on the right.\n\n\nReferences\n\nUNEP, GEO-2000. 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Publisher Full Text\n\nZhan G, Erich MS, Ohno T: Release of trace elements from wood ash by nitric acid. Water Air Soil Poll. 1996; 88(3): 297–311. Publisher Full Text\n\nAlice S, Thingbaijam BS: Post forest fire soil nutrient levels in burned and unburned areas of the Koubru Hills, Manipur. figshare. 2014. Data Source"
}
|
[
{
"id": "39546",
"date": "12 Nov 2018",
"name": "Xiankai Lu",
"expertise": [],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors addressed post-fire nutrient availability in a subtropical forest. They found that the forest wildfire resulted in a significant loss of most soil nutrients. Throughout the whole manuscript, however, the authors mainly described the results rather than mechanisms. In fact, there was lack of direct evidence to explain their findings. They pointed out that very few studies have evaluated post-fire effects on soil micro-nutrients, but did not show why they did this work. \"very few\" may mean that this work is not important, or it has been very clear. Hence, the major concerns are related to the novelty and the mechanism explanations.\nOthers:\nI am a little curious that the abstract mainly described the results without conclusion.\n\nIn the \"site description\", 3.99 mm for mean annual precipitation?\n\nTable 1, please show the sampling time, and better to unify the unit (e.g. N and P).\n\nThe authors mentioned element contents (such as SOC),the term \"content\" means stock. So it is better to show the responses of total soil nutrient stocks to fire for the whole soil layers.\n\nRepeated-measure ANOVA can be used to examine the overall fire effects on soil nutrient dynamics over the study period.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
}
] | 1
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https://f1000research.com/articles/3-30
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https://f1000research.com/articles/3-29/v1
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29 Jan 14
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{
"type": "Short Research Article",
"title": "Findings from a survey of wildlife reintroduction practitioners",
"authors": [
"Alexandra E. Sutton",
"Roel Lopez",
"Roel Lopez"
],
"abstract": "Wildlife reintroduction programs are a type of conservation initiative that seek to re-establish viable populations of a species in areas from which they have been extirpated or become extinct. Past efforts to improve the outcomes of reintroduction have focused heavily on overcoming ecological challenges, with little attention paid to the potential influence of leadership, management, and other aspects of reintroduction. This 2009 survey of reintroduction practitioners identified several key areas of leadership and management that may deserve further study, including: (i) the potential value of reintroduction partnerships for improving programmatic outcomes; (ii) the potential management value of autonomy vs. hierarchy in organizational structure; (iii) gaps in perceptions of success in reintroduction; and (iv) the need for improved evaluations of reintroduction programs and outcomes.",
"keywords": [
"conservation",
"leadership",
"management",
"reintroduction"
],
"content": "Objectives\n\nIn the fight to preserve global biodiversity, conservationists and biologists must make use of every available tool and approach. Reintroductions are a type of triage initiative; a last-ditch intervention when every other effort to keep a species present within its historic range has failed. They are employed only in cases of significant biodiversity loss, and are subsequently operating under more dire conditions than any other type of conservation initiative. Regardless, they maintain a low success rate, estimated in the past 12 years between 26% and 32% (Fischer & Lindemeyer, 2000; Jule et al., 2008). Efforts to improve this success rate have focused heavily on improving biological knowledge as an avenue toward greater success. However, we suggest that another, overlooked, area of significant influence might lie in the human dimensions of reintroduction - specifically, the types of leadership and styles of management under which reintroduction programs are operated. Reliable data on reintroduction management is limited and restricted almost entirely to the gray (i.e. informally published) literature, with the exception of (Clark & Westrum’s, 1989) paper on high-performance teams in wildlife conservation. This is unfortunate, as a slightly greater emphasis on the human dimensions of reintroduction would be to the benefit of both ecological and human communities. To that end, this survey is an exploratory effort to gain information about simple trends in reintroduction management and praxis, with the goal of informing future studies in this field.\n\n\nMethods\n\nThis survey was designed as an online-only, 47-question survey, presented via email between April and May 2009 and requiring approximately 20 minutes for completion. Emails of reintroduction practitioners were collected from the IUCN Reintroduction News online newsletter, the Reintroduction News Directory of Practitioners, and from the author contacts of reintroduction publications between 1999 and 2009, found through Google Scholar. There was no bias in participant selection relating to species, size or length of project, or budget. Invitations to participate in the survey were sent via email to 401 reintroduction practitioners worldwide.\n\n\nSurvey design\n\nThe survey was designed subsequent to a case study of the leadership and management of the Sea Eagle Recovery Project, undertaken from May 2008 to August 2009 (Sutton, unpublished data). The six sections of the survey included two introductory demographic sections and four project-based sections, within which questions were designed based on observations made during the 2009 case study. These sections were: (i) About Your Project, (ii) About You and Your Position, (iii) About Organizational Structure, (iv) About Goal-Setting, Meetings and Evaluation, (v) About Public Relations and Outreach, and (vi) About Success and Performance. General trends and descriptive statistics were drawn from the data using Qualtrics website software (Qualtrics, 2009).\n\n\nResults\n\nSixty-eight (16.95%) invitees responded to the survey. An additional 40 (9.98%) responded to email invitations and stated that (a) they no longer worked in the field; (b) they had only conducted retrospective analyses of reintroduction and not participated in a program; or (c) they did not, for other reasons, wish to share their experiences. An additional 25 (6.23%) were not contactable by email (i.e. email addresses were outdated). The remaining 268 invitees (66.83%) did not respond. Reminders were sent to invitees at the two-week and one-month mark.\n\n\nRespondent demographics\n\nMost respondents (45.95%) had served as senior employees or founders of reintroduction programs (Figure 1), with the majority of respondents (62.16%) also reporting less than three years’ experience at the time they took on that role with the reintroduction program (Figure 2).\n\n\nReintroduction phases and lengths\n\nQuestions about phase length revealed four reintroduction phases: (1) planning, (2) approval, (3) action, and (4) monitoring. “Planning phase” referred to the period of time used to conceive and plan the reintroduction project. “Approval phase” referred to the period of time used to gain permission from government agencies or leading organizations to reintroduce the focal species. “Action phase” referred to the period of time during which animals were actually captured, captive-bred, raised, and released into the wild. “Monitoring phase” referred to the period of time during which reintroduced animals were monitored post-release.\n\nResults indicated that planning phases most frequently took one to three years, while approval phases typically took nine months to one year. Both action and monitoring phases most commonly took more than four years (Figure 3).\n\n\nTask supervision and organizational structure\n\nRespondents indicated that tasks were ‘rarely’ monitored, either directly (43.24%) or indirectly (30.56%), by supervisors (Figure 4). Most respondents (32.43%) self-assessed their program as having been “somewhat autonomous”; however, a nearly-equivalent number self-assessed their program as having been “autonomous” (21.62%) or “very autonomous” (27.03%) (Figure 5). Most respondents also indicated that their assigned tasks and responsibilities were “frequently” shared with coworkers (47.22%).\n\nRespondents most frequently reported two levels of authority existed between the most senior and most junior employee, and one level of authority existed between the most senior volunteer and most junior volunteer (Figure 6). Most respondents (48.49%) self-assessed their projects as having been “somewhat hierarchical” (Figure 7).\n\n\nMeetings and goal-setting\n\nThe majority (56.00%) of all-staff, general meetings within reintroduction projects took place annually (Figure 8). Most meetings that specifically aimed to establish, modify, or augment goals for the project were held annually to discuss long-term goals (57.58%) and monthly to discuss short-term goals (54.55%) (Figure 9).\n\n\nEvaluation\n\nThe majority of respondents reported evaluations of employee performance as an annual event (64.52%), as were evaluations of overall program outcomes, both by internal employees (71.88%) and external authorities (41.38%) (Figure 10).\n\n\nPublic relations and outreach\n\nMost programs had no staff dedicated solely to public relations/media affairs (67.65%) or public education and outreach (64.71%) (Figure 11). Respondents indicated that projects were most likely to form partnerships with national wildlife organizations (77.42%) or local community groups (77.42%), and least likely to partner with corporations/businesses (43.75%) or other reintroduction programs (45.45%) (Table 1).\n\n\nSuccess and progress\n\nMost respondents self-assessed their projects as having been a success (57.14%); most also reported a formal evaluation as having determined that their project had been a success (62.86%). A wide majority of respondents self-assessed their project as having “made good progress” (74.29%); most also reported that a formal evaluation had determined their reintroduction to have made good progress (60%) (Figure 12, Figure 13).\n\n\nDiscussion\n\nThe survey results show several trends in reintroduction management and reveal a multitude of gaps in knowledge and management practice. The clear gaps in knowledge, expertise, partnerships and evaluation yield a bevy of interesting questions for further study – and demonstrate the lack of a best practices management protocol in this field.\n\nExpertise gap: despite respondents’ high-level roles as reintroduction founders or senior officers, they typically lacked reintroduction experience. Most respondents reported less than three years’ experience at the time they took on high-level roles; this is the same length of time typically required for planning and approval for a reintroduction, according to respondents’ reports. This overlap indicates that the majority of reintroduction founders and executives responding to this survey had never witnessed the full planning-approval-action-monitoring process of a reintroduction at the time they were placed in charge of one.\n\nPartnership and knowledge-sharing gap: overall, respondents reported very limited engagement between their reintroduction and partner organizations of any type. Partnerships that were reported skewed heavily toward national wildlife or conservation organizations and national news outlets, and very few partnered with either businesses or other reintroduction programs. The former gap is a missed opportunity to engage corporate partners in conservation and build a stronger sponsorship base for local projects; the latter may indicate a tragic lack of connectivity between parallel projects, and hints at a likelihood of redundant work and “learned lessons” that go unshared.\n\nEvaluation gap: the lack of established, recurrent evaluations conducted by external authorities was lamented by (Kleiman et al., 1999) in all areas of conservation, and is only too evident here. A trend toward frequent, informal, internal evaluations means that rigor is decreased; this decrease in rigor and shift toward informality has been recognized as a challenge to maintaining the value of program evaluation across all types of organizations (Roch & McNall, 2007). This type of weaker evaluation can lead to a loss of accurate perceptions, as suggested by the gaps between respondents’ self-assessment of their programs’ success or progress and the results of formal evaluations.\n\nAlthough the success-perception gap in our survey was not large (a 5.72% difference), the progress-perception gap was nearly triple (14.29%), and respondents reporting that they believed good progress had been made were common than those reporting that they believed success had been met (74.29% vs. 57.14%). This may suggest that respondents have a poor understanding of how to recognize markers of progress that lead to success – a problem that weak evaluation would only exacerbate.\n\n\nSummary\n\nThis survey, although preliminary, provided insight into several areas of conservation leadership and management that could be focal areas of future study. Understanding the gaps in expertise and evaluation, as well as the missed opportunities in partnership and knowledge-sharing, could be hugely beneficial in the future improvement of project management and reintroduction outcomes.",
"appendix": "Author contributions\n\n\n\nBoth authors contributed extensively to this work. A.E.S. designed and distributed the survey instrument and conducted analyses. R.L. advised the development of the instrument and interpretation of results. Both authors discussed the results and implications and commented on the manuscript at all stages.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nWe would like to thank Texas A&M University and the MSC L.T. Jordan Institute for International Awareness, for providing support for Alexandra Sutton’s research travel through the Jordan Fellows Program.\n\n\nAcknowledgements\n\nWe would like to thank Drs. Gillian Bowser, Gerard Kyle, and Jean Madsen for their input and guidance.\n\n\nReferences\n\nClark TW, Westrum R: High-performance teams in wildlife conservation: a species reintroduction and recovery example. Environ Manage. 1989; 13(6): 663–670. Publisher Full Text\n\nFischer J, Lindenmayer DB: An assessment of the published results of animal relocations. Biol Conserv. 2000; 96(1): 1–11. Publisher Full Text\n\nJule KR, Leaver LA, Lea SEG: The effects of captive experience on reintroduction survival in carnivores: A review and analysis. Biol Conserv. 2008; 141(2): 355–363. Publisher Full Text\n\nKleiman DG: Reintroduction of captive mammals for conservation. Bio Science. 1989; 39: 152–161. Reference Source\n\nRoch SG, McNall LA: An investigation of factors influencing accountability and performance ratings. J Psychol. 2007; 141(5): 499–523. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "3424",
"date": "05 Feb 2014",
"name": "David Norton",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting short note that addresses a useful question. While it is true that environmental factors play a key role in limiting the success of reintroduction programmes, management and leadership factors could also be an important issue. This note sought information on this issue through a survey of people involved in reintroduction programmes. I think the two key conclusions from this survey are useful, that there is a lack of long-term experience amongst those undertaking reintroduction programmes, and that there is a lack of formal evaluations of these programmes. This latter point also links to the observation that there is a lack of linkages between different organisations and different reintroduction programmes.There were, however, some issues that I felt did require further attention: I was unclear what the focus of the survey was - was it just in the USA or was it wider? This should be clarified. Was there a bias in the responses in that perhaps only those that had been involved with more successful reintroduction programmes responded? The survey results indicate success rates of around 60% which is much higher than the 26-32% cited in the Objectives section. This should be discussed further. I would also have liked to have seen more discussion in the conclusions on how the results of this survey might be used. How might these results be taken up and used to improve future re-introduction programmes? I also felt that the paper had too many figures and wondered if some of these could be removed with just the key results presented in the text (or perhaps tabulated). I thought the abstract was a good summary of the article, but was unclear what the difference is between extirpation and extinction.",
"responses": []
},
{
"id": "3423",
"date": "13 Feb 2014",
"name": "Gary Luck",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral commentsI agree with the authors that much of the assessment of reintroduction success is focused on the ecological aspects of reintroduction programs, with little attention paid to how humans manage reintroduction programs. I agree also that some of these management aspects can be critical to program success, although I think the authors could have elaborated on this in more detail in the Introduction (Objectives) section. A survey of wildlife practitioners involved in reintroduction programs is therefore a useful addition to the literature.The study employs a fairly basic survey instrument delivered via email. More information on the survey instrument (design, types of questions, justification for questions included etc.) could have been provided, including a copy of the survey published as supporting information. The response rate to the survey was low, and I wonder if this introduced any biases to the results? Finally, the Discussion doesn’t really elaborate on how the trends in the responses might actually impact on the success rate of reintroduction programs. Would greater expertise lead to more success? How much expertise might be needed and in what areas specifically? Would more partnerships with corporate bodies actually improve reintroduction success? Why? Specific comments Objectives:For the sentence ‘Regardless, they maintain a low success rate, estimated in the past 12 years between 26% and 32%’ it would be instructive to know how the cited authors measured ‘success rate’.This section could include a more detailed argument regarding why ‘types of leadership’ or ‘styles of management’ are likely to be so important to reintroduction success.Methods:Change ‘Emails of reintroduction practitioners…’ to ‘Email addresses of…’ to avoid confusion.‘There was no bias in participant selection relating to species, size or length of project, or budget.’ - Were practitioners asked about these things? It would have been instructive to know the spread of responses (e.g. types of species dealt with).Survey design:It would be useful to include more information about survey design including a copy of the actual survey published as supporting information. Additional information could include details of question design and type, and justification for inclusion.Results:Did the low response rate lead to any biases in the results? Task supervision and organizational structure:Please define ‘autonomous’ in this context.Success and progress:Again, a short definition of ‘success’ would be useful here. Was a definition included in the questionnaire or were practitioners just asked something like ‘Was your project successful?’ and just left to self-define the meaning of ‘success’?DiscussionA broader discussion of how specific results might influence reintroduction program success would be highly beneficial and help guide future research in this area.",
"responses": []
},
{
"id": "3425",
"date": "13 Feb 2014",
"name": "Ryan Chisholm",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have conducted a survey of reintroduction practitioners and they have analysed the distribution of different project statistics, such as degree of autonomy of and frequency of meetings. They also present data on project success rates.These data will no doubt be useful to people working in the field. I was surprised that the authors did not investigate whether any of the other factors, e.g., frequency of meetings, was correlated with project success. They obviously have the data to look at this. Why didn’t they?Minor comments:p2: \"Lindemeyer\" -> \"Lindenmayer\"p2: Citation formatting: “Clark & Westrum’s (1989)”p7: Heading of column 2 in Table 1 could just be “0”, because otherwise it could be read as “number of partnerships”.p8, Figure 13: “Formall Evaluated” -> “Formally Evaluated”p8: Citation formatting again: “Kleiman et al. (1999)”",
"responses": []
},
{
"id": "3422",
"date": "14 Feb 2014",
"name": "Mary Blair",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis short research paper presents the results of a survey of wildlife reintroduction practitioners. The paper is well-written and the results have relevance to future studies, but I suggest several revisions to further improve the paper.In the Summary, the authors state that the results provide insight into areas of conservation leadership and management, although the article really only focuses on reintroduction programs specifically. There are many ongoing discussions in the wider conservation management arena about leadership and evaluation of success and it might be important to give the paper some more context in relation to those ongoing discussions (e.g. by referring to Black & Groombridge 2010 Conservation Biology, several publications related to the CMP Open Standards http://www.conservationmeasures.org/initiatives/standards-for-project-management, Manolis et al. 2009 Conservation Biology).Also, further discussion on what sorts of responses to other questions resulted in a perception of \"success\" in a given program would greatly strengthen the claims of the paper. To do this the authors could add a correlation analysis among variables, or, perhaps the authors could detail a few case examples?Minor comments:In the Methods, it is stated that the survey was sent to practitioners worldwide, but of the 17% that responded, was there bias in terms of the countries respondents represented? A bit more elaboration on the potential of bias in the survey results (both geographic and other kinds of bias) would strengthen the paper. It would be very informative for future studies to include the survey instrument itself as an appendix. Table 1. It might be more informative if values in this table were represented as percentages or proportions to better illustrate the claims in the discussion section about how partnerships are \"very limited\" or \"very few\" with businesses or other reintroduction programs.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-29
|
https://f1000research.com/articles/2-194/v1
|
23 Sep 13
|
{
"type": "Observation Article",
"title": "Amblyomma tapirellum (Acari: Ixodidae) collected from tropical forest canopy",
"authors": [
"Jose R Loaiza",
"Matthew J Miller",
"Eldredge Bermingham",
"Oris I Sanjur",
"Patrick A Jansen",
"Jose R Rovira",
"Eric Alvarez",
"Eric Rodriguez",
"Philip Davis",
"Larissa C Dutari",
"James Pecor",
"Desmond Foley",
"Meghan Radtke",
"Montira J Pongsiri",
"Matthew J Miller",
"Eldredge Bermingham",
"Oris I Sanjur",
"Patrick A Jansen",
"Jose R Rovira",
"Eric Alvarez",
"Eric Rodriguez",
"Philip Davis",
"Larissa C Dutari",
"James Pecor",
"Desmond Foley",
"Meghan Radtke",
"Montira J Pongsiri"
],
"abstract": "Free-ranging ticks are widely known to be restricted to the ground level of vegetation. Here, we document the capture of the tick species Amblyomma tapirellum in light traps placed in the forest canopy of Barro Colorado Island, central Panama. A total of forty eight adults and three nymphs were removed from carbon dioxide–octenol baited CDC light traps suspended 20 meters above the ground during surveys for forest canopy mosquitoes. To our knowledge, this represents the first report of questing ticks from the canopy of tropical forests. Our finding suggests a novel ecological relationship between A. tapirellum and arboreal mammals, perhaps monkeys that come to the ground to drink or to feed on fallen fruits.",
"keywords": [
"Ticks",
"Amblyomma tapirellum",
"forest canopy",
"mosquito CDC light traps",
"monkeys"
],
"content": "Introduction\n\nIncreasing interest in tick-borne diseases in the Neotropics and particularly in Panama during the last decade has fuelled studies on tick biology, behavior and distribution in this region1–3. These studies have focused on tick species associated with humans and domesticated animals, likely due to their role as vectors of disease agents1,2,4. However, basic knowledge about tick natural history still remains largely unexplored, especially for those taxa that thrive in tropical forests. The tick species Amblyomma tapirellum5 predominates over Amblyomma cajennense as the primary human tick parasite in lowland forest ecosystems of central Panama and Darien6. Adults of A. tapirellum have Baird’s Tapir (Tapirus bairdii) as their primary host, but also opportunistically feed on other wildlife and domesticated mammals6,7, and also humans (Table 1). A. tapirellum is one of the most common species collected with a cloth dragged through the understory vegetation, but it is not known to be found arboreal mammals (Table 1), and in addition, a recent survey of tick occurrence on Panamanian birds found no evidence that this species feeds on birds (Miller et al., in prep.). Here, we report A. tapirellum collected from mosquito light traps placed in the canopy of old-growth lowland tropical forest on Barro Colorado Island (BCI) in central Panama. To our knowledge, this is the first report of ticks being collected in the canopy of Neotropical forests and highlights the potentially complex ecological relationships of likely vectors of zoonotic disease in undisrupted forest habitats.\n\n* This record is doubtful as the sample could have been pulled from the body of the collector\n\n\nMethods\n\nCenters for Disease Control and Prevention miniature light traps (CDC-LTs) baited with CO2 (dry-ice) and 1-octen-3-ol were placed in areas of old-growth forest on BCI, in the Panama Canal (9.16457 N; -79.86347 W), which has served as a field station for studies of Neotropical flora and fauna for over 100 years. Six traps were placed in the forest canopy (20–30 meters off the ground) and six at the ground level (1.5 meters of height) for seven consecutive days, every other month, from August 2009 to July 2010 (Figure 1). Arthropod samples were collected every morning upon removal of light traps and placed separately in 1.5 milliliters plastic tubes. Ticks were counted by trap and preserved as vouchers as part of the ectoparasite - cryological collection of the Smithsonian Tropical Research Institute (STRI).\n\nLeft side: the set up of one Centers for Disease Control and Prevention miniature light trap in the forest canopy of Barro Colorado Island (BCI). Right upper side: the dorsal view of the scutum of one female (left) and one male (right) of Amblyomma tapirellum collected from the forest canopy at BCI. Right lower side: image of an adult of Ateles geoffroyi panamensis (Black Spider Monkey) walking freely around the Smithsonian Tropical Research Institute field station at BCI.\n\n\nObservation\n\nForty eight adults and three nymphs of A. tapirellum were collected from CDC-LTs placed in the forest canopy at BCI (Table 2). All adults were identified using standard taxonomic keys6, while all three nymphs and one adult male and one adult female were confirmed as belonging to A. tapirellum based on a neighbor-joining tree generated from reference library of mitochondrial DNA barcoding (COI gene) sequences from Panamanian ticks (Miller et al., in prep.). We selected four individuals of A. gaeyi, one of A. longirostre, five of A. cajennense, three of A. oblongoguttatum, three of A. tapirellum, and two of Haemaphysalis juxtakochi (as an outgroup) to build the tree in MEGA48 with group support evaluated via 500 bootstrap replicates (Figure 2). Mean Kimura 2 parameter (K2P) genetic distance between all five canopy collected ticks and the reference library specimens of A. tapirellum was 0.1% (maximum K2P distance 0.6%), well below the typical 2% threshold for interspecific distances for most barcoding studies9. Specimen data, sequences, and sequencing trace files for the five canopy-collected ticks and the 13 reference specimens are archived in the BOLD barcoding database (dx.doi.org/10.5883/DS-TICKSCAN) and are available on the online global database of DNA barcode sequences (http://boldsystems.org). Genbank accession numbers for the five canopy ticks generated in this study are: KF370887–KF370891, whereas the Genbank numbers for the adult reference library are: KF200081, KF200091, KF200097, KF200098, KF200101, KF200103, KF200105, KF200119, KF200124, KF200130, KF200133, KF200135, KF200159, KF200160, KF200171.\n\nEach row contains information about the number of specimens collected in one trap during one night. The number of tick positive CDC-LTs out of the total number of canopy traps per month are as following: August (5/42 = 0.119), October (4/42 = 0.095), January (1/42 = 0.023), March (1/42 = 0.023), May (18/42 = 0.418), and July (1/42 = 0.02).\n\nNode support (as a percentage) was estimated from 500 bootstrap replicates. Taxa indicated with asterisks (*) represent canopy collected ticks from this study; otherwise tip labels refer to Genbank accession numbers.\n\nInterestingly, ticks were only extracted from CDC-LTs set up at the canopy level, no ticks were collected from traps at ground level, and only a few canopy traps were positive for ticks (Table 2). Our findings are unexpected because CDC-LTs are not commonly used to collect ticks, but rather blood-sucking insects such as mosquitoes, sand flies and biting midges. However, they reinforce the notion that ticks use CO2 to locate their hosts10. Other Amblyomma species have been previously collected with CO2 baited traps11, but no study has ever reported questing ticks collected in this fashion from the canopy of a tropical forest. This finding indicates that A. tapirellum is not restricted to the ground, but uses both vertical strata (e.g., canopy and ground) to seek hosts. The fact that adults of both sexes as well as nymphs were recovered from canopy traps suggests that A. tapirellum can complete its life cycle in the canopy, but this is most likely the result of foresia – the passive movement of one organism by another – by hosts moving vertically. Candidate vectors for movement into the canopy are two monkeys, Alouatta palliata (Mantled Howler Monkey) and Ateles geoffroyi panamensis (Black Spider Monkey), which were often seen near CDC-LTs, and on one occasion, destroyed a trap (Figure 1). Yet, at present there are no records of A. tapirellum collected from these monkeys or any other arboreal mammals in Panama (Table 1). In addition, the majority of ticks were collected at the beginning of the dry-wet transition period in May 2010 (Table 2), when ground populations of A. tapirellum are quite abundant and monkeys may come to the ground to feed on ripe and over-ripe fruits12. This possibility suggests that an association between arboreal monkeys and ticks is opportunistic, perhaps occurring principally at the peak of the fruiting season13. However, ticks were also collected during August and October of 2009, and so, tick-monkey ground interactions could also be the result of monkey behaviors such as drinking from terrestrial sources or chasing games14. Fairchild and collaborators6 noted that A. geayi and A. varium are practically confined to arboreal sloths. Sloths descend to the ground every three to eight days, dig a hole, defecate, and climb back up into the trees, a behavior that puts the animal at risk if predators are nearby15, and it may also increase the odds of getting ground ticks. Our findings highlight the lack of information on the basic ecology of some species of Neotropical ticks, and argue for an expanded vision of wildlife-tick relationships when planning and conducting disease ecology studies in the Neotropics. Further studies should include canopy catches to better understand the bionomics of A. tapirellum and its role in pathogen transmission to wildlife.",
"appendix": "Author contributions\n\n\n\nJRL, MJM, EB, OIS, JP, and MJP conceived the study. JRL, JP, and MJP designed the project.\n\nJRL, JRR, EA, ER, PD, LCD, JP, DF and MR carried out the research. MJM, PAJ, and DF contributed to the design of experiments and provided expertise in the field. JRL, MJM, PAJ, and DF prepared the first draft of the manuscript. EB, OIS, JP, MR, and MJP contributed to the experimental design and preparation of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nFinancial support was provided by Smithsonian Tropical Research Institute – Environmental Protection Agency grant DW33-92296801-0 “Mosquito Species Diversity and Landscape Change” to MJP and OIS; and the Secretariat for Science, Technology, and Innovation of Panama (SENACYT) – National Research Investigator award (SNI) granted to JRL. Funds for DNA barcoding came from an award to MJM, JRL, OIS and EB from the Smithsonian Institution’s Consortium for the Barcode of Life (2011 COB award: “DNA barcodes of arthropod disease vectors in Panama” grant FED/CBOL-2011010014-009).\n\n\nAcknowledgements\n\nWe are grateful to Oris Acevedo, Belkis Jimenez and Hilda Castañeda for logistical support at BCI; Celestino Aguilar for providing the DNA barcoding labwork; Eyda Gomez for helping managing the project logistics and for technical guidance; Mauricio Quintero, Anel J. Duncan, Denis Lezcano, Gaspar Ho and Apolonio Valdez for field assistance. The opinions and assertions contained herein are those of the authors and are not to be construed as official or reflecting the views of the supporting agencies.\n\n\nReferences\n\nBermúdez SE, Miranda R: Distribution of ectoparasites of Canis lupus familiaris L. (Carnivora: Canidae) from Panama. Revista MVZ Córdoba. 2011; 16: 2274–2282. Reference Source\n\nBermúdez C SE, Castro A, Esser H, et al.: Ticks (Ixodida) on humans from central Panama, Panama (2010–2011). Exp Appl Acarol. 2012; 58(1): 81–88. PubMed Abstract | Publisher Full Text\n\nMurgas IL, Castro AM, Bermúdez SE: Current status of Amblyomma ovale (Acari: Ixodidae) in Panama. Ticks Tick Borne Dis. 2013; 4(1–2): 164–166. PubMed Abstract | Publisher Full Text\n\nBermúdez SE, Eremeeva ME, Karpathy SE, et al.: Detection and identification of rickettsial agents in ticks from domestic mammals in eastern Panama. J Med Entomol. 2009; 46(4): 856–861. PubMed Abstract | Publisher Full Text\n\nDunn LH: Two New Species of Ticks from Panama (Amblyomma tapirellum and A. pecarium). Parasitology. 1933; 25(3): 353–358. Publisher Full Text\n\nFairchild G, Kohls G, Tipton V: The Ticks of Panama (Acarina: Ixodidae). In: Wenzel R.L., Tipton V.J. (Eds.), Ectoparasites of Panama. Field Museum of Natural History Chicago, Illinois, 1966; pp. 167–219. Reference Source\n\nFairchild GB: An annotated list of bloodsucking insects, ticks and mites known from Panama. Am J Trop Med Hygiene. 1943; 23(8): 569–591. Reference Source\n\nKumar S, Tamura K, Nei M: MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief Bioinform. 2004; 5(2): 150–163. PubMed Abstract | Publisher Full Text\n\nHebert PD, Cywinska A, Ball SL, et al.: Biological identifications through DNA barcodes. Proc Biol Sci. 2003; 270(1512): 313–321. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSonenshine DE: Pheromones and other semiochemicals of ticks and their use in tick control. Parasitology. 2004; 129(suppl): S405–S425. PubMed Abstract | Publisher Full Text\n\nKesinger BJ, Allan BF: Efficacy of dry ice-baited traps for sampling Amblyomma americanum (Acari: Ixodidae) varies with life stage but not habitat. J Med Entomol. 2011; 48(3): 708–711. PubMed Abstract | Publisher Full Text\n\nLeigh EG: Tropical Forest Ecology: A View from Barro Colorado Island. Oxford and New York: Oxford University Press. ISBN 0–19–509602–9/OCLC 3678102. 1999. Reference Source\n\nFoster RB: The seasonal rhythm of fruitfall on Barro Colorado Island. In Leigh, E.G., Ran A.S. & Windsor, D. (eds). The ecology of a tropical forest: seasonal rhythms and long-term changes. Smithsonian Institute Press, Washington. 1982; 151–172. Reference Source\n\nCampbell C, Aureli F, Chapman C, et al.: Terrestrial Behavior of Ateles spp. Int J Primatol. 2005; 26(5): 1039–1051. Publisher Full Text\n\nVoirin JB, Kays R, Lowman MD, et al.: Evidence for Three-Toed Sloth (Bradypus variegatus) predation by spectacled owl (Pulsatrix perspicillata). Edentata no. 8–10 2009. Reference Source"
}
|
[
{
"id": "2042",
"date": "17 Oct 2013",
"name": "Brian Allan",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn addition to being notable for describing a unique approach for sampling ticks in Neotropical forests, this article presents the first reported instance of the Amblyomma tapirellum tick found in the forest canopy. The authors suggest that this may indicate host transportation of these normally ground-dwelling ticks vertically through the canopy by an unreported host. Though the authors mention that the vertical movement of sloths may lead to ticks being acquired while on the ground, they primarily focus on two candidate host species for the vertical transport of ticks: the Mantled Howler Monkey and the Black Spider Monkey. They hypothesize that the monkeys acquire the ticks on the ground during the peak of fruiting season, and then transport the ticks vertically as they return to the canopy. This is a thoughtful insight that prompts additional questions regarding the natural history of this tick and differences in host acquisition between ground-dwelling and canopy-dwelling populations. One suggestion is to highlight the interesting contradiction that arose from finding no ticks in the ground CDC light traps (CDC-LTs) despite the statement that “ground populations of A. tapirellum are quite abundant” during the sampling period. The lack of ticks in ground traps compared to canopy traps could suggest differences in host acquisition strategies between the two environments. In addition, the capture of these ticks in the canopy may indicate that an arboreal life strategy in ticks is a more common phenomenon than previously thought. However, the authors take a different approach regarding stating the article’s significance. Instead of highlighting potentially interesting differences in host acquisition in ticks, the authors focus on implications for disease ecology. Though in this paper these ticks are suggested to be vectors for zoonotic disease, no mention is made of pathogens known to be associated with A. tapirellum, which leaves the implications for disease ecology or public health vague and ill-defined. Despite this, the findings in this paper are ecologically interesting and could be framed in a behavioral ecology context to emphasize the importance of finding ground-dwelling ticks actively seeking for hosts in the canopy. Additional, minor revisions could be incorporated to improve the clarity of the paper’s findings. In several instances, A. tapirellum is referred to as a vector yet the authors offer no evidence that these ticks transmit a known illness. By definition, a vector is an agent that transmits disease. Additionally, ticks captured by the CDC-LTs are described here as “questing”, but the correct term to describe this behavior is “hunting” or “host seeking”, since “questing” refers to a specific, relatively passive, host acquisition strategy. The authors state that the presence of ticks in the canopy traps “reinforce[s] the notion that ticks use CO2 to locate their hosts”, a characteristic which is true of many species of ticks. However, this raises the question of why ground traps did not capture ticks, a finding for which no explanations were proposed.",
"responses": [
{
"c_id": "675",
"date": "20 Jan 2014",
"name": "Jose Loaiza",
"role": "Author Response",
"response": "\"In several instances, A. tapirellum is referred to as a vector yet the authors offer no evidence that these ticks transmit a known illness.\"The sentence in the Introduction now reads: “…highlights the potentially complex ecological relationships of Neotropical ticks, which as a group, are potential vectors of zoonotic disease in undisrupted forest habitats.” \"Additionally, ticks captured by the CDC-LTs are described here as “questing”, but the correct term to describe this behavior is “hunting” or “host seeking”, since “questing” refers to a specific, relatively passive, host acquisition strategy\"References to “questing” were changed to “host-seeking”. \"The authors state that the presence of ticks in the canopy traps “reinforce[s] the notion that ticks use CO2 to locate their hosts”, a characteristic which is true of many species of ticks. However, this raises the question of why ground traps did not capture ticks, a finding for which no explanations were proposed\"We agree that it is curious that we did not find ticks on the understory traps. However, as we don’t have a plausible explanation, we defer from additional speculation, as it is apparent that simply reporting our finding has created enough controversy without additional conjecture."
}
]
},
{
"id": "2288",
"date": "05 Nov 2013",
"name": "Michael Levin",
"expertise": [],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors speculate that questing A. tapirellum ticks, which are normally feeding primarily on tapirs, could be transported and, moreover, dropped into carbon dioxide baited mosquito traps by primates. (Yes, “Questing” is the appropriate term, which describes a stage in the tick life cycle when it is ready to take a blood-meal, whether it is searching for food actively or passively.) This “delivery of questing ticks” is a tenuous conjecture at best.Unlike mosquitoes, ixodid ticks feed only once per life stage; they rarely attach to and get transported by unsuitable hosts, on which ticks would not feed. Ticks, that “by mistake” get onto a wrong target – be it a flagging device or an inappropriate host, get off of it within minutes unless they are frozen in dry ice. Ticks which do find a suitable host and begin feeding do not just suddenly drop off, but continue taking a blood-meal for several days until full engorgement (or until the host’s death). A. tapirellum has a 3-host life cycle; it means that at each stage engorged (blood-fed) ticks fall off the host into the substrate in order to molt into the next life stage or to lay eggs (unlike in mosquitoes, female ixodid ticks die after just one oviposition). It takes several weeks for an engorged tick to molt and start questing again. Therefore, it is highly implausible that unfed (questing) ticks would be delivered en masse into the traps by any hosts. Engorged ticks, on the other hand, might have been delivered in this way but only if an infested animal crawled into a trap for a rest and fed ticks had time to fall off of it. However, neither spider nor howler monkeys could possibly fit into miniature light traps, not to mention that neither species is known to serve as a host for A. tapirellum.It is much more likely that the appearance of questing Amblyomma ticks in mosquito traps resulted from a procedural (trap-handling) artifact. Authors placed their carbon dioxide baited traps either 1.5 meters above the ground (not really “at the ground level”), or 20-30 meters up in the trees. Obviously, traps hanging 1.5 meters above the ground could be serviced right where they were, without contact with the tick-infested ground cover. However, those traps placed 20 meters high would have had to be lowered to the ground daily for checking and replacement of the dry ice, and that would give ticks a chance to crawl onto the traps out of their own volition. Ticks are attracted to carbon dioxide and fast crawling ticks like Amblyomma spp. can be collected in large numbers quite quickly. Unfed questing ticks that crawled onto and eventually into dry ice container(s) would be discovered the next day to cause a “sensational discovery”. Uneven distribution and seasonal dynamics of ticks explain why some traps had ticks and others did not. Differences in placement of traps explain why only high-hanging traps caught ticks.The suppositions advanced in this account can only stem from ignorance regarding ticks, their biology, behavior, and even the standard terminology. It is usually advisable to consider simple explanations of unexpected results prior to making \"astounding\" claims.",
"responses": [
{
"c_id": "674",
"date": "20 Jan 2014",
"name": "Jose Loaiza",
"role": "Author Response",
"response": "\"Authors placed their carbon dioxide baited traps either 1.5 meters above the ground (not really “at the ground level”).\"To avoid confusion we have changed the text to read “understory.” \"Obviously, traps hanging 1.5 meters above the ground could be serviced right where they were, without contact with the tick-infested ground cover. However, those traps placed 20 meters high would have had to be lowered to the ground daily for checking and replacement of the dry ice, and that would give ticks a chance to crawl onto the traps out of their own volition. Ticks are attracted to carbon dioxide and fast crawling ticks like Amblyomma spp. can be collected in large numbers quite quickly. Unfed questing ticks that crawled onto and eventually into dry ice container(s) would be discovered the next day to cause a “sensational discovery”. Uneven distribution and seasonal dynamics of ticks explain why some traps had ticks and others did not. Differences in placement of traps explain why only high-hanging traps caught ticks.\"We appreciate the reviewer calling attention to our lack of detail about our field sampling practice. It is clear that the original draft of our manuscript was without sufficient detail to demonstrate that our finding is not likely to be the consequence of trap-handling artifacts. In fact, when we first found ticks on our traps, our team recognized the novelty of our finding and we took critical steps to ensure that the ticks originated in the canopy and were not accidentally sampled from the ground. We have added the following text to our manuscript:\"When our field team first discovered the presence of ticks on the outside of CDC-LTs, recognizing that this was a novel occurrence, we carefully reviewed and modified our field protocol to ensure that our observance of host-seeking ticks in the forest canopy was not an artifact of our field methods. To wit, each morning when the traps were lowered, field members, including the senior author, first checked carefully for the presence of ticks on the exterior of each CDC-LT. This was done while the trap was suspended. Any ticks were immediately removed and placed in ethanol and labeled with appropriate metadata (date, trap number, etc.). Subsequently, the netting containing mosquitoes was secured in a plastic box for processing in the indoor laboratory space of Barro Colorado Island. The umbrella and the cylinder containing the fan mechanism of each trap were also taken back to the lab, but the igloo cooler was sealed in a white garbage bag and re-suspended in the mid-canopy (free from by-passers and foliage) during the day. In the evening, CDC-LTs were carried pre-assembled in Rubbermaid-style plastic boxes to the field and were quickly re-assembled in each field site, with loading of the solid CO2 as the final step. At no time were either canopy or understory CDC-LTs placed on the ground while they were being serviced in the field.\" \"The suppositions advanced in this account can only stem from ignorance regarding ticks, their biology, behavior, and even the standard terminology. It is usually advisable to consider simple explanations of unexpected results prior to making “astounding” claims.\"We would like to emphasize that the entire point of F1000Research observational articles is that they are precisely designed to document unexpected results that may improve our understanding of the natural world:“Science’s most important breakthroughs, from the discovery of microorganisms to the theory of evolution, have come about through observation. As part of the scientific method, observations are made to record a fact or an occurrence to help either prove or disprove a hypothesis. However, we all know the unpredictable nature of science and the curve balls it can throw” (See more at: http://blog.f1000research.com/2013/09/12/the-observation-article-recording-phenomena-in-scientific-research/#sthash.QP2xbUJj.dpuf )Our observations are robust and repeatable. As a research team working at one of the world’s oldest tropical research institutions, we are acutely aware of just how much remains to be discovered in our tropical forests, especially in the extremely inaccessible tropical forest canopy. Given that nymphs and larvae of A. tapirellum typically cannot be identified to species in the field (e.g. Bermúdez et al. 2010), and that a Google Scholar search retrieves only 27 articles (including ours) when searching for “Amblyomma tapirellum”, certainly Dr. Levin is not suggesting that our scientific knowledge of the life history of this and other Neotropical ticks species is complete. Fortunately, third party teams are currently actively working in Panama to more fully document the frequency that host-seeking ticks exploit the tropical canopy and confirm our finding. We are grateful for the opportunity given to us by F1000Research to publish our observation that has encouraged those efforts."
}
]
},
{
"id": "2289",
"date": "08 Nov 2013",
"name": "Matias Pablo Juan Szabó",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe phenomenon presented by Jose Loaiza and collaborators is astonishing by several criterions and thus should be carefully evaluated to dispel any doubt. The mobility described for Amblyomma tapirellum is atypical and the explanations rather speculative (which is acceptable considering the absolute lack of knowledge on the matter, as clearly stated by authors). At the same time, finding 48 adult ticks (I will focus on the stage found in the highest numbers) on different occasions in different light traps suspended 20m above the ground indicates a standard. One has to consider that for regular access to traps, adult ticks either climbed trees by themselves, fed on an arboreal host and moulted to adults on trees, were taken to the trees as adults by hosts (monkeys) and detached from them or had access to the traps during handling. Apart from the last possibility all others are worthwhile considering and deserve further research. In fact, movement from tree to suspended mosquito trap is, by itself, a challenge for ticks. I am not acquainted with such traps but it seems from the picture that it is held by a string or something similar and thus ticks would have to crawl on a variable length of string to reach the trap. Regardless, before further speculation, it is essential to dismiss trap infestation during handling through review, and careful description of field procedures. Considering the uncommon finding and its ecological importance such information is of paramount importance for the article.According to the authors, this tick species is quite prevalent on the ground or on ground vegetation. For this reason, I strongly recommend describing the handling of traps in detail. Were they mounted /examined/maintained on the ground at any moment? By personal experience, it is common to find ticks crawling on boxes used to carry dry ice (CO2) for tick traps. After finding the first specimens in the traps was any specific care taken to avoid accidental access of ticks to the traps? Were there any differences in the handling of traps in the canopy in comparison to those closer to the ground?Should these doubts be eliminated in the text, the manuscript will be acceptable for further discussion and publication.",
"responses": [
{
"c_id": "673",
"date": "20 Jan 2014",
"name": "Jose Loaiza",
"role": "Author Response",
"response": "\"According to the authors, this tick species is quite prevalent on the ground or on ground vegetation. For this reason, I strongly recommend describing the handling of traps in detail. Were they mounted/examined/maintained on the ground at any moment? By personal experience, it is common to find ticks crawling on boxes used to carry dry ice (CO2) for tick traps. After finding the first specimens in the traps was any specific care taken to avoid accidental access of ticks to the traps? Were there any differences in the handling of traps in the canopy in comparison to those closer to the ground?\"We appreciate Dr. Szabó’s concerns, and as we mentioned above, we apologize for not providing greater detail in our description of our field methods. We hope that the added text will sufficiently describe our field techniques. As we point out, independent confirmation of our surprising finding is on-going in Panama.We have taken advantage during this revision to fix spelling and grammatical errors in the text and figures:In Figure 2, Amblyomma gaeyi was replaced by Amblyomma geayi"
}
]
}
] | 1
|
https://f1000research.com/articles/2-194
|
https://f1000research.com/articles/3-26/v1
|
27 Jan 14
|
{
"type": "Case Report",
"title": "Case Report: An unusual clinical presentation of Kikuchi’s disease: a case report",
"authors": [
"Singaram A.",
"Ramesh Menon P.",
"Cherian N. C.",
"Geetha P.",
"Rajesh T. V.",
"Ramesh Menon P.",
"Cherian N. C.",
"Geetha P.",
"Rajesh T. V."
],
"abstract": "Introduction Kikuchi’s disease is usually a benign condition presenting with fever and lymphadenopathy. Rarely, the severe form of this disease can have an unusual presentation. Case Report We report a case of 9 year old girl, a resident of Kerala (South India) with no significant past medical history, who presented with fever of 8 days with facial puffiness. She had tender cervical lymphadenopathy. Two days after admission, her condition worsened with venous congestion of the face and neck, throbbing headache, dyspnea and she developed seizures. Very soon, she developed pericarditis with cardiogenic shock and required adrenaline support. Lymph node biopsy confirmed Kikuchi’s disease. The possibility of superior vena cava (SVC) obstruction due to mediastinal lymphadenopathy was considered and treated with steroids following which the child showed a dramatic improvement. The child also had four of the American Rheumatism Association (ARA) diagnostic criteria for systemic lupus erythematosus (SLE) (viz., serositis, seizures, hematological abnormalities and antinuclear antibodies (ANA) positivity. Conclusion Although Kikuchi’s disease is a self-limiting condition, it is important to consider this possibility in any patient who fits the typical clinical scenario to avoid unnecessary workup. An unusually severe form of Kikuchi’s disease can have a presentation like SVC obstruction syndrome due to enlarged mediastinal lymph nodes. Hence, timely recognition of this condition and prompt institution of steroid therapy will result in a dramatic clinical response.",
"keywords": [
"blood",
"lymph nodes",
"fevers"
],
"content": "Introduction\n\nKikuchi’s disease is a rare disease that was originally reported in patients of Asian heritage. It usually occurs in 8–16 year old aged children and presents with fever accompanied with lymphadenopathy especially in posterior cervical region. The diagnosis is made through lymph node biopsy1. Here we report a case of superior vena cava obstruction as an unusual presentation of Kikuchi’s disease in a 9 year old Indian girl.\n\n\nCase history\n\nA 9 year old girl presented with fever of 8 days duration with poor appetite, nausea and occasional headache. She was a previously healthy child with no significant past medical or family history. She had facial puffiness and swellings in the neck noted two days previously. There was no history of decreased urine output, vomiting, rash, arthralgia, breathlessness, skin or mucosal bleeds, significant weight loss or contact with a case of tuberculosis. On physical examination, she was febrile (102°F) and had marked periorbital edema, along with multiple, enlarged and tender lymph nodes in the posterior cervical region with the largest node measuring 3 cm. There was no rash, bleeding manifestations or enlargement of other groups of lymph nodes. Examination of the gastrointestinal, respiratory, cardiovascular and nervous systems was normal. Investigations revealed the following: hemoglobin 10.1 g/dL, white blood cell (WBC) count 2760 cells/mm (neutrophils-55%, lymphocytes-41%) with platelet count of 82,000 cells/cu.mm, erythrocyte sedimentation rate (ESR) – 50 mm/hr and C-reactive protein (CRP) – 44 mg/dL. Renal and liver function tests were normal. Blood Widal and Dengue NS1 antigen tests were negative. She was started on cefotaxim (1 g intravenously q8 hourly). The patient’s condition worsened on the second day of admission with progressive dyspnea and increasing facial puffiness and she also complained of a throbbing headache. She had venous congestion of the head and neck region with conjunctival congestion. Since chest X-ray showed mediastinal widening, the possibility of enlarged mediastinal lymph nodes causing SVC obstruction was considered and she was scheduled for a CT chest examination but she developed multiple seizures at that time. Repeat investigations on the same day (day 2 of admission) showed a fall in hemoglobin (9.6 g/dL) and platelets (67,000 cells/mm3) from the baseline. Lumbar puncture findings were normal. Two hours later, she also developed pericarditis with pericardial effusion, went into cardiogenic shock and required adrenaline support (0.5 mcg/Kg/min continuous intravenous infusion). A cervical lymph node biopsy done showed necrosis with karyorrhexis, histiocytic infiltrates, crescentic plasmacytoid monocytes, and absence of neutrophils – a picture consistent with Kikuchi-Fujimoto disease. Her blood, CSF, urine cultures were sterile. Bone marrow aspiration studies were normal. Investigation also showed ANA-IF – 3+ positivity and anti-dsDNA was strongly positive. Epstein-Barr virus (EBV) DNA PCR was negative. In view of the child fulfilling four of the ARA criteria for systemic lupus erythematosus (SLE) (viz., serositis, seizures, hematological abnormalities and ANA positivity)8, a diagnosis of SLE with Kikuchi disease presenting as superior vena cava syndrome was made and was treated with intravenous methylprednisolone (600 mg over 30 minutes) for 3 days. The patient showed a dramatic improvement with treatment, both clinically as well as with laboratory parameters. Her general condition improved, congestion of the face and neck decreased, the lymph nodes decreased in size and she became afebrile and remained so thereafter. After 3 days of intravenous methylprednisolone, she was changed over to oral prednisolone (1 mg/Kg/day) and oral hydroxychloroquine (5 mg/Kg/day). She was discharged after 1 week with advice to continue prednisolone and hydroxychloroquine (at the same dose). At the time of follow up (after 2 weeks) she remained asymptomatic, blood count and disease activity were normal and she did not develop any features of steroid toxicity and had no evidence of adrenal insufficiency.\n\n\nDiscussion\n\nTo the author’s knowledge, this is the first report of SVC obstruction as a clinical presentation of Kikuchi’s disease (due to enlarged mediastinal lymph nodes).\n\nThe etiology of Kikuchi’s disease is not entirely known. It has been linked to sequelae of infection by human herpes virus 6 (HHV-6), cytomegalovirus (CMV), EBV and even human T lymphotropic virus 1 (HTLV-1)2–4. Serological tests conducted with our patient ruled out the possibility of an associated infection. The most common clinical presentation of Kikuchi's disease is fever and cervical lymphadenopathy in a previously healthy young woman5. In a literature review describing 244 patients affected by Kikuchi’s disease, the common presentations were fever, fatigue, and joint pain with cervical lymphadenopathy, leucopenia, elevated ESR, and anemia6. Our patient also had prolonged fever with constitutional symptoms and cervical lymphadenopathy with raised ESR.\n\nOur patient also presented with features of SLE (she fulfilled four of the ARA criteria for SLE viz., serositis, seizures, hematological abnormalities and ANA positivity). According to Kucukardali et al.6, SLE-associated Kikuchi’s disease is more common in cases from Asian countries than from Europe. Among the 28 cases studied by Kucukardali et al., 18 cases presented with both SLE and Kikuchi’s disease diagnosed simultaneously, 6 cases were diagnosed with SLE after being diagnosed with Kikuchi’s disease and 4 were previously diagnosed with SLE6. The clinical features of SLE and Kikuchi’s disease are very similar and definite discrimination between them is based on histopathological findings7. The absence of hematoxylin bodies and paucity of neutrophils indicate Kikuchi’s disease rather than SLE and our patient showed definite features of Kikuchi’s disease in the lymph node biopsy.\n\n\nConclusion\n\nKikuchi’s disease should be considered in any child, especially from Asian heritage, who presents with the typical clinical features, as prompt diagnosis of this condition will avoid further unnecessary investigations. An unusually severe form of Kikuchi’s disease can have a presentation similar to SVC obstruction syndrome due to enlarged mediastinal lymph nodes. Hence timely recognition of this condition and prompt institution of steroid therapy will result in a dramatic clinical response and a life saving measure.\n\n\nConsent\n\nWritten informed consent for publication of clinical details was obtained from the child’s parents.",
"appendix": "Author contributions\n\n\n\nDr. Singaram A. wrote the manuscript. Dr. Ramesh Menon P. revised the first draft. Dr. Cherian N.C. and Dr. Geetha .P. managed the patient in the pediatric ward. Dr. Rajesh T.V. managed the patient in the intensive care unit. All authors were involved in the revision of the manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nAcknowledgements\n\nThe authors would like to acknowledge Dr. Arun Preeth, Pediatric surgeon for his timely help in conducting the lymph node biopsy in this patient which helped us to confirm the diagnosis.\n\n\nReferences\n\nDorfman RF: Histiocytic necrotizing lymphadenitis of Kikuchi and Fujimoto. Arch Pathol Lab Med. 1987; 111: 1026–1029. PubMed Abstract\n\nLopez C, Oliver M, Olavarria R, et al.: Kikuchi-Fujimoto necrotizing lymphadenitis associated with cutaneous lupus erythematosus: a case report. Am J Dermatopathol. 2000; 22(4): 328–33. PubMed Abstract | Publisher Full Text\n\nMartinez-Vazquez C, Hughes G, Bordon J, et al.: Histiocytic necrotizing lymphadenitis, Kikuchi-Fujimoto’s disease, associated with systemic lupus erythematosus. QJM. 1997; 90(8): 531–3. PubMed Abstract | Publisher Full Text\n\nSpies J, Foucar K, Thompson CT, et al.: The histopathology of cutaneous lesions of Kikuchi’s disease (necrotizing lymphadenitis): a report of five cases. Am J Surg Pathol. 1999; 23(9): 1040–7. PubMed Abstract\n\nDorfman RF, Berry GJ: Kikuchi's histiocytic necrotizing lymphadenitis: an analysis of 108 cases with emphasis on differential diagnosis. Semin Diagn Pathol. 1988; 5(4): 329–345. PubMed Abstract\n\nKucukardali Y, Solmazgul E, Kunter E, et al.: Kikuchi-Fujimoto Disease: analysis of 244 cases. Clin Rheumatol. 2007; 26(1): 50–54. PubMed Abstract | Publisher Full Text\n\nKim SK, Kang MS, Yoon BY, et al.: “Histiocytic necrotizing lymphadenitis in the context of systemic lupus erythematosus (SLE): is histiocytic necrotizing lymphadenitis in SLE associated with skin lesions?” Lupus. 2011; 20(8): 809–819. PubMed Abstract | Publisher Full Text\n\nHochberg MC: “Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus”. Arthritis Rheum. 1997; 40(9): 1725. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "4854",
"date": "30 May 2014",
"name": "Stelios Assimakopoulos",
"expertise": [],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe histology of the lymph node in Kikuchi's disease can usually be easily differentiated from most known infectious conditions in the differential diagnosis of lymphadenopathy, but not from that seen in some patients with systemic lupus erythematosus (SLE). Even if we accept the histological diagnosis based on lymph node pathology, this does not necessarily explain the whole clinical picture if other more probable diagnoses have not been ruled out.Beyond the fact that the child fulfilled four of the American Rheumatism Association (ARA) diagnostic criteria for systemic lupus erythematosus (SLE), they tested positive for anti-double-stranded DNA (dsDNA) antibodies, which are highly specific for SLE.The diagnosis of superior vena cava syndrome was based on clinical manifestations (dyspnea, facial puffiness, throbbing headache and venous congestion of the head and neck region with conjunctival congestion). However, chest CT was not performed. Considering the fact that the child was definitely suffering by cardiac tamponade, which could also be manifested with dyspnea and congestion of face veins, I am not persuaded about the co-existence of SVC obstruction. In this specific clinical text this diagnosis should have been confirmed by chest CT.In conclusion, after reading this case I was rather convinced that it was a case of SLE complicated by cardiac tamponade, effectively responding to corticosteroid treatment.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-26
|
https://f1000research.com/articles/3-25/v1
|
24 Jan 14
|
{
"type": "Case Report",
"title": "Lentigo maligna mimicking invasive melanoma in Mohs surgery: a case report",
"authors": [
"Teresa Tsakok",
"Nisith Sheth",
"Alistair Robson",
"Catherine Gleeson",
"Raj Mallipeddi",
"Nisith Sheth",
"Alistair Robson",
"Catherine Gleeson",
"Raj Mallipeddi"
],
"abstract": "Lentigo maligna is a lentiginous proliferation of atypical melanocytes confined to the epidermis, typically on chronically sun-damaged skin. Following biopsy and exclusion of invasive disease, therapy may involve Mohs surgery, topical treatment or radiotherapy. However, lentigo maligna often involves adnexal structures, creating histological difficulty in distinguishing these foci from invasive melanoma. We present a case in which, during Mohs excision, a nodule of severely atypical melanocytes appeared to lie within the dermis, potentially altering treatment and prognosis. The use of laminin-5 provided a means of resolving this diagnostic dilemma, facilitating continuation of Mohs surgery until tumour clearance was achieved.",
"keywords": [
"Lentigo maligna is a form of melanoma in-situ that may be present in up to 1.17% of Caucasians aged 65–74 years1. The most significant risk factor is lifetime UV radiation exposure",
"accordingly",
"lentigo maligna often presents as an irregularly pigmented macule at sun-damaged sites in elderly individuals – particularly the head and neck. Left untreated",
"reported rates of progression to invasive melanoma vary between 2.2–50%2",
"and the prognosis of invasive disease is similar to that of other melanoma types after adjusting for tumour thickness3. However",
"controversy remains regarding the best therapeutic modality for lentigo maligna."
],
"content": "Introduction\n\nLentigo maligna is a form of melanoma in-situ that may be present in up to 1.17% of Caucasians aged 65–74 years1. The most significant risk factor is lifetime UV radiation exposure; accordingly, lentigo maligna often presents as an irregularly pigmented macule at sun-damaged sites in elderly individuals – particularly the head and neck. Left untreated, reported rates of progression to invasive melanoma vary between 2.2–50%2, and the prognosis of invasive disease is similar to that of other melanoma types after adjusting for tumour thickness3. However, controversy remains regarding the best therapeutic modality for lentigo maligna.\n\n\nReport\n\nA 76 year old British seaman presented in April 2010 with an unevenly pigmented brown macule on the left side of his neck, measuring 31 × 26 mm. This was at the site of a previous lentigo maligna that had been excised with a 5 mm margin.\n\nA diagnostic biopsy showed a lentiginous proliferation of atypical melanocytes tracking down adnexae, confirming recurrence. The patient was referred for Mohs surgery.\n\nIn addition to lentigo maligna in the epidermis, one Mohs section revealed a large nodule of severely atypical melanocytes, apparently lying mid-dermis.\n\nThis presented a significant interpretational problem. Lentigo maligna commonly involves hair follicle epithelia; thus, the nodule may reflect complete replacement of the follicular epithelial cells still confined by basement membrane (in-situ disease). Alternatively, it could represent a focus of invasive melanoma, in which case Mohs surgery would have to be aborted.\n\nTo clarify this important distinction, immunocytochemical staining was performed for laminin-5 (Dako, Z0097, 1:100), a component of basement membrane. This revealed immunopositivity of the basement membrane of the follicular epithelium, confirming that the atypical melanocytes were confined to the hair follicle.\n\nMohs surgery was therefore completed in a 2-stage, 10-section procedure. Following confirmation of negative margins, the defect measured 45 × 35 mm – substantially larger than the original lesion. This was closed using a rhombic transposition flap, which healed well. To date there has been no recurrence.\n\n\nDiscussion\n\nLentigo maligna presents several challenges in terms of diagnosis and management. Firstly, clinical and dermoscopic features may be strikingly similar to solar lentigo4. It is also prone to significant subclinical extension beyond the visible limits of the tumour. Following initial tumour debulk, single atypical melanocytes scattered irregularly along the dermo-epidermal junction may be the only histological feature of residual lentigo maligna5. This difficulty is compounded by the presence of single mildly atypical melanocytes in chronically sun-damaged skin6. Finally, lentigo maligna often spreads into deep adnexal structures, confounding both diagnosis and treatment6.\n\nThere are various management options for lentigo maligna. Excision is accepted as the gold standard and can consist of conventional excision with a 5 mm margin, or some form of Mohs surgery. Other therapies are destructive, including radiotherapy and imiquimod (Aldara), a topical immunomodulator.\n\nAn evidence-based comparison of treatment modalities supports Mohs surgery as first-line, with recurrence rates as low as 0.3% compared to up to 20% for standard excision6. The follow-up periods of these trials varied from 18 months to almost five years, but it is notable that the most favourable study for Mohs surgery included 625 patients and measured recurrence over 58 months7.\n\nThe impressive statistics for Mohs surgery are in part attributable to the superior histological margin control afforded by this technique. Traditional surgical excision uses ‘bread-loafing’ to check for residual tumour, but this allows visualization of less than 1% of the margin. By contrast, in Mohs surgery, tissue is excised tangentially before being processed into horizontal sections. This method – combined with precise mapping and orientation of tissue by the Mohs surgeon reading the pathology – allows all tumour extensions to be traced. The end benefits are two-fold: recurrence rates are significantly improved, as tumour clearance is optimised; and healthy tissue is conserved, preserving cosmesis and function – important considerations on the face. The significance of visualising the entire tumour is underscored by evidence showing that almost 25% of biopsy-proven melanoma in-situ lesions are found to contain invasive melanoma upon pathological examination after complete surgical removal6.\n\nFollicular involvement by lentigo maligna is a widely recognised phenomenon, and is a characteristic of lentiginous spread. Indeed, a key dermoscopic feature of lentigo maligna is asymmetrical pigmentation of follicular openings, reflecting uneven descent of melanoma cells into individual hair follicles. Over time the slate-grey dots and streaks surrounding the follicle progress into a rhomboidal pattern, and then to eventual obliteration of the follicle, visible on dermoscopy as black blotches highly specific for malignant growth.\n\nFollicular melanoma has been reported as a rare variant of melanoma, to be distinguished from lentigo maligna melanoma by its significantly smaller surface area (typically < 0.5 cm), the relative symmetry of the lesion and its similarity in appearance to a comedo or pigmented cyst8. A recent study investigating the growth characteristics of melanoma in-situ in relation to hair follicle microanatomy has suggested that the follicle itself represents a physiological barrier, restricting the intraepithelial spread of melanoma tumour cells at or beyond the level of the hair follicle bulge9.\n\nThe present case posed an interesting diagnostic dilemma – the difficulty in distinguishing between dermal invasion and melanoma in-situ within a hair follicle. To resolve this, we used immunocytochemistry for laminin-5 to delineate normal basement membrane surrounding the hair follicle, indicating in-situ disease and therefore justifying continuation of the Mohs procedure.\n\nLaminin-5 is a component of the adhesion complex in basement membrane10. It is thought to act as a ligand for the attachment of normal melanocytes to the basement membrane. Conversely, loss of laminin production by melanoma cells is thought to be a marker for malignant transformation10. To our knowledge, the use of laminin-5 staining during Mohs has not been previously reported.\n\n\nLearning points\n\nLentigo maligna presents significant challenges in diagnosis and treatment\n\nMohs surgery can be effective as long as disease remains in-situ, but this may be difficult to ascertain\n\nAlmost 25% of biopsy-proven melanoma in-situ lesions are upstaged to invasive melanoma upon pathological investigation, highlighting the importance of visualising the entire tumour\n\nSurgeons using the Mohs technique require a thorough understanding of histopathology using horizontal sections to achieve meaningful margin control\n\n\nConsent\n\nWritten informed consent for publication of clinical details and clinical images was obtained from the patient.",
"appendix": "Author contributions\n\n\n\nNS provided the clinical images, AR provided the histopathology, TT prepared the first draft of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nWeinstock MA, Sober AJ: The risk of progression of lentigo maligna to lentigo maligna melanoma. Br J Dermatol. 1987; 16: 303–10. PubMed Abstract\n\nWayte DM, Helwig EB: Melanotic freckle of Hutchinson. Cancer. 1968; 21: 893–911. PubMed Abstract\n\nSmalberger GJ, Siegel DM, Khachemoune A: Lentigo maligna. Dermatol Ther. 2008; 21(6): 439–46. PubMed Abstract | Publisher Full Text\n\nCohen LM: Lentigo maligna and lentigo maligna melanoma. J Am Acad Dermatol. 1995; 33(6): 923–936 quiz 937–40. PubMed Abstract | Publisher Full Text\n\nBarlow RJ, White CR, Swanson NA: Mohs' micrographic surgery using frozen sections alone may be unsuitable for detecting single atypical melanocytes at the margins of melanoma in-situ. Br J Dermatol. 2002; 146(2): 290–4. PubMed Abstract | Publisher Full Text\n\nDawn ME, Dawn AG, Miller SJ: Mohs surgery for the treatment of melanoma in-situ: a review. Dermatol Surg. 2007; 33(4): 395–402. PubMed Abstract | Publisher Full Text\n\nBricca GM, Brodland DG, Ren D, et al.: Cutaneous head and neck melanoma treated with Mohs micrographic surgery. J Am Acad Dermatol. 2005; 52: 92–100. PubMed Abstract | Publisher Full Text\n\nHantschke M, Mentzel T, Kutzner H: Follicular malignant melanoma: a variant of melanoma to be distinguished from lentigo maligna melanoma. Am J Dermatopathol. 2004; 26(5): 359–63. PubMed Abstract\n\nPozdnyakova O, Grossman J, Barbagallo B, et al.: The hair follicle barrier to involvement by malignant melanoma. Cancer. 2009; 115(6): 1267–75. PubMed Abstract | Publisher Full Text\n\nScott GA, Cassidy L, Tran H, et al.: Melanocytes adhere to and synthesize laminin-5 in vitro. Exp Dermatol. 1999; 8(3): 212–21. PubMed Abstract"
}
|
[
{
"id": "3331",
"date": "10 Feb 2014",
"name": "Eduardo Nagore",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nA nice case report - I have no comments.",
"responses": []
},
{
"id": "4667",
"date": "30 May 2014",
"name": "Rajiv Kumar",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nWell defined report. I have no further comments.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-25
|
https://f1000research.com/articles/2-274/v1
|
16 Dec 13
|
{
"type": "Short Research Article",
"title": "Cladonia lichens on extensive green roofs: evapotranspiration, substrate temperature, and albedo",
"authors": [
"Amy Heim",
"Jeremy Lundholm",
"Jeremy Lundholm"
],
"abstract": "Green roofs are constructed ecosystems that provide ecosystem services in urban environments. Shallow substrate green roofs subject the vegetation layer to desiccation and other environmental extremes, so researchers have evaluated a variety of stress-tolerant vegetation types for green roof applications. Lichens can be found in most terrestrial habitats. They are able to survive extremely harsh conditions, including frequent cycles of desiccation and rehydration, nutrient-poor soil, fluctuating temperatures, and high UV intensities. Extensive green roofs (substrate depth <20cm) exhibit these harsh conditions, making lichens possible candidates for incorporation into the vegetation layer on extensive green roofs. In a modular green roof system, we tested the effect of Cladonia lichens on substrate temperature, water loss, and albedo compared to a substrate-only control. Overall, the Cladonia modules had significantly cooler substrate temperatures during the summer and significantly warmer temperatures during the fall. Additionally, the Cladonia modules lost significantly less water than the substrate-only control. This implies that they may be able to benefit neighboring vascular plant species by reducing water loss and maintaining favorable substrate temperatures.",
"keywords": [
"Green roofs are constructed ecosystems",
"designed to provide ecosystem services such as the reduction of heat flux through the roof",
"the capture of storm water",
"and the provision of habitat for animals1",
"2. Green roofs consist of a vegetation and growing medium layer (substrate) over engineered layers that provide a root barrier",
"drainage",
"and/or water retention layers1. The majority of green roofs constructed in temperate climates are “extensive” green roofs",
"characterized by shallow growing media (<20cm)",
"which minimizes the weight added to the building. Shallow growing media in such systems have led to a reliance on succulent plant species in the vegetation layer",
"to ensure survival during drought conditions. Other plant growth forms have also been used on extensive green roofs",
"with grasses",
"forbs",
"and mosses among the most frequent3",
"4. Lichens are often found on conventional roof surfaces5 and form an important component of cryptogamic crusts in terrestrial ecosystems6. Cryptogamic crusts in arid environments perform a range of ecosystem services",
"such as stabilizing soil",
"fixing nitrogen",
"and enhancing soil water holding capacity7",
"but have not been intentionally planted in green roof ecosystems."
],
"content": "Introduction\n\nGreen roofs are constructed ecosystems, designed to provide ecosystem services such as the reduction of heat flux through the roof, the capture of storm water, and the provision of habitat for animals1,2. Green roofs consist of a vegetation and growing medium layer (substrate) over engineered layers that provide a root barrier, drainage, and/or water retention layers1. The majority of green roofs constructed in temperate climates are “extensive” green roofs, characterized by shallow growing media (<20cm), which minimizes the weight added to the building. Shallow growing media in such systems have led to a reliance on succulent plant species in the vegetation layer, to ensure survival during drought conditions. Other plant growth forms have also been used on extensive green roofs, with grasses, forbs, and mosses among the most frequent3,4. Lichens are often found on conventional roof surfaces5 and form an important component of cryptogamic crusts in terrestrial ecosystems6. Cryptogamic crusts in arid environments perform a range of ecosystem services, such as stabilizing soil, fixing nitrogen, and enhancing soil water holding capacity7, but have not been intentionally planted in green roof ecosystems.\n\nLichens are symbiotic organisms, an association between a fungus (the mycobiont) and one or more algal and/or cyanobacterial photobionts8,9. Typically the mycobiont forms 95% of the lichen body. They are the dominant plant life in harsh environments such as the Arctic, Antarctic, mountains, and dry land crusts8. Lichen species are widespread and can be found from the Arctic to deserts; they can survive frequent cycles of desiccation and rehydration, nutrient-poor soil, fluctuating temperatures, and UV light intensities8,10. They can survive and grow on the bare surface of rocks and in poor soils such as heathlands, peat lands, sand dunes, and toxic spoil heaps10. Selection of vegetation types appropriate for extensive green roofs often involves identifying local habitats that have characteristics in common with green roofs (shallow soil, harsh abiotic conditions)11; lichens are a common component of rock barrens, dunes, and heathland habitats that can be a source of plants for green roofing projects12,13.\n\nLichens are lightweight and can be found growing naturally on bare tile or slate rooftops1. This could make them a candidate for roofs on buildings with low weight-loading capabilities. Species of the genus Cladonia, large fruticose lichens that colonize bare soils, are common in cold temperate climates. These lichens produce bundles of hyphae that stabilize the soil and add organic matter. The light color of the lichen can reflect solar radiation, keeping the soil cool and moist14. These characteristics indicate the possible utility of Cladonia as a component of extensive green roofs. In this study, the performance of key green roof ecosystem functions in green roof modules planted with live Cladonia lichens was compared with substrate-only controls.\n\n\nMethods\n\nThe study site was located on the east side of the roof of the five-story Atrium building at Saint Mary’s University in Halifax, Nova Scotia, Canada (44°39′N, 63°35′W ) (Figure 1). Data on soil temperature and water loss were collected between July and October 2012. Albedo was measured in September 2013. During the 2012 study period, the weather station on the adjacent green roof testing facility (~50m away from the study site) recorded the minimum monthly temperature as 6.7–20.7°C and the monthly maximum as 12–30°C. The monthly precipitation recorded from the green roof weather station averaged between 1.7 and 11.59mm. Albedo was collected on September 28, 2013 (average temperature: 14.12°C, total precipitation: 0mm).\n\nIn order to quantify the influence of Cladonia on green roof substrate, a trial was set up to determine the effects of Cladonia on soil temperature, water loss, and albedo. Ten green roof modules were placed on the Atrium roof on top of the roof surface, which was made up of grey concrete pavers. Each module had a length and width of 36cm, with a freely draining base (Polyflat®, Stuewe & Sons Inc., Oregon, USA). Modules contained a root barrier/water retention fleece (length and width 36cm) over the base (EnkaRetain and Drain 3111®, Colbond Inc., North Carolina, USA) with 6cm depth of Sopraflor X substrate, purchased in 2011 (Soprema Inc., Drummondville, QC, Canada), over the root barrier/water retention layer. A soil test describing the composition of the Soprema X substrate can be seen in Supplementary Table 1. This experiment consisted of two substrate-only controls and eight modules covered 100% in Cladonia lichen approximately 6cm thick (Figure 2). The lichen was collected from a coastal barrens site (Chebucto Head (44°30′N, 63°31′W)) in May 2012 and placed on the surface of the substrate. A mix of two lichen species, C. terranova and C. boryi, was used; both species have similar colors and growth forms, and co-occur in lichen mats. Lichens were alive when transplanted and shoot tips were marked to determine incremental growth over the experiment, but any such growth was undetectable and these results are not included here.\n\nSubstrate temperature was recorded using a Taylor 9878 Slim-Line Pocket Thermometer Probe (Commercial Solutions Inc., Edmonton, Alberta, Canada) once a month throughout the growing season. The temperature was recorded from the center of each module approximately 2cm below the substrate surface when exposed to full sun, no more than two hours from solar noon. Only one measurement was recorded for each module on the day of measurement. These spot measurements represented maximal substrate temperatures and were collected during hot sunny conditions to provide maximum contrast between controls and planted green roof modules. This technique does not characterize the long-term provisioning of thermal benefits but has been used to contrast performance of different plant species or vegetation types during hot conditions15. Volumetric water content of the substrate (VWC: water volume/soil volume × 100%) was recorded one day after a rain event and again one day later if no new showers were observed. Water loss was determined by subtracting the day one VWC from the day two VWC, which provides an index of net evapotranspiration over 24 hours15. This variable is also used as an index of the hydrological performance of green roof systems. Measurements were taken once in August, September, and October 2012. VWC was measured using a GS3 probe with the ProCheck soil moisture control unit (Decagon Devices Inc., Pullman, Washington, USA) inserted into the center of each module adjacent to the target species to a depth of 5cm. Separate two sample t-tests were used to compare water loss and substrate temperature between control and lichen treatments A p-value <0.05 was considered significant. All statistical analysis was analyzed using R project for statistical engineering version 3.0.1 (http://www.r-project.org/index.html).\n\nAlbedo was not originally part of this trial, it was added later in order to get a better understanding of how Cladonia lichen could affect green roof substrates; visually, the lichens were very similar throughout both 2012 and 2013. Albedo was measured on September 28, 2013. The information was gathered from five lichen modules (100% cover) from the previous season and five substrate only controls created by removing vegetation from the old lichen modules (due to wind damage, only five lichen modules could be salvaged). Albedo was measured with a single LI-COR pyranometer sensor and LI-250A light meter (LI-COR Biosciences, Lincoln, Nebraska, United States) attached to a fixed position 35cm above the soil surface. The incoming solar radiation (Wm-2) was measured by pointing the sensor 180° from the roof surface. The reflected radiation was measured by rotating the sensor 180° to face each module. Albedo was calculated by dividing the reflected radiation by the incoming radiation15, and a one-tailed t-test was used to compare the control and lichen modules. A p-value <0.05 was considered significant.\n\n\nResults\n\nThe albedo of the Cladonia modules was significantly higher than the control modules (Table 1, Data Set 1). Substrate temperatures for July and August were significantly lower in the Cladonia modules compared to the controls (Table 1). In September, there was no significant difference in soil temperature between the Cladonia modules and the controls. In October, the Cladonia modules had significantly higher substrate temperatures than the controls (Table 1, Data Set 2). Regarding water loss, there was no significant difference between the Cladonia modules and the control for August, but in September and October, Cladonia modules lost significantly less water than the controls (Table 1, Data Set 3).\n\nAn (*) indicates a significant difference (>0.05) between the substrate-only control and Cladonia treatment determined by a t-test.\n\nAlbedo was calculated by dividing the reflected radiation (Wm-2) by the incoming radiation (Wm-2).\n\n\nDiscussion\n\nCompared to the substrate-only controls, the Cladonia modules were cooler during hot conditions and retained more moisture in the substrate. These cooler temperatures may have been a result of shading and higher albedo. Even though albedo was only measured in 2013, the Cladonia lichen always had a lighter coloring than the bare substrate. Lighter colors are associated with higher albedo. Likewise, the lichen cover resulted in less net evapotranspiration compared with the bare substrate treatment. Interestingly, the soil temperature during October (average air temperature on date of measurement: 17.9°C) was significantly warmer in the Cladonia modules compared to the controls. This implies that a Cladonia mat may help reduce heat loss during the winter and help cool the roof during the summer.\n\nPlants surrounded by Cladonia may benefit from such temperature regulation (cooler temperatures in the summer, warmer temperatures in the winter) and greater water availability. Other studies have shown that plants with mat-forming growth can benefit less drought-tolerant species16. In addition to this, Cladonia lichens do not appear detrimental to plant growth. Vascular plant species growing out of these lichen mats is a natural occurrence on the coastal barrens of Nova Scotia and, during the trial, seedlings of trees and grasses were observed growing out of these modules, although control modules also had such seedling growth. Some lichen species also play a key role in soil development in conditions of high abiotic stress8,17. If lichen inclusion on a green roof could improve the substrate properties for neighboring species, the entire system would benefit.\n\nIn this study, while the lichens remained in the roof modules for the duration of the experiment, it is not clear that the lichens remained alive. More research is required to determine whether this method of transplanting lichens onto green roof substrates results in viable lichen populations. Further, our transplanting method involved sourcing lichens from the wild and while these species are very common in the region this kind of collection would clearly not be feasible on an industrial scale. Finally, while green roofs are becoming more popular in all regions, lichens, in varying degrees, are sensitive to air pollution and many species are likely to perform poorly where air quality is low14. Our study establishes that Cladonia lichens could provide roof cooling and water retention services in a green roof environment, but more work is necessary to explore the long-term viability of fruticose lichens in these systems.",
"appendix": "Author contributions\n\n\n\nA. Heim contributed to experimental design, collected the data, analyzed data, wrote the paper; J. Lundholm contributed to experimental design and wrote the paper. Both authors checked and agreed to the final publication of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research was funded by NSERC Discovery Grant 311788-2010 to J. Lundholm.\n\n\nAcknowledgments\n\nWe thank Emily Walker for critical edits on the manuscript.\n\n\nSupplementary material\n\n\n\n\nReferences\n\nDunnett N, Kingsbury N: Planting green roofs and living walls. Timber press: Portland, Oregon. 2004; 75. , 104–114. Reference Source\n\nGetter KL, Rowe DB: The Role of Extensive Green Roofs in Sustainable Development. Hortscience. 2006; 41(5): 1276. Reference Source\n\nEmilsson T, Kaj R: Comparison of establishment methods for extensive green roofs in southern Sweden. Urban Forestry and Urban Greening. 2005; 3(2): 103–111. Publisher Full Text\n\nDvorak B, Volder A: Green roof vegetation for North American ecoregions: A literature review. Landsc Urban Plan. 2010; 96(4): 197–213. Publisher Full Text\n\nRichardson DHS: The transplantation of lichen thalli to solve some taxonomic problems in Xanthoria parietina (L.) Th. Fr. The Lichenologist. 1967; 3: 386–391. Publisher Full Text\n\nEldridge DJ, Greene RSB: Microbiotic soil crusts: a review of their roles in soil and ecological processes in the rangelands of Australia. Australian Journal of Soil Research. 1994; 32: 389–415. Publisher Full Text\n\nBelnap J, Prasse R, Harper KT: Influence of biological soil crusts on soil environments and vascular plants. Belnap J and Lange OL (eds.) Biological Soil Crusts: Structure, Function, and Management. Springer-Verlag, Berlin. 2003; 281–300. Publisher Full Text\n\nBeckett RP, Zavarzina AG, Liers C: Oxidoreductases and cellulases in lichens: Possible roles in lichen biology and soil organic matter turnover. Fungal Biol. 2013; 117(6): 431–438. PubMed Abstract | Publisher Full Text\n\nScheidegger C, Werth S: Conservation strategies for lichens: insights from population biology. Fungal Biology Reviews. 2009; 23(3): 55–66. Publisher Full Text\n\nSeymour F, Crittenden D, Dyer P: Sex in the extremes: lichen-forming fungi. Mycologist. 2005; 19(2): 51–58. Publisher Full Text\n\nLundholm JT: Green roofs and facades: a habitat template approach. Urban Habitats. 2006; 4: 87–101. Reference Source\n\nClayden S, Bouchard A: Structure and dynamics of conifer-lichen stands on rock outcrops south of Lake Abitibi, Quebec. Can J Bot. 1983; 61(3): 850–871. Publisher Full Text\n\nOberndorfer EC, Lundholm JT: Species richness, abundance, rarity and environmental gradients in coastal barren vegetation. Biodivers Conserv. 2009; 18(6): 1523–1553. Publisher Full Text\n\nBrodo I, Sharnoff SD, Sharnoff S: Lichens of North America. Yale University Press: New Haven. 2001; 3, 9, 50. Reference Source\n\nMacIvor S, Lundholm J: Performance evaluation of native plants suited to extensive green roof conditions in a maritime climate. Ecol Eng. 2011; 37(3): 407–417. Publisher Full Text\n\nButler C, Orians CM: Sedum cools soil and can improve neighboring plant performance during water deficit on a green roof. Ecological Engineering. 2011; 37(11): 1796–1803. Publisher Full Text\n\nAsta F, Orry F, Toutain B, et al.: Micromorphological and ultrastructural investigations of the lichen–soil interface. Soil Biol Biochem. 2001; 33(3): 323–337. Publisher Full Text"
}
|
[
{
"id": "2795",
"date": "17 Dec 2013",
"name": "Richard Sutton",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe Title is appropriate, the Abstract is useful and the article clearly constructed. The experimental design also appears appropriate. I am pleased to see complete data collection information, enabling the experiment to be duplicated, however, although it is required as a part of the F1000Research format, the raw data could be more easily understood if graphed. I have the following comments about the Conclusion/Discussion section: The last part of last sentence in the Methods section should go into the Discussion and lead on to a more detailed discussion of the lichen’s growth (or death). This following sentence is unclear. It implies that the non-planted controls had seedling growth. How could this be possible if it was simply straight substrate? “[D]uring the trial, seedlings of trees and grasses were observed growing out of these modules, although control modules also had such seedling growth.” How would the Sopraflora-X get seeds? While beyond the scope of the study, could a line be added about transplanting/regrowth-rate/propagation? Those features have an important impact on the future use of lichens. In my experience, Lichen growth under “normal” conditions is very slow. The authors should talk more about the wind damage which might be an indication that the lichens have not attached themselves. This is important because most green roofs are very windy places. It is also worth noting that displaced lichen cover would function less effectively.",
"responses": []
},
{
"id": "2790",
"date": "23 Dec 2013",
"name": "Martin Hermy",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe title and abstract cover the contents of this original work well. The study design, methods and analysis seem appropriate. Overall I do agree with the conclusions. However, one crucial element should be mentioned (which is recognized by the authors).The fact that it is not sure that the lichens still were alive after the experiment puts considerable constraints about the applicability of these organisms in extensive green roof systems. My experience with lichens is that they normally grow on nutrient poor, dry, acidophilous soils. I've seen Cladonia's enter extensive green roof systems after a number of years (when substrate pH dropped). Green roof substrate used here (which is the standard) is indeed nutrient poor, but definitely not acid (see info). That makes the point whether these lichens are still alive or not even more important. One could eventually use dead lichen material and or other dead materials with similar properties (e.g. albedo), e.g. white pebbles.",
"responses": [
{
"c_id": "3019",
"date": "12 Sep 2017",
"name": "Jeremy Lundholm",
"role": "Author Response",
"response": "The lichens were alive after all. We have since observed the same species colonizing an adjacent green roof from fragments originating in the experiment described in this paper. (there are no other local sources of these species)"
}
]
}
] | 1
|
https://f1000research.com/articles/2-274
|
https://f1000research.com/articles/3-24/v1
|
23 Jan 14
|
{
"type": "Case Report",
"title": "Bilateral adrenal hemorrhage due to heparin-induced thrombocytopenia following partial nephrectomy – a case report",
"authors": [
"Ashley G. Winter",
"Ranjith Ramasamy",
"Ashley G. Winter"
],
"abstract": "Heparin-induced thrombocytopenia (HIT) can cause severe life-threatening events such as bilateral adrenal hemorrhage (BAH). A 48-year-old female developed a pulmonary embolus (PE) following partial nephrectomy. The anticoagulation treatment for her PE was complicated by HIT and subsequent BAH. To the author’s knowledge, this is the first reported case of HIT-associated BAH following renal surgery.",
"keywords": [
"bilateral adrenal hemorrhage",
"heparin-induced thrombocytopenia",
"HIT",
"partial nephrectomy"
],
"content": "Case presentation\n\nA 48-year-old Caucasian female with a past medical history of hypothyroidism presented to us with an incidentally discovered 3 cm enhancing right renal mass. Her home medications included levothyroxine and oral contraceptives. She underwent an uncomplicated robotic-assisted laparoscopic right partial nephrectomy. The patient received routine perioperative deep venous thrombosis (DVT) prophylaxis with sequential compression devices, (SCD). On postoperative day (POD) 2, she developed new-onset sinus tachycardia and showed depressed pulse-oximetry readings. As subsequent chest computed tomography (CT) scans demonstrated multiple large pulmonary emboli, she was started immediately on a continuous intravenous unfractionated heparin infusion, titrated to target an activated partial thromboplastin time (aPTT) of 64–97 seconds. The patient was transitioned to therapeutic anticoagulation with low-molecular-weight heparin (LMWH) at 1mg/Kg q12hrs on POD 3 and discharged the next day.\n\nOn postoperative day 6, she developed syncope and hypotension and was admitted to the hospital. Work up in the emergency room was remarkable for a hematocrit (HCT) of 19%, and a CT scan demonstrated a bleeding pseudoaneurysm in the nephrectomy bed. She responded to red blood cell transfusion (4 units) and embolization of the pseudoaneurysm, becoming normotensive with a HCT of 26.2%. Anticoagulation with LMWH at 1mg/Kg was resumed the next day. She was discharged from the hospital on POD 10.\n\nThe patient presented to the surgeon’s office on postoperative day 13 with progressive lethargy and subjective fevers. She was normotensive but tachycardic, and was admitted with concern for recurrent hemorrhage. Laboratory analysis at that time was remarkable for a leukocytosis to (16,000/µL), thrombocytopenia of 49,000/µL (from a pre-operative value of 234,000/µL), hyponatremia to 125mEq/L (baseline of 139mEq/L) and a relatively normal HCT of 31.7%. CT scan of the adrenal glands demonstrated bilateral adrenal gland enlargement and hyperdensity, consistent with BAH (Figure 1). Within 2 hours of initiating 50 mg intravenous hydrocortisone every 8 hours, her pulse decreased from 128 beats per minute (bpm) to 107bpm, and her blood pressure increase from 89/54mmHg 115/63mmHg. A percutaneous supra-renal inferior-vena-cava filter (IVCF) was placed by interventional radiology and an argatroban infusion started. The patient underwent careful titration to an outpatient regimen of fludrocortisone, hydrocortisone, and fondaparinux. She was discharged with a platelet count of 141,000/µL and serum sodium of 139mEq/L. After 12 months follow-up, the patient remains on non-heparin anticoagulation and steroid supplementation.\n\nCT scan of adrenal glands; white arrows indicate severely enlarged, hyperdense, adrenal glands bilaterally consistent with adrenal hemorrhage.\n\n\nDiscussion\n\nHeparin-induced thrombocytopenia is an immune-mediated complication occurring infrequently following exposure to unfractionated or low-molecular weight heparin. HIT occurs when a complex between IgG antibodies and heparin-platelet-factor-4 forms. The antibodies activate platelets by binding to the FcɣIIa receptor, which results in systemic thrombosis and consumptive thrombocytopenia1.\n\nWith their rich arterial supply and single central vein, the adrenal glands are particularly susceptible to congestive hemorrhage following venous thrombosis. Adrenal hemorrhage associated with HIT tends to be bilateral, resulting in severe clinical manifestations from abdominal pain, fever and lethargy, to catastrophic hemodynamic collapse2. In the post-surgical patient, these symptoms can be confused with more common complications, such as sepsis or hemorrhage. As undiagnosed cases tend to be fatal, a prompt recognition is crucial2.\n\nFew cases of HIT-associated BAH have been previously described in the literature2–4. Approximately 90% of the patients are post-surgical, with the most common operations being orthopedic. HIT may be an important cause of BAH, accounting for up to 10% of the cases4. To our knowledge, this is the first reported case of HIT-associated BAH following partial nephrectomy in particular, or urologic surgery in general. Our case highlights two critical points; 1) the diagnostic complexity of HIT-associated BAH, and 2) the possibility of a successful clinical outcome when rapid diagnosis is made.\n\n\nConsent\n\nWritten informed consent for publication of clinical details was obtained from the patient.",
"appendix": "Author contributions\n\n\n\nAshley Winter performed the case research and wrote the initial manuscript draft.\n\nRanjith Ramasamy performed manuscript edits and review.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe would like to acknowledge Joseph Del Pizzo for the referral of this patient.\n\n\nReferences\n\nNamias N: Chance favors the prepared mind: The association between heparin-induced thrombocytopenia and bilateral adrenal hemorrhage. Crit Care Med. 2011; 39(4): 912–3. PubMed Abstract | Publisher Full Text\n\nRosenberger LH, Smith PW, Sawyer RG, et al.: Bilateral adrenal hemorrhage: the unrecognized cause of hemodynamic collapse associated with heparin-induced thrombocytopenia. Crit Care Med. 2011; 39(4): 833–838. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChow VW, Abnousi F, Huddleston JI, et al.: Heparin-Induced Thrombocytopenia After Total Knee Arthroplasty, With Subsequent Adrenal Hemorrhage. J Arthroplasty. 2012; 27(7): 1413.e15–e18. PubMed Abstract | Publisher Full Text\n\nKetha S, Smithedajkul P, Vella A, et al.: Adrenal haemorrhage due to heparin-induced thrombocytopenia. Thromb Haemost. 2013; 109(4): 669–75. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "3326",
"date": "24 Jan 2014",
"name": "Ryan Smith",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have composed an interesting case report with a good review of the literature. Anticoagulation requirements before and after Urologic surgery have significant importance and can result in a myriad of post-operative complications. Prompt recognition of these is essential to prevent additional patient morbidity. HIT-associated BAH represents a less commonly seen complication following anticoagulation and it is important for surgeons to be aware of this possibility.",
"responses": []
},
{
"id": "3791",
"date": "24 Feb 2014",
"name": "Boback Berookhim",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors provide a report of an interesting clinical situation following robotic assisted partial nephrectomy. The difficulties of diagnosis and non-specific presentation of potentially life-threatening bilateral adrenal hemorrhage in a patient with previously diagnosed pseudoaneurysm are clearly spelled out. An excellent literature review is provided. This article serves as a reminder to the urologist of the untoward and life threatening potential effects of heparin induced thrombocytopenia, and the importance of prompt diagnosis and treatment of BAH.",
"responses": []
},
{
"id": "4138",
"date": "17 Mar 2014",
"name": "Joyce Popoola",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper highlights a relatively uncommon complication which could easily be overlooked if no thought is given to it.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-24
|
https://f1000research.com/articles/3-22/v1
|
22 Jan 14
|
{
"type": "Case Report",
"title": "Case report: congenital dislocation of the radial head –a two-in-one approach",
"authors": [
"Raju Karuppal",
"Anwar Marthya",
"Rajendran V Raman",
"Sandhya Somasundaran",
"Anwar Marthya",
"Rajendran V Raman",
"Sandhya Somasundaran"
],
"abstract": "Background: Congenital dislocation of the radial head of the elbow is rare. It is genetically transmitted in some cases and is often associated with syndromes, such as Nail-Patella syndrome, antecubital pterygium and ulnar dysplasia. About two thirds are posterior, with the remainder being either anterior (15%) or lateral (15%). The natural history of the condition is that symptoms are relatively benign, with only some limitation of motion and deformity. Treatment either involves early attempts at reconstruction or delayed intervention at skeletal maturity with radial head excision. We evaluated the radiographic and functional results of a two-in-one procedure (radial shortening and open reduction) in the treatment of congenital dislocation of the radial head of an eight year old girl.Objective: To describe a technique for easy reduction and maintenance of normal radiocapitellar joint anatomy in cases of congenital dislocation of the radial head.Method: We have introduced one modification to the Sachar’s method of open reduction by adding radial shortening. This can be described as a ‘two incision approach’ with the first incision for the radial shortening and the second for the open reduction of the radiocapitellar joint. The radial shaft was osteotomised first before we performed the radial head relocation. Then the overlapping part of radial shaft was trimmed. It was stabilized with a transarticular K wire fixation.Results: At one year follow up, the elbow is stable with no valgus or fixed flexion deformity. Supination has increased to 40 degrees from zero degrees. An X-ray showed reformation of the radial head with good congruity of the radiocapitellar joint and correction of the radial bow.Conclusion: As far as the authors are aware, this is the first report of congenital dislocation of the radial head being treated by radial shortening and open reduction of radiocapitellar joint through a two incision approach (two-in-one approach). This paper describes this new technique, which we implemented for easy reduction maintenance of normal radiocapitellar joint anatomy.",
"keywords": [
"Congenital dislocation of the radial head",
"Valgus deformity",
"Open reduction",
"Reconstruction",
"Two in one approach"
],
"content": "Report of a new method of treatment\n\nAn eight year old, otherwise healthy girl, born out of non consanguineous marriage presented in 2012 with swelling of her left elbow joint, valgus deformity, limitation of movement, and occasional pain. The patient had no previous history of trauma or significant illness. Her elbow was apparently normal until the age of 4 years. At this point her mother noticed swelling and progressing deformity. On physical examination the radial head was dislocated posterolaterally and was not reducible. There was cubitus valgus (30 degrees) and 15 degrees of fixed flexion deformity. She had full flexion at her elbow but no supination of her forearm.\n\nAn X-ray showed posterior dislocation of the radial head with a domed articular surface. McLaughlin’s line had not bisected the capitellum. The radius was bowed anteriorly and was relatively longer in relation to ulna. The capitulum was hypoplastic and flat. There was no evidence of any previous fracture (Figure 1). An MRI showed posterior dislocation of the radial head and formation of a pseudo-joint with the adjacent margin of the ulna (Figure 2). Due to relative radial lengthening and radial bow, it would have been difficult to reduce the radial head in the radiocapitellar joint. Hence we planned for reconstruction surgery by radial shortening and open reduction of the radiocapitellar joint through two incisions. Pre-operative templating was done to assess the amount of shortening required for the easy reduction and maintenance of adequate joint space.\n\nThe capitulum is hypoplastic.\n\nThe radial bow is corrected. The radial head is fixed with a transarticular pin.\n\n\nOperative technique\n\nThe novel two incision/two-in-one technique described here involves one modification to the Sachar’s method1 of open reduction by adding a radial shortening step. Under tourniquet, we performed the surgery through a two incision approach. The first incision on the anterior aspect of the proximal forearm was for radial shortening (Henry approach) and the second incision on the lateral part of the elbow was for open reduction of the radiocapitellar joint (Kocher’s approach). The proximal radial shaft was osteotomised first before we performed the radial head relocation to facilitate the reduction, which reduces the risk of osteonecrosis2. Then the overlapping part of radial shaft was trimmed with a bone nibbler, similar to the procedure of femoral shortening in the treatment of a dysplastic dislocated hip joint3. The radial shaft was stabilized by a transradiocapitellar fixation with a 1.8 mm K wire with the elbow in flexed position.\n\nPost-operatively, the elbow was immobilized with an above elbow plaster splint, in 90° of flexion, for six weeks at which point the K-wire was removed and the elbow was mobilized. The patient was further managed with controlled elbow movement exercises and physiotherapy for the next three months to improve the range of movement and muscle strength.\n\nAt one year follow up in 2013, the patient’s elbow was stable with no valgus or fixed flexion deformity. Supination had increased to 40 degrees from zero degrees. An X-ray showed reforming of the radial head articular surface with good congruity of the radiocapitellar joint with no deformity of the radial shaft (Figure 3).\n\n\nDiscussion\n\nCongenital dislocation of the radial head is the most common congenital elbow abnormality1 and usually occurs in association with other conditions (60% of the time), but can also occur in isolation4. The more common associated conditions include lower extremity anomalies, scoliosis, mental retardation, and nail-patella and Klippel-Feil syndromes5. The condition is usually bilateral, but some unilateral cases have been described5,6. When unaccompanied by other radial or systemic conditions, it is almost always bilateral. The majority of radial head dislocations are posterior (65% of cases), followed by anterior (~15%) and lateral (~15%)7. It is often not noted until the age of four or five at which time some limitation of motion or deformity becomes evident7. Our case is an isolated unilateral congenital dislocation of the radial head noted at the age of four years.\n\nIn our case, we anticipated difficulty in reducing the radial head to the radiocapitellar joint, due to relative radial lengthening and radial bow. Hence we planned for reconstruction surgery by radial shortening and open reduction of the radiocapitellar joint through two incisions. Pre-operative templating was done to assess the amount of shortening required for the easy reduction and maintenance of adequate joint space.\n\nThe dislocation of radial head and its associated features are now believed to be triggered by failure of development of a normal capitulum, which deprives the developing radial head of the contact pressure required for normal development and results in malformation of the radiocapitellar joint8. The symptoms are relatively benign, with only some limitation of motion and deformity9. Early radiographic findings are subtle due to the absence of the capitulum and radial head ossification centers9. Before radial head ossification (~ five years) a line drawn along the shaft of the radius should normally bisect the capitellum ossification center (McLaughlin’s line) but did not in this case10.\n\nMcFarland (1936)11 described the radiological signs which he believed distinguished the congenital from the traumatic in unilateral dislocations. However the convex radial head, flattening of the capitellum and anterior angulation of the ulna are regarded as characteristic of congenital dislocation are clearly seen as a result of the injury as well12.\n\nGenerally, patients become symptomatic by adolescence and are treated by radial head resection. Surgery at an earlier age with open reduction and ligament reconstruction may offer advantages over late radial head resection1. Early reconstruction may prevent the long term complication of pain, loss of motion, deformities and osteochondral loose bodies13. Ideally the care of congenital dislocation of the radial head would involve open reduction and restoration of normal anatomy. The logic is that if the radial head can be reduced early, the deformity of the capitellum and the forearm may not occur or remodel with growth13.\n\nWe agree with De Boeck14 that reconstruction of the annular ligament seems unnecessary, and that unreduced radial head dislocations, therefore, may be treated by simple open reduction and fixation for six weeks with a transarticular pin. This is why the annular ligament was not reconstructed in our case. Although much of the literature refers to the treatment of radial head dislocation by means of an ulnar osteotomy, as described by Hiramaya and coworkers15, the main indication of this procedure is the presence of residual deformity of the ulna or radius with a concave radial head articular surface16. In our case there was no deformity of the ulna, but there was lengthening of the radius with a domed radial head.\n\nWe were cautious not to excise the radial head in our case because of the risk of secondary subluxation of the distal radio-ulnar joint due to proximal migration of the radius17. We believe on the basis of our results that surgical correction is fully justified in irreducible dislocation of the radial head. We also believe that shortening of the radius would have been necessary to achieve reduction in cases where there is radial lengthening.\n\n\nConclusion\n\nNo reported case of congenital dislocation of the radial head treated by radial shortening and open reduction of the radiocapitellar joint through two incision approaches (two-in-one approach) has hitherto been available, as far as the authors are aware. This paper describes such a case, where we used this technique for the easy reduction of the radiocapitellar joint in order to maintain normal joint anatomy.\n\n\nConsent\n\nConsent was obtained from the patient and her mother for the novel technique to be conducted. We also obtained the consent from them for use of their information and images for publication in this article.",
"appendix": "Author contributions\n\n\n\nRaju Karuppal: Described the idea of this novel technique and performed the surgery, post operative evaluation, design, writing and editing of the case report. Anwar Marthya: Planning of the surgery, analysis and interpretation of the result, design and editing of the manuscript. Rajendran V. Raman: Interpretation of result and editing of manuscript. Sandhya Somasundaran: Interpretation of the results, writing and editing of the manuscript. All authors critically revised the manuscript and approved the final manuscript for publication.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nSachar K, Mih AD: Congenital Radial Head Dislocations. Hand Clin. 1998; 14(1): 39–47. PubMed Abstract\n\nZionts LE, MacEwen GD: Treatment of congenital dislocation of the hip in children between the ages of one and three years. J Bone Joint Surg Am. 1986; 68(6): 829–46. PubMed Abstract\n\nKarakas ES, Baktir A, Argun M, et al.: One-stage treatment of congenital dislocation of the hip in older children. J Pediatr Orthop. 1995; 15(3): 330–6. PubMed Abstract\n\nJoseph PI, Richard P: The Netter Collection of Medical Illustrations: Musculoskeletal System Volume 6, Part I - Upper Limb: Elsevier Health Sciences, Medical. 2012; (6): 104. Reference Source\n\nAgnew DK, Davis RJ: Congenital unilateral dislocation of the radial head. J Pediatr Orthop. 1993; 13(4): 526–8. PubMed Abstract\n\nEchtler B, Burckhardt A: Isolated congenital dislocation of the radial head. Good function in 4 untreated patients after 14–45 years. Acta Orthop Scand. 1997; 68(6): 598–600. PubMed Abstract\n\nHughes T, Chung CB: Bone and Cartilage Injury, Chapter 12. In Chung CB and Steinbach LS (editors). MRI of the Upper Extremity: Shoulder, Elbow, Wrist, and Hand. Lippincott Williams & Wilkins; Philadelphia, USA. 2010; 487–488.\n\nMaresh MA: Linear growth of long bones of extremities from infancy through adolescence; continuing studies. AMA Am J Dis Child. 1955; 89(6): 725–42. PubMed Abstract | Publisher Full Text\n\nKelly DW: Congenital dislocation of the radial head: spectrum and natural history. J Pedatr Orthop. 1981; 1(3): 295–8. PubMed Abstract | |Publisher Full Text\n\nMiles KA, Finlay DB: Disruption of the radiocapitellar line in the normal elbow. Injury. 1989; 20(6): 365–7. PubMed Abstract\n\nMcFarland B: Congenital dislocation of the head of the radius. Br J Surg. 1936; 24(93): 41–49. Publisher Full Text\n\nOka K, Murase T, Morimoto H, et al.: Morphologic evaluation of chronic radial head dislocation: three-dimensional and quantitative analyses. Clin Orthop Relat Res. 2010; 468(9): 2410–2418. PubMed Abstract | |Publisher Full Text | |Free Full Text\n\nWood WL, Robert BW, Raymond TM, et al.: Weinstein. Lovell and Winter’s Pediatric Orthopaedics Volume 2; Lippincott Williams & Wilkins, Medical. 2006; 2. : 935. Reference Source\n\nDe Boeck H: Treatment of chronic isolated radial head dislocation in children. Clin Orthop Relat Res. 2000; (380): 215–9. PubMed Abstract | |Publisher Full Text\n\nHirayama T, Takemitsu Y, Yagihara K, et al.: Operation for chronic dislocation of the radial head in children: Reduction by osteotomy of the ulna. J Bone Joint Surg Br. 1987; 69(4): 639–42. PubMed Abstract\n\nSeel MJ, Peterson HA: Management of chronic posttraumatic radial head dislocation in children. J Pediatr Orthop. 1999; 19(3): 306–12. PubMed Abstract\n\nWatson-Jones R: Fractures and Joint Injuries. Fourth edition, Volume 2, 579. Edinburgh and London: E. and S. Livingstone. 1955."
}
|
[
{
"id": "5769",
"date": "13 Aug 2014",
"name": "Nikolaos Gougoulias",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a nicely presented case report of the relatively uncommon condition of congenital elbow dislocation. The authors describe the rationale of management and the surgical technique with clarity. They also provided evidence of follow up. The discussion is nicely written, providing information regarding the background of the condition they treated, putting the reader, who is not a specialist in the field, in the correct context. I approve the article's indexation. Its current version could be further improved if the authors provided clinical pictures of the patient's forearm postoperatively in different positions, indicating for example the ability to perform supination/pronation movements (if such photos were available).",
"responses": []
},
{
"id": "10118",
"date": "03 Sep 2015",
"name": "Kishore Puthezhath Menon",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nApart from the rarity, the case report attempts to convey that the treatment options available for congenital dislocation of radial head are either early reconstruction or late radial head excision and the former is ideal. But the fact of the matter is that, this is not in alignment with established evidence. \"A congenitally dislocated radial head is irreducible manually or surgically because of adaptive changes in the soft tissues and the absence of normal surfaces for articulation with the ulna and humerus. Consequently, open reduction of the dislocation and reconstruction of the annular ligament in childhood are not advised. Any impairment of function usually is caused by restriction of rotation of the forearm; in children, physical therapy to improve this motion is the only treatment indicated. If pain persists into adulthood, the radial head and neck can be excised.\" Campbell's Operative Orthopaedics, 12e.Though not recommended by standard textbooks, if a reconstruction is planned as in the present report, annular ligament reconstruction is necessary as per available literature. De-Boeck's recommendations against this in case of chronic acquired isolated radial head dislocations do not seem to be applicable for congenital dislocations. Further more, as per Sachar, reconstruction is better than radial head excision in adulthood only if it is performed before 2 years.Figure 2 is not a MRI picture. Do MRIs have any role in determining whether a rotation osteotomy is to be done or not? How can we measure cubitus valgus angle in patients with fixed flexion deformity? A detailed explanation on method of pre operative templating may help curious surgeons.",
"responses": []
},
{
"id": "13345",
"date": "04 May 2016",
"name": "Sandeep Vijayan",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPositive points:The article is well designed and authored mentioning the surgical management of a rare congenital entity. The case report and surgical steps are clearly described. As per the report, early reduction of the radial head helps in better remodeling of the radio-capitellar joint similar to early reduction of hip in developmental dysplasia of the hip.Some of my queries and suggestions are as follows:Even though, authors are unaware of radial shortening and open reduction of the radiocapitellar joint through two incisions to the best of their knowledge, I wish to draw their attention to the paper by Hui Taek Kim in Journal of Paediatric Orthopaedics. On 3 elbows with congenital dislocation of radial head (2 patients) they did open reduction of radial head through a posterior approach and radial shortening through a midshaft approach. As the natural history of congenital dislocation of the radial head is benign, the readers would be interested and benefited to know what are / were the indications for surgical intervention in these / this case. Authors could discuss briefly about situations where surgical management is not needed. Even though, authors have mentioned about MRI findings (figure 2), the article does not carry any MRI picture. This minor mistake might be rectified and the role of MRI, if any, in the management of congenital dislocation of the radial head could be discussed in brief. Anatomic relationship between ulnohumeral, radio-capitellar and proximal radio-ulnar joint must be maintained in a skeletally immature patient for normal development to occur. It would be worthwhile if authors can mention about the status of ulno-humeral and proximal radio-ulnar joints in the pre and post operative radiographs. Readers would be interested to know if any rotation of the proximal fragment was done to improve supination before transfixation with K wire and plate fixation. It would be worth mentioning in the ‘operative technique’ about fixation of the osteotomy site with plate and screws. What was the position of the forearm in the cast postoperatively? Cadaveric studies have shown that the annular ligament is the most important ligament in stabilizing the radial head and the proximal radio-ulnar joint during all phases of rotation while the interosseous membrane contribute significantly only during the terminal phase of supination. So the claim made by authors that annular ligament reconstruction is not needed warrants further justification. The main problem in congenital dislocation of the radial head is the hypoplasia of capitullum and the mismatch between the dome shaped radial head and capitellum and alteration in the radial neck-head angle leading to re-dislocation/subluxation. It would be more informative if the authors could provide a lateral view of the elbow at final follow up to show the maintenance of the reduction and remodeling of the radio-capitellar joint. At one year follow up, the radiograph shows a positive ulnar variance. Authors could mention about the function of the wrist as well. It would be worth mentioning that a longer follow up is required to know the final outcome of the surgery as the child is skeletally immature.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-22
|
https://f1000research.com/articles/3-21/v1
|
22 Jan 14
|
{
"type": "Case Report",
"title": "A case of severe psychosis induced by novel recreational drugs",
"authors": [
"Filippo Dragogna",
"Lucio Oldani",
"Massimiliano Buoli",
"A. Carlo Altamura",
"Filippo Dragogna",
"Massimiliano Buoli",
"A. Carlo Altamura"
],
"abstract": "Introduction: The use of novel recreational drugs is becoming of public interest, especially after recent international alerts about their cardiovascular and neurological toxicity. Additionally, little is known about the psychiatric consequences of the long-term use of these compounds.Case presentation: We describe a case of severe psychotic episode likely induced by chronic use of a combination of new recreational drugs (methylenedioxypyrovalerone, mephedrone, butylone and alpha-pyrrolidinopentiophenone). The patient had no psychiatric history and showed poor response to conventional antipsychotic treatment (haloperidol).Conclusions: This case illustrates the potential negative effects of recreational drugs that cannot be limited to an acute psychotic episode but might determine a condition of prolonged paranoid psychosis. Although the use of these compounds is currently increasing, such molecules might often pass undetected in patients accessing the emergency room, leading to misdiagnosis (e.g. schizophrenic episode) and lack of appropriate treatment.",
"keywords": [
"We report the case of a 46-y.o. Italian Caucasian man (1.70 m",
"70 kg)",
"working as a full-time legal consultant."
],
"content": "Case description\n\nWe report the case of a 46-y.o. Italian Caucasian man (1.70 m, 70 kg), working as a full-time legal consultant.\n\nHe was brought to the emergency room of our Psychiatric Department in 2013, after having ingested a large quantity of zolpidem, a prescription drug indicated for insomnia, with suicidal intent. His family history was negative for psychiatric disorders, but he presented a positive medical history for liver disease (active chronic hepatitis C and Gilbert's syndrome). He had never suffered from any psychiatric symptoms in his childhood, adolescence or early adulthood. He had no family history of psychiatric disorders.\n\nThe patient’s psychopathological onset occurred three months prior to admission when, after continuous use (from one to three times a week) of a non-specified recreational drug since July 2012, provided to him by a friend who used to buy it online. He developed a persecutory delusion, characterized by the conviction of being spied upon by some unknown people who placed video cameras around his house. In addition, he believed that sexual activity he had with his wife was being filmed and then spread on pornographic websites. For this reason, in the following weeks, he made a written complaint against unknown persons and presented it to the local police office. In addition, one night he called the police force to his own home, as he saw suspicious movements outside his window. On that occasion, the police found out that the patient had a gun, which was then confiscated due to security concerns. Thus, the police then became part of patient’s delusional plot: the patient started thinking his daughter was involved in prostitution and he blamed the police for that. These events, along with two formal warnings received from his employer (since he did not show up for work for many days in a row), and a prolonged condition of global insomnia, generated a state of severe discouragement and embarrassment in the patient. On account of this he attempted suicide by ingesting a large quantity of zolpidem tablets (about 40 tablets of 10 mg, as related by the patient himself). Zolpidem had been prescribed four months prior to admission by a general practitioner to treat the patient’s insomnia.\n\nIn the emergency room of our hospital, the patient was initially drowsy and slow in his movements, although he was eupneic and had stable vital parameters. He underwent a physical examination, an electrocardiogram, a chest X-ray and a brain computer tomography, which were all normal. Though initially considered appropriate, no gastric lavage with activated charcoal was conducted as the patient had a spontaneous episode of vomiting. The patient was rehydrated with intravenous physiological saline and then admitted to our ward.\n\nSince the first day of admission, during the daily interviews with the medical staff, the patient showed the same delusional ideas described above and showed a very poor insight about his psychopathological condition [Brief Psychiatric Rating Scale (BPRS) score=50]. He admitted that the drug he had been taking during the previous months might have had a role in the development of his thoughts, but this did not change his belief that he was being persecuted by the police and his colleagues and managers at work. During his hospitalization, which lasted 17 days, his prolonged insomnia improved. He developed a mild criticism of his delusion, doubting some of the events he had reported, but contemporarily maintained a suspicious behavior towards the nurses and the doctors and an overall persecutory ideation. At discharge, he reported that he intended to sue the policeman who visited him at home as he had no right to suspend his gun license, showing overall poor insight and a very solid and structured delusion (BPRS score=31).\n\nAs soon as the patient was admitted to our ward, we collected blood and urine samples, which were analyzed by the central laboratory of our hospital. Haematological and chemistry tests highlighted a condition of normocytic anemia (Hb 11.2 mg/dl, Ht 33.4%, mean cell volume (MCV) red cells 3.55 × 106/mmc) and a mild hepatic distress [alanine transaminase (ALT) 136 U/l]. Routine screening for psychotropic drugs gave negative results. A positive emission tomography (PET) was performed on the 10th day of admission, which showed some unspecific findings consisting of an increased glucose metabolism in the basal nuclei but normal levels in the cortical regions.\n\nDuring the hospitalization period, a sample of the drug the patient had used for some months, in the form of white powder, was found at his home and delivered to the medical staff. This powder sample, along with further blood and urine samples, were sent to the Legal Medicine laboratory in the Forensic Toxicology section of our University, after having obtained a regular written consent from the patient. A specific analysis on the three samples was conducted by means of gas chromatography/mass spectrometry and liquid chromatography/high resolution mass spectrometry. The following molecules were found by the analysis on the powder: methylenedioxypyrovalerone, mephedrone, butylone and alpha-pyrrolidinopentiophenone (a-PVP) (the proportion of the each was not provided by the laboratory) (Table 1). Traces of methylenedioxypyrovalerone were found in the urine sample. A test for psychotropic drugs gave negative results for the blood sample.\n\nWith regards to the psychopharmacological therapy, since the beginning of his hospitalization the patient was treated with haloperidol 5 mg daily, showing an overall good medication adherence although his response was very poor. For this reason, we decided to administer an injection of haloperidol decanoate at a dosage of 150 mg (to be repeated every 4 weeks, as part of an outpatient regimen). One month after his discharge, in a follow-up visit, the patient showed a BPRS score of 29, corresponding to a slight improvement in persecutory delusion but no change in insight.\n\n\nDiscussion\n\nIn contrast to other reports that show hallucinatory delirium, agitation, anxiety and sleep disturbances following an acute intake of more popular substances of abuse (e.g. cannabis, cocaine), we describe a clinical picture of a persistent substance-induced psychosis, secondary to a prolonged intake of methylenedioxypyrovalerone, mephedrone, butylone and alpha-pyrrolidinopentiophenone (a-PVP). The correlation of symptoms with the intake of these substances is supposed in the light of a negative psychiatric history and no concomitant medical treatments. Of note, increased glucose metabolism in the basal nuclei at PET was probably due to the effects of haloperidol than to the substances, as documented by other reports1. In addition, the moderate hepatic failure of the patient could be responsible for the increased availability of the drugs and increased toxicity, as hepatic metabolism is involved in the excretion of all these substances.\n\nThe present report shows the danger of these novel drugs that are often bought as apparently safe and legal on the internet. However a number of case reports have documented deaths related to the ingestion of such substances, especially for mephedrone2 and methylenedioxypyrovalerone3,4. A further problem is that these substances are not detected by standard blood and urine tests so that the diagnosis of intoxication is often delayed. This means that patients may not receive the appropriate treatment due to the lack of diagnostic tools and may be misdiagnosed as schizophrenic or manic bipolar patients. This issue is particularly relevant in the emergency department, where a fast and precise diagnosis and management of patients is required. In addition, patients leaving the emergency department without being correctly diagnosed might continue their abuse and delay specialized follow-up visits in an outpatient context, finally developing a condition of chronic treatment-resistant psychosis.\n\nThese drugs are very popular among young clubbers and mephedrone was reported as the sixth most commonly used drug after alcohol, tobacco, cannabis, MDMA and cocaine in the dance music scene, night clubs and gay bars5–7. Educational campaigns about the risks related to these substances are needed in light of the negative effects that can be sustained over time8. In addition, clinical pictures similar to the one described should not be confused with schizophrenic episodes despite the atypical presentation and good pre-morbid social functioning.\n\n\nConsent\n\nConsent was obtained from the patient for use of their information for publication in this article.",
"appendix": "Author contributions\n\n\n\nDr Dragogna and Dr Oldani followed the case and wrote the case report. Dr Buoli revised the paper and constructed the table. Professor Altamura revised the paper. All authors agreed the final version of the manuscript for publication.\n\n\nCompeting interests\n\n\n\nProf. A. Carlo Altamura is a Merck consultant, Astra Zeneca consultant, Sanofi-Aventis speaker’s bureau member, Lilly speaker’s bureau member, Pfizer speaker’s bureau member and a Roche consultant. Dr Buoli is a Roche consultant. Dr Dragogna and Dr Oldani have no competing interests to disclose.\n\n\nGrant information\n\n\n\n\nReferences\n\nCorson PW, O'Leary DS, Miller DD, et al.: The effects of neuroleptic medications on basal ganglia blood flow in schizophreniform disorders: a comparison between the neuroleptic-naïve and medicated states. Biol Psychiatry. 2002; 52(9): 855–62. PubMed Abstract\n\nMaskell PD, De Paoli G, Seneviratne C, et al.: Mephedrone (4-methylmethcathinone)-related deaths. J Anal Toxicol. 2011; 35(3): 188–91. PubMed Abstract | Publisher Full Text\n\nMurray BL, Murphy CM, Beuhler MC: Death following recreational use of designer drug \"bath salts\" containing 3,4-Methylenedioxypyrovalerone (MDPV). J Med Toxicol. 2012; 8(1): 69–75. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKesha K, Boggs CL, Ripple MG, et al.: Methylenedioxypyrovalerone (\"bath salts\"), related death: case report and review of the literature. J Forensic Sci. 2013; 58(6): 1654–9. PubMed Abstract | Publisher Full Text\n\nZawilska JB, Wojcieszak J: Designer cathinones--an emerging class of novel recreational drugs. Forensic Sci Int. 2013; 231(1–3): 42–53. PubMed Abstract | Publisher Full Text\n\nWinstock AR, Mitcheson LR, Deluca P, et al.: Mephedrone, new kid for the chop? Addiction. 2011; 106(1): 154–161. PubMed Abstract | Publisher Full Text\n\nLea T, Reynolds R, De Wit J: Mephedrone use among same-sex attracted young people in Sydney, Australia. Drug Alcohol Rev. 2011; 30(4): 438–40. PubMed Abstract | Publisher Full Text\n\nHunter LJ, Dargan PI, Benzie A, et al.: Recreational drug use in men who have sex with men (MSM) attending UK sexual health services is significantly higher than in non-MSM. Postgrad Med J. 2014; [Epub ahead of print]. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "3493",
"date": "06 Mar 2014",
"name": "John Mendelson",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDragogna and colleagues describe a case of persistent psychosis associated with the ingestion of mephadrone and related compounds. These cathinone derivatives have similar risks as other phenethylamine stimulants such as methamphetamine and MDMA and are widely available and easily abused. It is therefore not surprising that persistent psychosis, as described in this case report, can be seen with prolonged use. Although the authors show an association of cathinone use with admission for a persistent psychosis the relationship between cathinone abuse and the development of psychosis is less clear - this patient may have become psychotic well before using any illicit drugs. However, the case report makes the essential point that urine drug screens with currently available immunoassay are likely to miss cathinone-like drugs. Thus, if cathinone use is suspected, a laboratory work up of biospecimes will need to be performed by groups specializing in medical toxicology. Due to the increasing abuse of drugs in this class clinicians can expect to see more toxicity. Overall, this report adds to the literature.",
"responses": []
},
{
"id": "6396",
"date": "13 Oct 2014",
"name": "George Woody",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting case report that is clearly written and documents an instance in which a persistent and treatment resistant psychotic disorder developed after taking an unknown amount of hallucinogenic and stimulant drugs. This case differs from the typical course of substance induced psychoses in that it did not resolve with abstinence and pharmacotherapy. I have no suggestions for modifications.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-21
|
https://f1000research.com/articles/3-19/v1
|
21 Jan 14
|
{
"type": "Web Tool",
"title": "Visualizing and quantifying movement from pre-recorded videos: The spectral time-lapse (STL) algorithm",
"authors": [
"Christopher R. Madan",
"Marcia L Spetch",
"Marcia L Spetch"
],
"abstract": "When studying animal behaviour within an open environment, movement-related data are often important for behavioural analyses. Therefore, simple and efficient techniques are needed to present and analyze the data of such movements. However, it is challenging to present both spatial and temporal information of movements within a two-dimensional image representation. To address this challenge, we developed the spectral time-lapse (STL) algorithm that re-codes an animal’s position at every time point with a time-specific color, and overlays it with a reference frame of the video, to produce a summary image. We additionally incorporated automated motion tracking, such that the animal’s position can be extracted and summary statistics such as path length and duration can be calculated, as well as instantaneous velocity and acceleration. Here we describe the STL algorithm and offer a freely available MATLAB toolbox that implements the algorithm and allows for a large degree of end-user control and flexibility.",
"keywords": [
"movement",
"tracking",
"path analysis",
"video visualization",
"video summarization",
"image analysis"
],
"content": "Introduction\n\nStudies of animal behaviour in open environments yield rich datasets. While behaviour can often be summarized through simple measurements (e.g., first target approached within an array, sequence of targets approached, timings of these behaviours), these measures are not always sufficient. A widely-used solution to this problem was introduced three decades ago, with a methods paper describing the use of video recordings to study animal behaviour (Godden & Graham, 1983). Although some researchers use commercial tracking equipment, movements are sometimes recorded using standard video cameras without markers on the animal and the data are manually scored. Using simple pre-recorded video recordings, we sought to summarize both spatial and temporal information of movements within a two-dimensional image representation. Specifically, we developed spectral time-lapse (STL) images that code the animal’s position with a time-specific color and overlay them on a frame of the video to produce a summary image (Figure 1A). We also incorporate automated tracking of the animal’s path and provide summary statistics (Figure 1B), as well as plotting velocity and acceleration over time (Figure 1C). Here, we describe the algorithm and offer a MATLAB toolbox that implements it, while allowing for substantial end-user control.\n\n(A) Spectral time-lapse (STL) image of the trial, sampled at 1 pps. First bar in bottom left corresponds to 10 seconds; second bar illustrates which frames highly overlapped with adjacent frames; third bar shows time-color mapping used. (B) Path overlaid on the STL image, sampled at 6 pps. (C) Velocity-acceleration plot of same movement data.\n\nThe challenge of visualizing movements within a two-dimensional image is not new. Although many solutions have been discussed (Jensenius, 2012, 2013), none integrate both spatial and temporal information to sufficiently characterize a path within a single image. Time-lapse images (illustrated in Jensenius, 2013, Figure 1) concatenate a series of still images adjacently, and do not present the images within the same spatial frame. Motion history and motion average images (illustrated in Jensenius, 2013, Figure 4–Figure 7) show movements within the same spatial frame, but lose temporal information. Our solution was to color images of the target using a time-specific color, and overlay these on the background, see Figure 1A.\n\nOur second goal was to obtain path data, specifically x- and y-coordinates of the animal at each time point. While solutions for this purpose already exist, many have drawbacks. EthoVision (Noldus et al., 2001, 2002; Spink et al., 2001), a widely used movement-tracking software package, needs to be adjusted for each set-up (e.g., animal to track and type of arena). Other methodological drawbacks include requiring markers on the animal during video acquisition (e.g., Chen et al., 2008), specification of templates of the animal’s shape (e.g., Kalafatić, 2003; Xu et al., 2009), or the ability to only process low-resolution videos (reducing precision; e.g., Crispim Junior et al., 2012). Although solutions exist that do not have these limitations (e.g., Khan et al., 2006; Perner, 2001; Tort et al., 2006; Tweed & Calway, 2002), our implementation of the STL toolbox in MATLAB allows the end-user to easily extract path data within the MATLAB environment (e.g., Figure 1B). To glean additional information from the path, we also calculate instantaneous velocity and acceleration (see Figure 1C).\n\n\nMaterials and methods\n\nAnimal research was conducted in accordance with Canadian Council on Animal Care guidelines and with approval from the University of Alberta Animal Welfare Policy Committee. Pigeons (Columba livia) were kept on a 12:12 h light:dark cycle with light onset at 6 AM. Birds were housed individually in metal cages and kept at 85% of their free feeding weight on a diet of Kee Tee pigeon pellets and vitamin supplement. Water and grit were available ad libitum.\n\nHere we present a spectral time-lapse (STL) image and describe the algorithm used to create the image. Figure 1A illustrates a single trial of a pigeon (Columba livia) entering an arena, moving to and eating from four food cups, and returning to the starting box. The STL image allows the researcher to observe the behaviour (e.g., sequence of cups visited, efficiency of path taken) without needing to watch the video. This is particularly useful as videos are often longer in duration than the movement; in this particular trial, the raw video lasts 45 sec., while the pigeon is only visible for 25 sec. The STL image in Figure 1A was generated to show one position-per-second (pps), in other words, one colored position (i.e., pigeon) is plotted for each second. The raw video for this particular trial is included as Data File 1.\n\nVideo data was acquired using a standard video camera connected to a PC running Microsoft Windows 7 (Redmond, WA) and recorded as a MPEG-2 transport stream file using the WinTV hardware and software package (Hauppauge Computer Works Inc., Hauppauge, NY). (Note: It is not necessary for the STL method that the videos be recorded with WinTV or that the videos be saved as MPEG-2 transport stream files, this was just how we chose to digitize our video recordings). We converted the video to an uncompressed AVI format using MPEG Streamclip (Squared 5 S.R.L., Rome, Italy), but other software could be used as well. These uncompressed AVI files can be read directly into the STL toolbox.\n\n\n\n\nThe STL algorithm\n\nThe steps comprising the STL algorithm are illustrated in Figure 2. Settings that can easily be adjusted by the end user are noted in parentheses and italicized throughout. These names refer to the variable names within the STL toolbox and are found within the configuration file (config.m, see Supplementary Materials).\n\n(A) Loading the raw video. (B) Pre-processing. (C) Colorizing the frames. (D) Creating the STL image. (E) Outputting the STL image. (F) Path analysis method. (G) Velocity-acceleration plot.\n\nThe raw video file is read in and only every i-th frame is sampled (sampling), as video is often acquired at higher rates than needed for the STL image. For instance, the animal’s position might be sampled at 1 pps, whereas video cameras often record at 24 or 30 frames-per-second (fps). If the original video speed has been adjusted, such as videos originally from a high-speed camera, then this can be accommodated and calculations adjusted (videospeed). The STL toolbox reports the video’s original acquired fps and the STL’s pps. The sampled video frames are converted to grey-scale, as color will be used to code for time. The folder containing the raw video must be specified in the configuration file (path_raw).\n\nTo allow the STL images to be based on only a portion of a video, start and end frames can be specified, (startFrame, endFrame). An additional MATLAB function called showFrameK is included to facilitate in determining start and end frames.\n\nIn this stage, the reference frame is also defined, which is often either the first or last frame of the video, or a ‘moving average’ (refFrame). The reference will be subtracted from all other frames to isolate the target animal, i.e., the change in the video frame, in the next stage. A moving average is useful when the background changes over time (e.g., lighting, bedding materials; refSmooth).\n\nThe STL algorithm implements a pre-processing stage to isolate movement data and reduce noise. Here, five pre-processing calculations were done for each frame:\n\nFirst, the reference is subtracted from the given frame, to isolate changes in the frame that corresponds to the target.\n\nSecond, the difference image is spatially smoothed to reduce noise. This is implemented by convolving a two-dimensional Gaussian kernel with the given frame. Ideally, the user will calibrate the kernel size to the image, based on the animal’s size, as viewed by the camera, and video resolution (smooth).\n\nThird, if the animal is lighter colored than the background, intensity values are negative. To produce consistent color mapping in the next stage, we reverse these values so that intensity of the target is always positive.\n\nFourth, irrelevant portions of the frame are masked out to improve the signal-to-noise ratio and later target detection. Two approaches are used to do this, a pre-made static mask (doMask) and a dynamic detection of an overlay (cleanWhite). For the pre-made mask, the filename to the mask image must be provided (maskName). For the overlay, any pixels with an intensity value above a set threshold are ignored (white). This is useful if a timestamp or other overlay is hard-coded into the video, as in Figure 1A.\n\nFifth, we trim frames from the start and end of the video that did not contain the target; this feature can be disabled by the end user (disableTrim). Frames are only retained if they are sufficiently different from the reference, based on thresholds (threshMask, threshTrim). At this point, only frames containing temporal information about the movement are retained.\n\nFigure 2 shows example images of the frames after these calculations.\n\nA mapping of time-to-color is created for each of the retained frames. This mapping is adjustable, but usually corresponds to one or two color cycles (cmap). A mask is then created such that only pixel intensities that surpass a threshold are retained (threshMask), further removing noise. At this point, the spatial information corresponding to the target has been isolated. The color specific to the given frame is then applied, see Figure 2.\n\nAll colorized frames are averaged to produce a single frame that is essentially the STL image. To improve color visibility after averaging, the saturation of the averaged frame is amplified (oversatCol).\n\nTo produce the final STL image, we overlay the averaged frame on the reference (refFrame). To further improve visibility of the colors, we increase the saturation of the reference (oversatRef). Legend bars are added to the image to show (a) actual time, (b) indicate overlapping frames as would occur if the target pauses, and (c) time-specific color mapping. The actual time bar denotes the length, relative to the other bars, of a fixed amount of time, e.g., 1 second (timeBar). The overlap bar is white if the frames overlapped more than a threshold amount (threshAdjac), and is otherwise black. The size of all three bars can also be adjusted (barSize).\n\nThe final STL image is exported as an image file to the specified folder (path_out). The image can also be viewed immediately (showSTL).\n\nIf path analysis is enabled (doPath), the STL toolbox uses a simple but efficient method to obtain x- and y-coordinates of the target at regular intervals (pathSampling), which is often a higher sampling frequency than used for the STL image. In our example (Figure 1B) we used 6 pps. These positions are plotted in a separate path image, which can either be overlaid on the STL image or the reference frame (pathBack).\n\nThe path analysis method takes advantage of the same thresholds used in the STL algorithm to isolate the target and remove spatial and temporal noise. The coordinates of the target are determined by calculating the x- and y-coordinates for the center of the largest centroid, after the image has been intensity thresholded (threshTrim). A minimum area for the largest centroid (areamin) is also used to re-determine the start and end frames for the path analysis.\n\nThe obtained x- and y-coordinates for the target across all retained frames can be plotted over the STL or reference image. A color map is applied, along with the STL image, and the marker’s border and arrows can be modified in the configuration (pathCol; usually black or white, depending on the background). The path image is saved in the same folder as the STL image (path_out). Along with the x- and y-coordinates for each frame, two summary statistics are calculated: total path length and duration. If the pixels-to-meters conversion is specified (px2m), coordinates and path length will be outputted in meters.\n\nUsing the distances travelled between time points, as calculated for the path analysis, we can readily also calculate the instantaneous velocity and acceleration (doVel). To reduce noise in these measures, a weighted average is taken across adjacent values (velSmooth). The plot is saved in the same folder as the STL image (path_out).\n\n\nGeneralizability of the STL algorithm\n\nSo far we have described the STL algorithm (Figure 2) and presented images for one trial of a pigeon study (Figure 1). To demonstrate the generalizability of the method, we tested it on videos of other animals.\n\nThe first video, of a mouse in a radial-arm maze (http://www.youtube.com/watch?v=y7zQgz0vmWo), was downloaded as a MPEG-4 file from YouTube and converted to an uncompressed AVI with MPEG Streamclip. We cropped the video to isolate the maze. As the video represented multiple trials, we chose a video segment from after the mouse had been trained, spanning from 1 min 46 sec to 1 min 59 sec; this temporal trimming was done through the STL toolbox by specifying the start and end frames (3178 and 3568, respectively). Several settings were modified to suit the video, such as the smoothing kernel size, color map cycles, and the target being lighter than the background. We sampled the mouse’s position at 3 pps for the STL image and 30 pps for the path analysis. We plotted the path over the reference frame. The resulting images are presented in Figures 3A–3C.\n\n(A) STL image of a mouse in a radial arm maze (available from http://www.youtube.com/watch?v=y7zQgz0vmWo, with permission of Anže Starič (University of Ljubljana)), sampled at 3 pps. First bar in bottom left corresponds to 1 second; second bar illustrates which frames highly overlapped with adjacent frames; third bar shows time-color mapping used. (B) Path of same movement data as shown in panel A, overlaid on the reference frame, sampled at 30 pps. (C) Velocity-acceleration plot of same movement data as panels A and B. (D) STL image of an ant in an open environment, sampled at 10 pps (after adjusting for use of high-speed camera; available from http://www.youtube.com/watch?v=u7LaPjMtmYM with permission of Antoine Wystrach, Paul Graham, and Andrew Philippides (University of Sussex)). First bar in bottom left corresponds to 10 seconds; second bar illustrates which frames highly overlapped with adjacent frames; third bar shows time-color mapping used. (E) Path of same movement data as panel D, overlaid on the STL image, sampled at 100 pps. (F) Velocity-acceleration plot of same movement data as panels D and E.\n\nThe second video was of an ant in a simple open environment demonstrating scanning behaviour, where the ant is searching for visual landmarks (http://www.youtube.com/watch?v=u7LaPjMtmYM). The video was also downloaded from YouTube and converted. Note that this video was recorded using a high-speed camera and had been slowed down by a factor of 10 (as stated in the video’s description). Settings were customized for differences in the video resolution and speed, as well as target size. Here we sampled the ant’s position at 10 pps, for the STL image and 100 pps for the path image. The resulting images are presented in Figures 3D–3F.\n\n\nResults and discussion\n\nHere we presented a novel method of visualizing and quantifying animal movement from pre-recorded videos acquired with standard video equipment. The STL images accurately summarize an animal’s position at a given time, within a single two-dimensional image representation, and allow researchers to observe movement patterns without needing to watch full videos for every trial. We incorporated a simple but efficient path analysis method into the algorithm to quantify properties of the movement, including instantaneous velocity and acceleration. The STL toolbox implementing the STL algorithm in MATLAB is available freely from the authors. (For an introductory guide to MATLAB, see Madan, 2014).\n\nAs the path analysis method implemented in the STL toolbox is fairly simple, it has a few limitations: the method can only be used for a single target and it cannot correct for partially occluded targets or lens distortions. Several methods could be incorporated to allow for the tracking of multiple targets, such as placing unique markers on each target (e.g., Sakiyama et al., 2006), identifying separable targets and calculating movement vectors or “limited-radius” searches for each (e.g., Perner, 2001; Tort et al., 2006; Xu et al., 2009), using shape templates (e.g., Kalafatić, 2003; Xu et al., 2009), or using a particle-based approach (e.g., Khan et al., 2006; Tweed & Calway, 2002). Future versions could use methods to correct for occlusions (e.g., Perner, 2001), which can include video artifacts such as timestamps embedded in the video (as in Figure 1). Estimates of path length may also be affected by lens distortions, e.g., if a fish-eye lens was used. These distortions can be corrected by combining manually-acquired known distances (i.e., a calibration grid) with the observed video data. (Lind et al., 2005) provide equations to compensate for lens distortions. Nonetheless, the path analysis method implemented here efficiently tracks a single target and requires no markers or shape templates.\n\nOther fields have also demonstrated interest in movement-tracking methods. Most notably, many papers outlining methods for tracking movements have been published in the Journal of Neuroscience Methods, driven by interest in how neurological lesions or pharmacological manipulations influence movement. Our methods offer a simple, readily-available tool to complement existing techniques. These methods may also prove useful in other domains such as tracking humans from stationary surveillance cameras (e.g., Buono, 2011) or tracking vehicles over large areas (e.g., van Dommelen et al., 2013).\n\n\nData and software availability\n\nfigshare: Data File 1. Raw video used in Figure 1 and Figure 2. doi: http://dx.doi.org/10.6084/m9.figshare.900359 (Madan & Spetch, 2014).\n\nCopies of the YouTube videos have been deposited with F1000Research for archival purposes. Should the videos no longer be available from the respective YouTube links provided in the article, please contact F1000Research.\n\nZENODO: Spectral time-lapse (STL) Toolbox. doi: 10.5281/zenodo.7663 (Madan & Spetch, 2014).",
"appendix": "Author contributions\n\n\n\nBoth authors developed the approach. CRM implemented the method within MATLAB. Both authors read and approved the final version of the manuscript.\n\n\nCompeting interests\n\n\n\nThe 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.\n\n\nGrant information\n\nThis research was partly funded by a Discovery grant and a Canada Graduate Scholarship, both from the Natural Science and Engineering Research Council of Canada, held by MLS and CRM, respectively.\n\n\nAcknowledgements\n\nWe would like to thank Ariel Greiner for feedback while developing the STL algorithm. We would also like to thank Anže Starič (University of Ljubljana) for allowing us to use his video of the mouse in the radial arm maze and Antoine Wystrach, Paul Graham, and Andrew Philippides (University of Sussex) for allowing us to use their video of ant behaviour.\n\n\nSupplementary materials\n\nSample text feedback from running the STL toolbox (stltool) on video file ''S−Video_20110718_1132.avi'', the video of the single trial from the pigeon study. The outputted images are shown in Figure 1. (Output is text wrapped to fit on page.)\n\n\n\nData output from same example.\n\n\n\nLegend of the outputted data's structure.\n\n\n\nExample configuration settings code (config.m).\n\n\n\nExample custom configuration code (configCustom.m).\n\n\n\n\nReferences\n\nBuono P: Analyzing video produced by a stationary surveillance camera. In Proceedings of the International Conference on Distributed Multimedia Systems (DMS 2011). 2011; pp. 140–145. Reference Source\n\nChen YJ, Li YC, Huang KN, et al.: Video tracking algorithm of long-term experiment using stand-alone recording system. Rev Sci Instrum. 2008; 79(8): 085108. PubMed Abstract | Publisher Full Text\n\nCrispim Junior CF, Pederiva CN, Bose RC, et al.: ETHOWATCHER: Validation of a tool for behavioral and video-tracking analysis in laboratory animals. Comput Biol Med. 2012; 42(2): 257–264. PubMed Abstract | Publisher Full Text\n\nGodden DH, Graham D: 'Instant' analysis of movement. J Exp Biol. 1983; 107: 505–508. PubMed Abstract\n\nJensenius AR: Evaluating how different video features influence the visual quality of resultant motiongrams. In Proceedings of the Sound and Music Computing Conference. 2012; pp. 467–472. Reference Source\n\nJensenius AR: Some video abstraction techniques for displaying body movement in analysis and performance. Leonardo. 2013; 46(1): 53–60. Publisher Full Text\n\nKalafatić Z: Model-based tracking of laboratory animals. In Proceedings of EUROCON 2003: Computers as a Tool. 2003; 2: pp. 175–178. Publisher Full Text\n\nKhan Z, Blach T, Dellaert F: MCMC data association and sparse factorization updating for real time multitarget tracking with merged and multiple measurements. IEEE Trans Pattern Anal Mach Intell. 2006; 28(12): 1960–1972. PubMed Abstract | Publisher Full Text\n\nLind NM, Vinther M, Hemmingsen RP, et al.: Validation of a digital video tracking system for recording pig locomotor behaviour. J Neurosci Methods. 2005; 143(2): 123–132. PubMed Abstract | Publisher Full Text\n\nMadan CR: An Introduction to MATLAB for Behavioral Researchers. Thousand Oaks, CA: Sage. 2014. Reference Source\n\nMadan CR, Spetch ML: Data File 1. Raw video used in Figure 1 and Figure 2 F1000Research. Figshare. 2014. Data Source\n\nMadan CR, Spetch ML: Spectral time-lapse (STL) Toolbox. ZENODO. 2014. Data Source\n\nNoldus LP, Spink AJ, Tegelenbosch RA: EthoVision: A versatile video tracking system for automation of behavioral experiments. Behav Res Methods Instrum Comput. 2001; 33(3): 398–414. PubMed Abstract | Publisher Full Text\n\nNoldus LPJJ, Spink AJ, Tegelenbosch RAJ: Computerised video tracking, movement analysis and behaviour recognition in insects. Comput Electron Agric. 2002; 35(2–3): 201–227. Publisher Full Text\n\nPerner P: Motion tracking of animals for behavior analysis. In Proceedings of the International Workshop on Visual Form (IWVF-4). 2001; pp. 779–786. Publisher Full Text\n\nSakiyama Y, Sujaku T, Furuta A: A new automated method to estimate the behavioral responses of a small animal using a multicolor detection technique. In Proceedings of the SICE (Society of Instrument and Control Engineers)–ICASE (Institute of Control, Automation, and Systems Engineers) International Joint Conference. 2006; pp. 2905–2910. Publisher Full Text\n\nSpink AJ, Tegelenbosch RAJ, Buma MOS, et al.: The EthoVision video tracking system—A tool for behavioral phenotyping of transgenic mice. Physiol Behav. 2001; 73(5): 731–744. PubMed Abstract | Publisher Full Text\n\nTort ABL, Neto WP, Amaral OB, et al.: A simple webcam-based approach for the measurement of rodent locomotion and other behavioural parameters. J Neurosci Methods. (2006); 157(1): 91–97. PubMed Abstract | Publisher Full Text\n\nTweed D, Calway A: Tracking multiple animals in wildlife footage. In Proceedings of the International Conference on Pattern Recognition. 2002; 2: pp. 24–27. Publisher Full Text\n\nvan Dommelen W, van de Laar P, Noldus LPJJ: Extending track analysis from animals in the lab to moving objects anywhere. In Situation Awareness with Systems of Systems. 2013; pp. 89–103. Springer: New York. Publisher Full Text\n\nXu J, Yu H, Liu Y: A method to quantify movement activity of groups of animals using automated image analysis. In Proceedings of the International Conference on Photonics and Image in Agriculture Engineering (PIAGENG 2009). 2009; pp. 74891C. Publisher Full Text"
}
|
[
{
"id": "3748",
"date": "27 Feb 2014",
"name": "Suzette Astley",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article, Madan and Spetch describe a tool for monitoring animal movement in an open field that will be extremely useful to many researchers. Not only does the software monitor the animal's path through the environment, it also offers time-based information, and can provide information about velocity and acceleration of the movement over time. MATLAB software is in wide use in behavioral research, and Madan and Spetch have made the STL algorithm freely available in MATLAB. This is the sort of new tool that can lead researchers to ask new questions and to look in new directions to answer current questions. This algorithm will undoubtedly make a significant contribution to our understanding of animal behavior.",
"responses": []
},
{
"id": "3744",
"date": "24 Mar 2014",
"name": "Michael Mangan",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverviewThis paper describes a MatLab toolbox for tracking single animals in video data, quantifying basic movement properties (path-length, velocity profile, etc), and then displaying overall movement pattern in a single figure using colours to encode animal position at different times. The animal tracker presented comprises an elementary background subtraction approach augmented with masking, thresholding, and filtering methods to extract the location of the animal. As the parameters for these techniques are manually defined, and then held constant across the video, this method seems best suited to laboratory recordings where the environment changes little. For outdoor recordings, where there are likely illumination changes, occlusions etc a more robust tracking method is required e.g. TLD tracker (Z. Kalal, K. Mikolajczyk, and J. Matas, “Tracking-Learning-Detection,” Pattern Analysis and Machine Intelligence, 2011). The authors summarise the movement of the animal in a \"spectral-time-lapse\" image. The STL overlays the animal position, colour coded with respect to time, on a reference image. I believe the primary benefit of this method is rapid visualisation of the position and orientation of the animal across its path. Although personally, I do not see the benefit of displaying the entire animal shape - I deduced more from the higher resolution track shown in Fig 1B for example. I also suspect that on longer or overlapping paths the STL would become cluttered and confusing.The techniques used are mostly technically sound (see specific comments below). I foresee this work being of some use to behavioural researchers tracking animals in laboratory settings. Specifically: as the code is available as a Matlab toolbox, it can be easily installed, the parameters tuned for the specific case, and data visualised easily.Specific CommentsThe authors describe a \"moving average\" reference frame, but do not describe how this is generated exactly. I think expanding upon this point to make it clear to a reader is important. The 3rd step in the Pre-processing algorithm inverts the negative pixel intensities. I think this step could be omitted by taking either a sum-squared or root-mean-square difference between reference and current frame in step 1. In the 4th step in the Pre-processing algorithm it is stated that the masking method \"is useful if timestamp or overlay is hard-coded in the video\". I would have thought this would be removed in the background subtraction step as it is constant across frames? The mouse data tracks the animal from 1 min 46 seconds until 1 min 59 seconds. Was there a reason for using this specific portion of the video?",
"responses": []
}
] | 1
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https://f1000research.com/articles/3-19
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https://f1000research.com/articles/2-30/v1
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01 Feb 13
|
{
"type": "Web Tool",
"title": "Construction and accessibility of a cross-species phenotype ontology along with gene annotations for biomedical research",
"authors": [
"Sebastian Köhler",
"Sandra C Doelken",
"Barbara J Ruef",
"Sebastian Bauer",
"Nicole Washington",
"Monte Westerfield",
"George Gkoutos",
"Paul Schofield",
"Damian Smedley",
"Suzanna E Lewis",
"Peter N Robinson",
"Christopher J Mungall",
"Sandra C Doelken",
"Barbara J Ruef",
"Sebastian Bauer",
"Nicole Washington",
"Monte Westerfield",
"George Gkoutos",
"Paul Schofield",
"Damian Smedley",
"Suzanna E Lewis",
"Peter N Robinson"
],
"abstract": "Phenotype analyses, e.g. investigating metabolic processes, tissue formation, or organism behavior, are an important element of most biological and medical research activities. Biomedical researchers are making increased use of ontological standards and methods to capture the results of such analyses, with one focus being the comparison and analysis of phenotype information between species.We have generated a cross-species phenotype ontology for human, mouse and zebra fish that contains zebrafish phenotypes. We also provide up-to-date annotation data connecting human genes to phenotype classes from the generated ontology. We have included the data generation pipeline into our continuous integration system ensuring stable and up-to-date releases.This article describes the data generation process and is intended to help interested researchers access both the phenotype annotation data and the associated cross-species phenotype ontology. The resource described here can be used in sophisticated semantic similarity and gene set enrichment analyses for phenotype data across species. The stable releases of this resource can be obtained from http://purl.obolibrary.org/obo/hp/uberpheno/.",
"keywords": [
"Research on model organisms is crucial for discovering the function of genes and DNA elements and for understanding the phenotypic effects of mutations on these genes",
"which is leading to a better understanding of the pathobiology of human disease1",
"2. The amount of phenotypic information derived from targeted mutations and hypothesis-driven studies is increasing rapidly",
"and is now being further augmented by high-throughput international efforts to systematically analyse the effects of genomic variation on model organism phenotypes. For example",
"the International Mouse Phenotyping Consortium (IMPC3)",
"is undertaking systematic phenotyping studies of the knockouts generated by the International Knockout Mouse Consortium (IKMC4). This means that there will soon be structured phenotype data for loss-of-function mutants for every protein-coding gene in the mouse. Similar approaches are being taken in zebrafish (Danio rerio) by the Zebrafish Mutation Project (ZMP",
"http://www.sanger.ac.uk/Projects/D_rerio/zmp/) and the data is being made available through the The Zebrafish Model Organism Database (ZFIN5)."
],
"content": "Introduction\n\nResearch on model organisms is crucial for discovering the function of genes and DNA elements and for understanding the phenotypic effects of mutations on these genes, which is leading to a better understanding of the pathobiology of human disease1,2. The amount of phenotypic information derived from targeted mutations and hypothesis-driven studies is increasing rapidly, and is now being further augmented by high-throughput international efforts to systematically analyse the effects of genomic variation on model organism phenotypes. For example, the International Mouse Phenotyping Consortium (IMPC3), is undertaking systematic phenotyping studies of the knockouts generated by the International Knockout Mouse Consortium (IKMC4). This means that there will soon be structured phenotype data for loss-of-function mutants for every protein-coding gene in the mouse. Similar approaches are being taken in zebrafish (Danio rerio) by the Zebrafish Mutation Project (ZMP, http://www.sanger.ac.uk/Projects/D_rerio/zmp/) and the data is being made available through the The Zebrafish Model Organism Database (ZFIN5).\n\nModel organism phenotype/genotype datasets are extremely valuable as they can provide clues to human gene functions and involvement in disease processes where no data is available for the human ortholog. At the time of writing, 2,358 human genes are associated with Mendelian phenotypes, but more importantly there are 5,492 human genes with no such phenotype associations, where an orthologous mouse or zebrafish gene does have phenotype data (Data obtained by analysing the file HSgenes_crossSpeciesPhenoAnnotation.txt from http://purl.obolibrary.org/obo/hp/uberpheno/). We have previously demonstrated the power of this approach in determining likely pathogenicity of genes within the intervals of recurrent copy number variation (CNV) diseases6 and it can be applied much more widely in, for example, prioritizing candidate genes identified through human genome wide association studies (GWAS)7,8. Historically, a major problem has been the lack of common semantics across databases, with each project using some combination of free-text descriptions or in-house vocabularies. Thus, phenotype information is not easily integrated across different species. This inhibits comparisons based on phenotype alone, and where orthology is useful phenotypic comparisons cannot be used to their full potential. This is made even more complicated by different conceptualizations of phenotypes in different species and the impact of species-specific anatomies. As the ability of investigators to mobilise this growing collection of model organism data has become more important, it is crucial to develop appropriate ontologies and computational strategies to describe phenotypes such that phenotype descriptions can be objectively related to each other, both within and between species. This becomes even more important as the divergence between the number of human genes with phenotype information and the amount of systematically phenotyped model organism genes is expected to increase in the near future due to high throughput-screens1.\n\nThe application of controlled vocabularies and ontologies has accelerated over recent years; the Gene Ontology (GO9) being probably the most successful example in the field of biomedical ontologies. Many other ontologies exist, each of which has been developed for a specific domain in biomedicine. Now a major goal is to increase semantic and syntactic interoperability between those ontologies (e.g. the Open Biomedical Ontologies (OBO) Foundry10). One approach is to develop ontologies by defining complex (\"pre-composed\") classes in terms of other more elementary (atomic) classes (building blocks) that are species-agnostic. If several ontologies make use of shared building block ontologies, interoperability can be facilitated across a larger domain. For example ontologies that contain classes concerned with DNA-replication in different organisms or cells should refer to a shared class representing DNA-replication-process, enabling computers to detect that the same class is referenced.\n\nWe have previously shown how phenotype information can be linked and used in cross-species phenotype analyses11–15. A crucial part of this strategy is the use of logical definitions to render ontology terms in a way that is computable. Recently, logical definitions of terms representing classes of phenotypic deviations have been developed by several groups. Developers of OBO Foundry ontologies, such as the GO16, the Mammalian Phenotype Ontology (MPO17), the Human Phenotype Ontology (HPO18,19), the Worm Phenotype Ontology20, and also the Cell Ontology21, are now creating logical definitions of their ontology-classes using terms from other building block ontologies. In this effort the Phenotype, Attribute and Trait Ontology (PATO), an ontology of phenotypic qualities, is a key tool19,22. Examples for building block ontologies that are used for the representation of classes of phenotypic abnormalities are given in the upper part of Table 1.\n\n\nObjectives\n\nGiven that logical definitions exist for most classes of an ontology, automatic reasoners can be applied. These implement algorithms for computing the logical consequences that can be inferred from a set of asserted axioms. An example can be seen in Figure 1 a), where logical definitions are used to automatically infer that Hypoglycemia is a subclass of Decreased aldohexose concentration (blood) based on the asserted subclass relationship between 'glucose' and 'aldohexose' in ChEBI. This means that reasoners are able to use computable, logical definitions to infer the positions of classes in a subsumption hierarchy. Thus, those definitions can be helpful tools for the development and maintenance of ontologies16,23.\n\nFigure 1. Part a) illustrates the main idea how logical definitions and building block ontologies (left) cooperate in order to allow for reasoning procedures to infer new knowledge (right). Note that for the purpose of increased readability, only the term labels are shown and the ontology Uniform Resource Identifier (URIs) are skipped. Part b) illustrates an excerpt of the Uberpheno ontology to show how information on phenotype abnormalities in different organisms can be combined. It also illustrates how the annotations of genes can be transferred across different species by means of orthology relationships of genes. For example, after reasoning one could easily request all genes that are known to be related to the phenotype description \"Bilateral microphthalmos\" from the HPO. In Uberpheno \"abnormally hypoplastic eye\" from zebrafish (ZP) and \"posterior microphthalmia\" from MPO, are inferred to be subclasses of \"Bilateral microphthalmos\". These inferences can be used to infer that the genes tcf7l1a (zebrafish) and PRSS56 (mouse) are annotated to the phenotype \"Bilateral microphthalmos\" as well.\n\nAlthough several methods, ideas, and applications on cross-species phenotype integration have been presented before11,12,16,24,25, accessing such data resources has been complicated by the lack of consistent documentation and distribution of data across heterogenous resources. For example, some ontologies are provided in the Web Ontology Language (OWL26) and others in the Open Biomedical Ontologies (OBO) format. Although the OBO-format focuses especially on human readability and ease of parsing, OWL is often needed to enable complex reasoning tasks. Unfortunately, the power and complexity of OWL may discourage some researchers.\n\nFor example, the OWLSim package (http://owlsim.org) provides the ability to execute a number of standard semantic similarity techniques. Although access to the results of OWLSim in phenotype analyses is available (25, http://www.mousemodels.org), there is at the moment no single set of gene annotations linked to a single integrated ontology.\n\nThe Uberpheno-ontology is similar to the \"phene.owl\" ontology distributed as part of the phenomeblast-project (http://code.google.eom/p/phenomeblast/) and generated as part of a phenotype data analysis executed within PhenomeNET24. These two ontologies differ in a number of characteristics. The first characteristic is the underlying OWL model, and the set of external ontologies that are brought in to enrich the ontology - it is not yet clear how far the OWL model or some of these external ontologies affect the resulting structure of the ontology. Also it is likely that both Uberpheno and \"phene.owl\" will converge on the same model and a standard set of imported ontologies. The second characteristic is the breadth of species covered, with \"phene.owl\" including fly, worm and yeast; in contrast, Uberpheno focuses on human, mouse and zebrafish, yielding a smaller more focused ontology. Further investigations are required to determine the extent to which the adding of more distant organisms help or hinder analyses. Another difference is that Uberpheno is intended for a wide range of biomedical researchers, some of who may be unfamiliar with OWL or OWL reasoning.\n\nOur objective here is to provide an OBO-format ontology (Uberpheno), which we update at regular intervals and which can easily be used for downstream analysis, e.g. by applying semantic similarity measures27 or gene set enrichment analyses28. Of similar importance are the data that link into such an ontology by means of the annotation relation. To the best of our knowledge, no single integrated cross-species ontology together with annotation of all genes in human and model organisms (here mouse and zebrafish) has been made easily available for researchers and kept up-to-date on a regular basis.\n\n\nMaterials and methods\n\nCross-species ontology-based approaches offer a promising new methodology to reliably detect phenotypic similarities between human disease manifestations and model organism phenotypes6,11,24,25. They can pave the way to gain clinically relevant insights from the almost 5,500 genes for which, currently, only mouse and zebrafish phenotype information is available. Both the Mouse Genome Informatics (MGI) and the ZFIN data resources provide manually curated assignments of their model organism genes to human genes. They are available from the corresponding website (see Table 2).\n\nThese files are especially important for Step 4 in Figure 2.\n\nThe annotation of genes to phenotypes are also accessible online. Zebrafish genes are annotated by Entity-Quality (EQ) statements. Mouse genes are annotated with terms from the MPO and are downloadable from the MGI website. To associate human genes with terms from the HPO, the annotation of human diseases is required. By using further files from OMIM (http://omim.org) and Orphanet39, (http://www.orphadata.org/) diseases can be mapped to the disease-causing genes. These two steps allow the transfer of phenotype information to the underlying genes. All required files and their corresponding links are summarized in Table 2.\n\nThe approach taken to logically define phenotype descriptions is termed the Entity-Quality approach (EQ), in which phenotype descriptions can be partitioned into (minimally) two parts. The first part represents the affected entity, i.e. the thing for which an observation is made. This can be entities of various domains, e.g., a chemical or an anatomical structure. The second part represents the quality of the entity and is described in a qualitative or quantitative way22. In the typical setting, a phenotype is described using a class expression consisting of a PATO quality class differentiated by a bearer entity class using the inheres_in relation from the OBO Relation Ontology40. To give an example for logical definitions, consider the HPO term Hypoglycemia and its EQ definition, specified in OWL as shown in Figure 1 (center).\n\nThe word Hypoglycemia refers to an abnormally decreased concentration of glucose in the blood. The logical definition uses relations and follows the pattern described in previous work on the definition of phenotypes16. The logical semantics are made explicit when translating the definitions to OWL. Currently, the translation to OWL is performed using a \"has_part some\"-semantics implemented in the OBO-format library (http://code.google.com/p/oboformat). The translation is shown in Manchester syntax in Figure 1 a). In the example, the class Hypoglycemia is defined as the equivalent of the intersection of all classes of things that are \"A concentration which is lower relative to the normal\" (decreased concentration), \"deviate from the normal or average\" (abnormal), with respect to (towards) glucose, and inhering in \"blood\" (using the term portion of blood from the FMA). More details can be found in16 or23. Automated reasoning logically infers then that the asserted knowledge in ChEBI induces Hypoglycemia to be a subclass of Decreased aldohexose concentration (blood). The files used to define phenotype classes are summarized in Table 3.\n\nHPO and MPO files downloaded from http://code.google.com/p/phenotype-ontologies. Behaviour files downloaded from http://code.google.com/p/behavior-ontology. GO-xp file downloaded from http://obofoundry.org/cgi-bin/detail.cgi?id=biological_process_xp_uber_anatomy.\n\nThe general work- and data-flow of the cross-species ontology generation is illustrated in Figure 2. In steps one to three, the aforementioned EQ definitions are used to generate a single cross-species phenotype ontology (Uberpheno) for human, mouse, and zebrafish phenotypes. Step four generates files that make it very convenient to use the generated data for several research purposes, because genes are linked to the terms of the generated cross-species phenotype ontology, which is very lightweight and available in the convenient OBO-format.\n\nStep 1. Logical definitions are being developed for GO16, MPO12, and HPO19. Almost all logical definitions refer to classes from other ontologies. A set of logical definitions is again an ontology itself. These bridging ontologies (also called cross-product files) are available on the main OBO Foundry website, as well as from the individual repositories for each of the projects. An example for a logical definition is presented in the previous section and in Figure 1. A major fraction of HPO and MPO terms are currently defined by means of EQ statements and a summary of the logical definition files that are used can be found in Table 3. These files provide axioms that connect phenotype classes to multiple classes in most of the ontologies listed in Table 1.\n\nThe HPO and MPO logical definitions were augmented with pairwise equivalence axioms generated by lexical matching. These mappings are represented in a file mp_hp-align-equiv.owl (see the phenotype ontologies archive on Google code at http://code.google.com/p/phenotype-ontologies). A total of 1,064 such lexically derived equivalence axioms were derived in this way and used to supplement the semantic analysis.\n\nIn step one, all of the required files are pulled from the web (see Table 1 and Table 3). Note, that there are ontologies that are required in their entirety (denoted (B) in Figure 2). In contrast, several building block ontologies (denoted (A) in Figure 2) are only referred in parts by the logical definitions.\n\nWhen defining phenotypes using the EQ model, the affected entity can either be a biological function or process from GO, or an anatomical entity. Some of the ontologies used to create the definitions are largely species-independent (GO, ChEBI). However, anatomical entities are mostly defined by referring anatomy ontologies that are specific for one species. In order to enable reasoning across these vertebrate anatomies, the metazoan, species-independent Uberon ontology is used in constructing anatomically-based cross-products36. In order to construct Uberpheno, an equivalence axiom was generated between every class in Uberon that contains a cross-reference to a species anatomy ontology class. Note that very general terms from Uberon such as tissue are excluded, which can be identified by their membership to the subset upper_level in Uberon. The generated file is called uberonbridge.owl.\n\nOne of the files (see Table 3) defines GO process terms by the anatomy term to which the process is related. For example,\n\nClass: eye pigmentation\n\nEquivalentTo:\n\npigmentation and\n\noccurs_in some eye\n\nHere, the GO process eye pigmentation (G0:0048069) is logically defined as being equivalent to everything that is a pigmentation (GO:0043473) and also \"occurs_in\" an eye (UBERON:0000970). In order to use these definitions, the different relationships used therein, such as occurs_in, are made interpretable for the reasoner. For our purposes, an additional ontology called http://compbio.charite.de/svn/hpo/trunk/misc/go_xp_misc/extra_equiv.owl was created in which these relationships are made a subPropertyOf of inheres_in.\n\nStep 2. In step two a data preprocessing is required, because for zebrafish no pre-composed ontology of phenotype abnormalities exists (e.g. no phenotype term such as abnormally hypoplastic eye exists). Instead, the ZFIN project makes use of so-called \"post-composed\" annotations, using a combination of classes in the EQ model. The ZFIN-file pheno.txt (Table 2) contains lines such as\n\nZDB-GENE-980605-30;83439;tcf7l1a;ZFA:0000107;eye;PATO:0000645;hypoplastic;abnormal\n\nFor legibility the tab-separators are replaced in this example by the semicolon. In order to use these annotations for reasoning, a translation table was implemented, as described before12, which generates the ontology denoted as zp.owl. For every modified gene, a set of post-composed phenotype annotations is stored in pheno.txt. For every unique annotation for zebrafish genes, a class in the ZP identifier space is created. Again, the aforementioned \"has_part some\"-translation to OWL is applied. For example, a zebrafish gene annotation with\n\nEntity=ZFA:0000107 (eye),\n\nQuality=PATO:0000645 (hypoplastic) and\n\nQualifier=PATO:0000460 (abnormal)\n\ngenerates an OWL class:\n\nClass: ZP_0003395\n\nAnnotations: label \"abnormal(ly) hypoplastic eye\"\n\nEquivalentClassOf:\n\nhas_part some:\n\nPATO_0000645 and\n\ninheres_in some ZFA_0000107 and\n\nqualifier some PATO_0000460\n\nBeside generating the ZP-ontology, the annotation relation between the zebrafish genes and ZP-term is written to a file called zp.annot, which is also available for download.\n\nSince some logical definitions of phenotypes are lacking the qualifier abnormal we ensure consistency, by adding this qualifier to all of the definitions. We also remove the inconsistently used ontology-tags from the xp-files.\n\nSteps 3 and 4.At first, a single, merged OWL ontology is created from all the ontologies and bridging axioms. The ELK reasoner41 is used to calculate subclass and equivalence relationships between classes. These steps are implemented within the GULO framework23.\n\nTo increase the usability of the ontology, the Ontologizer API28 was used to merge all clusters of equivalent classes together into a single class. The HPO identifier is taken as the primary identifier if present and the identifiers of other phenotype classes are stored under alt_id-tag for the term. For example, the HPO-term Gallbladder dysfunction (HP:0005609) has as alt_id the ZP-term abnormal(ly) decreased functionality gall bladder (ZP:0004170). The resulting ontology in OBO-format is named crossSpeciesPheno.obo and contains only phenotype classes from the HPO, MPO, and ZP.\n\nFinally a cross-species annotation file is generated, in which all human genes are associated with terms from the Uberpheno. The annotations are either stemming from human or model organisms, whereby the model organism annotations are stemming from the ortholog gene.\n\n\nResults and discussion\n\nAll of the above described methods are integrated into a single pipeline. This pipeline automatically downloads required files, preprocesses the data and applies a reasoning procedure to the obtained set of ontology classes. The ontologies used to construct Uberpheno are summarized in Table 1.\n\nThe construction pipeline is set up as a job in our continuous integration system accessible at http://compbio.charite.de/hudson, which is already used for data related to the HPO42. The job (called hpo.ontology.uberpheno) is configured to run once a week, ensuring that the most recent version of all ontologies and annotation files are used. Only stable releases of the generated files are made available to the users and errors are immediately forwarded to us via email. The generated build artifacts are available at http://purl.obolibrary.org/obo/hp/uberpheno/, whereas the file crossSpeciesPheno.obo contains the cross-species phenotype ontology in OBO-format. The resulting ontology has a light footprint (3.5 MB) and can easily be explored by using tools such as example OBO-Edit43. Note that only phenotype classes are present in the ontology and classes from the referenced building block ontologies are filtered out. Each build also generates the file HSgenes_crossSpeciesPhenoAnnotation.txt, which contains the annotation of all human genes to terms of HPO, MPO, and ZP. A summary of the data contained in the two files is given in Table 4.\n\n'Phenotype classes' denotes the number of classes that are either from the Human Phenotype Ontology (HPO), Mammalian Phenotye Ontology (MPO), or zp.owl (ZP). Note that the sum of HPO-, MPO-, and ZP-IDs is higher than the total number total 'Phenotype classes' because some MPO- and ZP-IDs are listed as alt_id of an HPO-class and are not listed as separate 'Phenotype class'. Also, the number of human annotations is less than the sum of annotations supported by OMIM or Orphanet entries, because some annotations have evidence from both databases.\n\nAn excerpt of the Uberpheno ontology is shown in (Figure 1 b), demonstrating how the phenotype descriptions from different ontologies are combined and automatically organised into a single, integrated hierarchy. For instance, the fact that the mouse term posterior microphthalmia is inferred to be a subclass of the human term Bilateral microphthalmos can be used to transfer the information that the mouse gene PRSS56 is known to cause Bilateral microphthalmos. This implies that querying the cross-species ontology for genes related to Bilateral microphthalmos will return the human gene TCOF1, the mouse gene PRSS56 and the zebrafish gene tcf7l1a.\n\nIn total, the annotation file contains approx. 235,000 annotations of human genes with phenotype classes (see Table 4). For example the human gene TCF7L1 is associated with the zebrafish phenotype abnormal(ly) hypoplastic eye because the ortholog zebrafish gene (tcf7l1a, ZDB-GENE-980605-30) is annotated with this phenotype. Thus, the generated file HSgenes_crossSpeciesPhenoAnnotation.txt contains the line:\n\n83439;TCF7L1;abnormal(ly) hypoplastic eye (ZP:0003395);tcf7l1a (ZDB-GENE-980605-30/ZEBRAF)\n\n\nConclusions\n\nThe phenotype resources for mouse, zebrafish, and human are used by several research projects44–46.\n\nThe problem of comparing phenotypes between species can be overcome by using formal logical definitions that make use of species agnostic ontologies together with a multi-species anatomy ontology, Uberon. The approach to implementing the paradigm that we report in this paper constructs a single, integrated, cross-species phenotype ontology, Uberpheno, based on the logical definitions of human and the main model species, mouse and zebrafish. The resulting construct is continuously updated and automatically constructed as the constituent ontologies are updated and augmented, making it a dynamic and current resource available to the community.\n\nIncreasingly model organism data are being used for gene set enrichment, pathogenicity prediction and semantic similarity analyses27 and the high throughput phenotyping projects newly underway promise rich genome-wide phenotypic coverage within a decade. This will complement the new initiatives to systematically gather high precision, formally coded, phenotype data from clinical studies47. The promise that all this data holds can only be realized if the informatics tools are available to handle and analyse this rich resource and we believe that Uberpheno is an accessible and widely applicable resource with which this may be achieved.",
"appendix": "Author contributions\n\n\n\nSK, CJM, SEL, PS and PNR conceived the study. SK, SB, CJM and DS set up the code to create the ontology and the annotation files. BJR, SCD, DS, NW, GVG, PS and MW helped with the data preparation and processing. SK, CJM, PS, BJR, PNR and NW wrote the manuscript. All authors read and approved the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests declared.\n\n\nGrant information\n\nThis work was supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231, and by grants of the Deutsche Forschungsgemeinschaft (DFG RO 2005/4-1), the Bundesministerium fur Bildung und Forschung (BMBF project number 0313911), the MGD grant from the National Institutes of Health, HG000330, the ZFIN grant from the National Institutes of Health, U41-HG002659, and the grants from the National Institutes of Health, R01-HG004838 and R24-OD011883.\n\n\nAcknowledgements\n\nWe would like to thank Anika Oellrich for extensive proofreading of the draft version of the manuscript.\n\n\nReferences\n\nRosenthal N, Brown S: The mouse ascending: perspectives for human-disease models. Nat Cell Biol. 2007; 9(9): 993–9. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nYevgeny Kazakov, Markus Krötzsch, František Simancík: Concurrent classification of EL ontologies. In Lora Aroyo, Chris Welty, Harith Alani, Jamie Taylor, Abraham Bernstein, Lalana Kagal, Natasha Noy, and Eva Blomqvist, editors, Proceedings of the 10th International Semantic Web Conference (ISWC'11). volume 7032 of LNCS. Springer, 2011. Reference Source\n\nMungall CJ, Dietze H, Carbon SJ, et al.: Continuous Integration of Open Biological Ontology Libraries. Bio-Ontologies 2012. Reference Source\n\nDay-Richter J, Harris MA, Haendel M, et al.: OBO-Edit--an ontology editor for biologists. Bioinformatics. 2007; 23(16): 2198–200. PubMed Abstract | Publisher Full Text\n\nBayés A, van de Lagemaat LN, Collins MO, et al.: Characterization of the proteome, diseases, evolution of the human postsynaptic density. Nat Neurosci. 2011; 14(1): 19–21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAmberger J, Bocchini C, Hamosh A: A new face and new challenges for Online Mendelian Inheritance in Man (OMIM®). Hum Mutat. 2011; 32(5): 564–7. PubMed Abstract | Publisher Full Text\n\nSmith CL, Eppig JT: The Mammalian Phenotype Ontology as a unifying standard for experimental and high-throughput phenotyping data. Mamm Genome. 2012; 23(9–10): 653–68. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCommittee on a Framework for Development a New Taxonomy of Disease, National Research Council: Toward Precision Medicine: Building a Knowledge Network for Biomedical Research and a New Taxonomy of Disease. The National Academies Press, 2011. PubMed Abstract"
}
|
[
{
"id": "757",
"date": "08 Feb 2013",
"name": "Lawrence E. Hunter",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is really nice work, clearly demonstrating the value of logical definitions of ontology terms and of inference over those definitions. The automatic updating of the inference, ensuring that UberPheno reflects current annotations (and definitions, which change less frequently) should be adopted as a 'best practice' by other providers of derived information.",
"responses": []
},
{
"id": "761",
"date": "11 Feb 2013",
"name": "Francisco Couto",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe availability of a cross-species phenotype ontology built as a mashup of different other ontologies is a great contribution to the community and results from the implementation of a well-designed integration process. The process combines knowledge from different sources taking in account their provenance, which guarantees the continuous update of the proposed ontology. The successful exploitation of logical definitions is also a very good contribution to promoting the usage of more formal definitions which I believe will, for example, help to enhance the reliability of semantic similarity and enrichment analyses.",
"responses": []
},
{
"id": "764",
"date": "12 Feb 2013",
"name": "Nicola J. Mulder",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper describes a cross-species phenotype ontology, which promises to be extremely useful. It was not clear to me how much of the data preparation was purely computational versus some biological input. One has to be wary of trying to make too many terms apply to multiple species, but given the collective experience of these authors I am sure this has been taken into consideration.",
"responses": []
}
] | 1
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https://f1000research.com/articles/2-30
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https://f1000research.com/articles/3-17/v1
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21 Jan 14
|
{
"type": "Web Tool",
"title": "Ranking the quality of protein structure models using sidechain based network properties",
"authors": [
"Soma Ghosh",
"Saraswathi Vishveshwara",
"Soma Ghosh"
],
"abstract": "Determining the correct structure of a protein given its sequence still remains an arduous task with many researchers working towards this goal. Most structure prediction methodologies result in the generation of a large number of probable candidates with the final challenge being to select the best amongst these. In this work, we have used Protein Structure Networks of native and modeled proteins in combination with Support Vector Machines to estimate the quality of a protein structure model and finally to provide ranks for these models. Model ranking is performed using regression analysis and helps in model selection from a group of many similar and good quality structures. Our results show that structures with a rank greater than 16 exhibit native protein-like properties while those below 10 are non-native like. The tool is also made available as a web-server(http://vishgraph.mbu.iisc.ernet.in/GraProStr/native_non_native_ranking.html), where, 5 modelled structures can be evaluated at a given time.",
"keywords": [
"Proteins are known to take up unique well defined structures that allow them to function efficiently under a given condition1. This becomes much more fascinating when one considers the time taken by a protein to fold in vivo2. Studies over the past decades have facilitated the preparation of a blueprint of the rules that govern protein folding3–6. The roles of hydrophobic residues in structural packing",
"e.g. proline and glycine as helix breakers",
"are now very well established7",
"8. Details of the various pair-wise interactions that hold the structure intact are also available in the literature9. However",
"even with the wealth of resources available",
"determining the structure of a protein from its amino-acid sequence still remains a challenging task."
],
"content": "Introduction\n\nProteins are known to take up unique well defined structures that allow them to function efficiently under a given condition1. This becomes much more fascinating when one considers the time taken by a protein to fold in vivo2. Studies over the past decades have facilitated the preparation of a blueprint of the rules that govern protein folding3–6. The roles of hydrophobic residues in structural packing, e.g. proline and glycine as helix breakers, are now very well established7,8. Details of the various pair-wise interactions that hold the structure intact are also available in the literature9. However, even with the wealth of resources available, determining the structure of a protein from its amino-acid sequence still remains a challenging task.\n\nTo begin with, protein structure prediction requires understanding of the differences that exist between a well-folded protein structure and a modelled structure. Many large scale decoy structures that mimic a native protein structure, but with minor variations (such as the sidechain orientations, hydrogen bonds and so on), are now freely available10–12. Such datasets are generated using various computational approaches such as molecular dynamics13–15 and discrete state models16. Decoy structures can be compared with a large number of available native structures, hence, forming an important resource to understand patterns that are unique to natively folded proteins.\n\nFor many years now, proteins structures have been represented as networks, with residues forming nodes with edges representing various factors that are important for protein structures, such as hydrogen bonds17, and Cα distances18. Although these networks help in understanding the structure of a protein at the level of secondary structures and backbone atoms, determining the subtle changes that occur at the level of sidechain interactions are not captured. We have been working on Protein Sidechain Network (PSN) for a number of years19,20 and have done various rigorous analyses at different levels to show its usefulness21–26. Generating networks at the level of a sidechain not only takes care of the geometry but also the chemistry that is encoded in the sidechain atoms of every amino acid in the polypeptide chain.\n\nSupport vector machine (SVM) is a machine learning algorithm mainly used for the purpose of classification27. The algorithm uses a training dataset to learn patterns and finally use those patterns to classify new cases. Given the complexity of biological systems, machine learning algorithms are widely used in biology to predict cellular locations28,29, cancer tissue classifications based on gene expression data30–32 and further in cases of protein structures to identify SCOP classes33, binding sites34,35 and also the quality of protein structures using features, such as secondary structures and hydrophobicity36,37.\n\nRecently, we have demonstrated the capabilities of PSNs to distinguish native structures from decoy models. We started with comparing the network properties of PSNs from native and decoy models where we established the unique network features exhibited by native structures38. This work was further followed by an in-depth analysis, where PSNs at different interaction strengths (Imin = 0%–7%) and SVM were used in tandem to classify the protein as native or non-native like. Further, the method was validated using a large number of CASP 10 [10th community wide experiment on the Critical Assessment of Techniques for Protein Structure Prediction] predicted models. Overall, an accuracy of 94% was achieved by this method39.\n\nAs an extension of our previous work, where a simple binary classification was carried out39, here we have developed a method to rank the quality of model structures through probability estimates. This advance is particularly important in cases where one needs to select the best quality structures from a set of many similar and good quality models. Many tools have now been developed that can successfully generate many possible structure candidates from a sequence; however, predicting the best from this list is still a demanding task and needs attention. In the present study we have observed that the structures with a rank greater than 16 generally show native like properties and hence this method provides a good measure for the rank and quality of a model.\n\n\nMethods\n\nThe main aim of this work was to obtain a ranking for a set of modeled structures and to select the best modeled structure that closely resembles a native structure. To achieve this goal, we obtained a large number of native and non-native structures and generated PSNs. The network parameters from the PSNs are combined with SVM to build a mathematical model and the ranking of each structure is determined using logistic regression analysis. Details of each step are provided below.\n\nTwo sets of data were used for this study;\n\na) a positive dataset (PSN-QA_positive), that consisted of 5422 protein crystal structures with resolution < 3A, R-factor < 0.25 and PDB size > 100 This dataset was curated using PISCES40,\n\nb) a negative dataset (PSN-QA_negative) that considered different decoys as well as modelled structures from various publicly available resources and databases.\n\nDetails of the individual datasets are provided in Table 1. Finally, a total of 29543 non-native structures were obtained.\n\nTable modified from39\n\n\n\nAs mentioned above, our laboratory has been working extensively on protein structure networks19, specifically generated at the level of non-covalent interactions of sidechains. Details to generate PSNs are available in our previous work20 and a brief description is provided here.\n\nPSNs are generated by considering amino acids as nodes and edges are constructed between these nodes based on the non-covalent interaction strengths between them. Interaction strengths between any two residues is calculated as follows,\n\n\n\nwhere, Iij = strength of interaction between residues i and j, where |i – j| ≥ 2; nij = number of distinct interacting atom pairs between i and j within a distance cut-off of 4.5 Å (excluding the backbone atoms); Ni and Nj are the normalization values for residues i and j obtained from a statistically significant dataset of proteins, as defined in our previous work20. Based on the interaction strengths between these residues, PSNs can then be generated at different interaction strength cutoffs (Imin), with a lower cutoff generating a dense network and including even the weaker interactions, while a higher cutoff signifies a network made of very strong non-covalent interactions and hence sparse. For this study, PSNs were generated at different Imins ranging from 0% to 7%.\n\nVarious network parameters such as number of non-covalent interactions (NCov), size of the largest cluster (SLClu), clustering coefficient (CCoe), size of the largest k-1 and k-2 communities, are calculated for each PSNs generated. Furthermore, the differences between these parameters at consecutive Imins are also considered in this study. In our previous studies39, we have discussed the importance of the transition profile of the various network parameters as a function of Imin to characterize the native structures and therefore distinguish them from the non-native ones. Along with the network parameters, main chain hydrogen bonds (MHB)41 were also analysed and included in the study. Table 2 provides a detailed list of all the network parameters that have been used in this study.\n\nTable adapted from39\n\nAs described before, SVMs are machine learning algorithms that learn patterns from a training dataset and further use that pattern to classify new datasets. In this study, we have built an SVM classifier based on the patterns that are specific for a native PSN. First, we randomly divided the datasets into a training set and a test set, so that the training set contained 3000 native structures and 3000 non-native structures. Remaining structures were set aside to form the test set. This was repeated 10 times to generate 10 random test sets and training sets. Compared to our previous study, we here went one step further and used the liblinear package of LibSVM42,43, to obtain the probability estimates (using –s8 option in the liblinear package) of each data point and thereby to obtain ranks for each of them. Furthermore, since the different network parameter values have different ranges, the values were scaled between -10 to +10 before the analysis.\n\n\nResults\n\nTwelve network features (at different Imins) (Table 2) and MHB are combined to get a total of 94 features that best characterize a PSN. Details about these parameters and the characteristic transition curves specific to PSNs generated from native structures are discussed in detail in our previous work39. Briefly, the transition profiles (Figure 1) as obtained by plotting the network features of native protein structures as a function of Imin show three specific features, a) higher value at lower Imin, (b) lower value at higher Imin and finally (c) steep transition between Imin = 1%–4%. Figure 1 shows the transition profile of 7 randomly selected native protein structures and their corresponding 981 model structures. A clear difference between the transition profiles of a native protein structure and decoy/modelled structures is visible. These differences are observed in all the datasets used in this study and forms the basis of the method developed here.\n\nTransition profile of one of the network features (SLClu; see Table 2) as a function of Imin is shown for 7 randomly selected native structures [green] and their corresponding decoy structures [red]. A clear distinction between the two transition profiles is visible, highlighting the 3 characteristic features that are uniquely displayed by native protein structures. X axis represents Imin from 0% to 7% and Y axis represents the average value of the SLClu obtained by native and decoy/modelled structures.\n\nThe main aim of this work was to obtain a ranking scheme for structure quality prediction. The 94 network features were combined into SVM using the liblinear package to obtain a ranking model. Specifically, for model generation, ‘L2-regularized L2-loss ranking support vector machine’ solver and cost value (c) equal to 2 was used43. As mentioned in the Methods section, 10 random training and test sets were obtained and the ranking model was generated for all the train sets. Finally, the model which showed the best pairwise accuracy of 98.2% was selected for further analysis.\n\nFigure 2 shows the percentage distribution of the ranks obtained by the 5422 native protein structures and 29543 non-native structures. These ranks represent the quality of the structures as determined by the network parameters using the SVM trained model. From figure 2, it is now quite evident that native structures almost always score above 16, while the scores of the non-native structures range from -70 to 20 with the majority being ≤ 16. It should be pointed out here that the dataset of decoy structures is taken from databases such as CASP and Rosetta and therefore in many cases might also contain structures very close to native or almost native like, thereby leading to some structure scoring beyond 16, but always ≤ 20. From Figure 2, it can now be safely assumed that structures scoring above 16 show native like properties and scores of bad, unrefined models are generally very low.\n\nThe figure shows the percentage distribution of ranks for the 5422 native structures (blue) and 29543 decoy/modelled structures (red). X-axis represents ranks while Y-axis represents the percentage distribution. It is clear that native structures have higher ranks (> 16) as compared to the decoy/modelled structures.\n\nThis tool is now made freely available for public use in the form of a web-server, http://vishgraph.mbu.iisc.ernet.in/GraProStr/native_non_native_ranking.html. Figure 3 shows the home page of the web-server (Figure 3a) and the output format (Figure 3b). A test case (PDB Id: 1CG5 and its decoy structures from Rosetta) is also provided with its scores as an example. Figure 4 shows the screenshot of the example test case. The tool can analyse five structures at a given time. For structures with multiple chains, individual chains are treated as different structures for the analysis. The tool accepts files in PDB formats as input and outputs the ranks for each model in a tabular format.\n\nThe figure shows screenshots of the a) home page and b) results page for structure ranking. At a given time, 5 structures can be uploaded. For structures with multiple chains, each chain would be treated individually. The output would be provided in a tabular format.\n\nFor easy understanding, a test case of native structure (PDB Id: 1CG5) and its two decoy structures (from Rosetta) is also made available. The page shows the structures and the PSN scores obtained by them. PDB files are also available for download.\n\n\nDiscussion\n\nProper folding of protein structures is imparted by various energetic and topological features1,3–9. While the secondary structures are stabilized by backbone hydrogen bonds, the mutual orientation of the secondary structures are uniquely determined by the sidechain interactions. Although studies at the backbone level have contributed enormously to the understanding of the protein structure17,18, they are not sufficient to understand the subtle balance at the atomic level. Our previous studies have highlighted the role of non-covalent interactions of the sidechain atoms in functioning23,25,26 as well as stability22,24 of protein structures. Protein structure networks are designed to account for sidechain interactions and therefore the network captures not only the geometric but also the chemistry encoded in the sidechain.\n\nIn our earlier studies, we had exploited protein structure networks to discriminate the native structures from the non-native ones. This is mainly done at the level of sidechain with only one important feature, MHB, representing the properties of the backbone atoms. In all these studies38,39, discrimination between the two sets is done qualitatively, with the method simply classifying the structures as native or non-native. Such qualitative analysis becomes ineffective when used for closely related and almost native like structures. However, given the current state of art in the field of protein structure prediction, we believe that expertise has been attained to predict near native like structures and more work is required now to select the best structure from a set of very similar structures.\n\nThe present work is an extension of our earlier work, where we have addressed the issue described above in a quantitative manner. Here, we have built a model that would score the structures based on how closely they mimic a native structure, instead of providing a simple binary classification. We were able to use the liblinear package of libSVM to build such a model. The model was further tested on a set of 5422 native structures and 29543 decoy/modelled structures. The ranking scheme (Figure 2) is clearly able to discriminate good structures from the bad ones. All the 5422 native structures get a rank greater than 16, while the scores for decoy/modelled structures range from -70 to 20. Overall, it can be concluded that structures with score > 16 display native like properties as evaluated from a network perspective and the models below the score of 12 are definitely show non-native like properties and do not mimic native structures.\n\n\nConclusion\n\nIn summary, large numbers of native as well as decoy/modelled structures have been used to build an SVM model. This model was trained using 94 features that included 93 network parameters and main chain hydrogen bonds. The model has an overall accuracy of 98.2% and can successfully rank structures based on their quality as determined from protein structure networks. Generally, structures with rank > 16 display native like properties and can be regarded as good quality structures. This is an important advancement from the previous qualitative assessments and would be helpful in cases where one needs to extract the best structure from a set of closely related structures.\n\n\nData and software availability\n\nData\n\nFigshare: Protein Structure Network : Quality Assessment (PSN-QA), doi: 10.6084/m9.figshare.90283847.\n\nSoftware\n\nProtein Structure Network Quality Assessment (PSN-QA) tool: http://vishgraph.mbu.iisc.ernet.in/GraProStr/native_non_native_ranking.html",
"appendix": "Author contributions\n\n\n\nSV conceptualized the idea and supervised the project. SG performed the analysis and developed the web server. Both authors wrote and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nSG is supported by DBT fellowship [DBTO/BMB/SV/364], Department of Biotechnology (DBT), Government of India.\n\n\nAcknowledgements\n\nWe thank Subhojyoti Chatterjee who participated in the earlier work of network analysis on decoy structures.\n\n\nReferences\n\nAnfinsen C: Principles that govern the folding of protein chains. Science. 1973; 181(4096): 223–230. PubMed Abstract | Publisher Full Text\n\nDaggett V, Fersht A: The present view of the mechanism of protein folding. Nat Rev Mol Cell Biol. 2003; 4(6): 497–502. PubMed Abstract | Publisher Full Text\n\nChan HS, Dill KA: The protein folding problem. Phys Today. 1993; 46(2): 24. 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PubMed Abstract | Publisher Full Text\n\nBrinda K, Vishveshwara S: A network representation of protein structures: implications for protein stability. Biophys J. 2005; 89(6): 4159–4170. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrinda KV, Kannan N, Vishveshwara S: Analysis of homodimeric protein interfaces by graph-spectral methods. Protein Eng. 2002; 15(4): 265–277. PubMed Abstract | Publisher Full Text\n\nKannan N, Vishveshwara S: Aromatic clusters: a determinant of thermal stability of thermophilic proteins. Protein Eng. 2000; 13(11): 753–761. PubMed Abstract | Publisher Full Text\n\nNoble WS: What is a support vector machine? Nat Biotechnol. 2006; 24(12): 1565–1567. PubMed Abstract | Publisher Full Text\n\nPark KJ, Kanehisa M: Prediction of protein subcellular locations by support vector machines using compositions of amino acids and amino acid pairs. Bioinformatics. 2003; 19(13): 1656–1663. PubMed Abstract | Publisher Full Text\n\nHua S, Sun Z: Support vector machine approach for protein subcellular localization prediction. Bioinformatics. 2001; 17(8): 721–728. PubMed Abstract | Publisher Full Text\n\nKhan J, Wei JS, Ringner M, et al.: Classification and diagnostic prediction of cancers using gene expression profiling and artificial neural networks. Nat Med. 2001; 7(6): 673–679. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGuyon I, Weston J, Barnhill S, et al.: Gene selection for cancer classification using support vector machines. Machine Learn. 2002; 46(1–3): 389–422. Publisher Full Text\n\nFurey TS, Cristianini N, Duffy N, et al.: Support vector machine classification and validation of cancer tissue samples using microarray expression data. Bioinformatics. 2000; 16(10): 906–914. PubMed Abstract | Publisher Full Text\n\nCai YD, Liu XJ, Xu Xb, et al.: Prediction of protein structural classes by support vector machines. Comput Chem. 2002; 26(3): 293–296. PubMed Abstract\n\nKoike A, Takagi T: Prediction of protein–protein interaction sites using support vector machines. Protein Eng Des Sel. 2004; 17(2): 165–173. PubMed Abstract | Publisher Full Text\n\nBradford JR, Westhead DR: Improved prediction of protein–protein binding sites using a support vector machines approach. Bioinformatics. 2005; 21(8): 1487–1494. PubMed Abstract | Publisher Full Text\n\nKryshtafovych A, Fidelis K: Protein structure prediction and model quality assessment. Drug Discovery Today. 2009; 14(7): 386–393. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDong Q, Chen Y, Zhou S: A machine learning-based method for protein global model quality assessment. Int J Gen Syst. 2011; 40(04): 417–425. Publisher Full Text\n\nChatterjee S, Bhattacharyya M, Vishveshwara S: Network properties of protein-decoy structures. J Biomol Struct Dyn. 2012; 29(6): 606–622. PubMed Abstract | Publisher Full Text\n\nChatterjee S, Ghosh S, Vishveshwara S: Network properties of decoys and CASP predicted models: A comparison with native protein structures. Mol Biosyst. 2013; 9(7): 1774–1788. PubMed Abstract | Publisher Full Text\n\nWang G, Dunbrack RL: PISCES: a protein sequence culling server. Bioinformatics. 2003; 19(12): 1589–1591. PubMed Abstract | Publisher Full Text\n\nMcDonald I, Naylor D, Jones D, et al.: HBPLUS computer program. Department of Biochemistry and Molecular Biology, University College, London, UK. 1993. Reference Source\n\nChang CC, Lin CJ: LIBSVM: a library for support vector machines. ACM Trans Intell Syst Technol (TIST). 2011; 2(3): 27. Publisher Full Text\n\nFan RE, Chang KW, Hsieh CJ, et al.: LIBLINEAR: A library for large linear classification. J Machine Learn Res. 2008; 9: 1871–1874. Publisher Full Text\n\nLeiserson CE, Rivest RL, Stein C, et al.: Introduction to algorithms. The MIT press. 2001. 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}
|
[
{
"id": "4283",
"date": "14 Apr 2014",
"name": "Rahul Banerjee",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe title is appropriate, and the abstract represents a suitable summary of the work. Although the design and methodology of the calculation are appropriate for the subject under\n\nstudy, some aspect of the calculation could have been explained in greater detail. This is discussed in greater detail in the report below. The conclusions are justified on the basis of the results obtained in the study. Enough information has been provided to replicate the calculations.The authors provide a novel method to validate protein structures based on the network properties of non-bonded side chain contacts within proteins. The method could find extensive application in the structural validation of both experimentally determined protein structures by x-ray crystallography, or modeled structures. Thus as a validation tool it could prove to be an extremely valuable addition to other existing methods. The authors have also installed a web server, thus making the facility available to a wide cross section of potential users.The success of any validation method depends on the scoring (or ranking) scheme adopted to sort structures based on some criteria. Unfortunately, the details regarding the ranking scheme are extremely terse or assume that the reader will be conversant with the details of support vector machines (SVM) and the relevant software (LibSVM). That need not be the case, as potentially work such as this should have a wide appeal. Although the authors do cite previous work, they could discuss this in somewhat greater detail. What do the terms or options used in the sentence ‘Specifically, for model generation, ‘L2-regularized L2-loss ranking support vector machine’ solver and cost value (c) equal to 2 was used.’, actually signify? Why was the specific option (s8) chosen?The authors could also compare their methodology with currently available validation packages such as Procheck or Molprobity on a small database consisting of native protein and decoys. Given the fact that experimentally determined erroneous structures occasionally seep through the currently available validation filters, this method could provide crucial information in error detection, where other methods consistently fail.",
"responses": []
},
{
"id": "4284",
"date": "06 May 2014",
"name": "Soumen Roy",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper aims at ranking protein structures in order to differentiate native protein structures from non-native/decoy models. For this, the authors employ machine learning approaches (Support Vector Machines) and assign ranks, based on regression analysis, to these models using Protein Structure Networks. This study includes the side chain interactions of amino acid residues unlike the previous network based approaches for detecting the most native-like structures from a huge set of decoys. The authors have built upon their earlier work employing Protein Structure Networks (PSN) to differentiate the native conformations from decoy/modelled structures; PSN parameters (93 network features) along with main chain hydrogen bonds were used to built the SVM classifier. The web tool provides wide and simple accessibility of the aforementioned methods to the larger community. Non-specialists should find it useful. We have the following comments:Use of network metrics – mostly based on “size” of higher communities and largest cluster, and the average clustering coefficients have been discussed by the authors. A discussion as to why these particular metrics have been chosen over so many other available network metrics would certainly be helpful for researchers with a keen interest in network theory. The methodology of ranking structures could certainly be presented in more detail. The part preceding “Finally, the model which showed the best pairwise accuracy of 98.2% was selected for further analysis” would do much better with a more detailed explanation, especially about how LibSVM is useful here. It would be good to know the computational complexity as well as advantages of the present approach over other accessible packages. A table summarizing this would be a highly desirable addition especially because the authors state in the Conclusion that this is an important advancement from the previous qualitative assessments and would be helpful in cases where one needs to extract the best structure from a set of closely related structures.",
"responses": []
}
] | 1
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https://f1000research.com/articles/3-17
|
https://f1000research.com/articles/3-16/v1
|
20 Jan 14
|
{
"type": "Case Report",
"title": "Surface arthroplasty in a cerebral palsied patient with hip dysplasia and dislocation",
"authors": [
"Carlos G. Rubín",
"Pablo G. Rubín",
"Pablo G. Rubín"
],
"abstract": "Hip dysplasia is considered to be an etiological factor in the progress of developmental osteoarthrosis of the hip. In patients with cerebral palsy, hip dislocation plus other anatomical alterations such as acetabular dysplasia, coxa valga, excessive femoral anteversion and muscle imbalance can be factors leading to coxarthrosis and pain. Total hip arthroplasty is a valuable method of treatment in some of these patients. However, sometimes young age, bone anatomy and shape, and the possible lack of stability of conventional designs in the context of big surgeries with multiple muscle releases and possible osteotomies make this arthroplasty a very difficult indication to reproduce as a surgeon. Hip resurfacing is a good option because it preserves as much bone stock as possible, doesn’t need corrective osteotomies and gives the prosthesis improved stability. The main concern about this type of prosthesis is the suspected lower survival rate of the femoral component, compared with regular total hip arthroplasties. We present a cerebral palsied patient with hip dysplasia who underwent resurfacing surgery. At a follow up one year after surgery, the patient was pain free, had recovered his motion and functionality, and was able to walk.",
"keywords": [
"Cerebral palsy. Hip dysplasia. Hip resurfacing."
],
"content": "Introduction\n\nCerebral palsy is a syndrome caused by a non-progressive upper motor neuron lesion, originating before the central nervous system is mature. Patients suffer abnormal control of motor and sensory function and sometimes abnormal intellectual development. There are different etiological factors but perinatal anoxia is one of the most common. The spastic type is the most frequent and is characterized by rigid muscles with an inability to relax that produces a variety of anatomical and clinical signs. The incidence of hip dysplasia is very high in these patients, including adduction contractures, increased femoral anteversion, coxa valga and acetabular dysplasia. Pain is highly associated with these abnormalities and up to 50% of patients have hip dislocation1. A dislocated painful hip in a patient with cerebral palsy can be treated by muscle releases, femoral or pelvic osteotomies and reduction, Girdlestone, arthrodesis or total hip arthroplasty1,2. In a young patient with a good quality of life who was previously able to walk, we think that the best option is a total hip arthroplasty1–4 in order to relieve pain, allow perineal care and facilitate mobility and the ability to walk independently.\n\n\nCase report\n\nIn 2011, a 15-year-old boy, Caucasian with triplegic cerebral palsy came to the clinic in a wheelchair, accompanied by his family. He complained of intense pain in his right hip that had been increasing over the last years. Unfortunately, the pain had translated to poor function. He had sparing of his upper left extremity of the hip. He described his hip function as limited and poor, and as continuously worsening. He tried not to bear weight on his right leg, although he was still able to walk around and transfer himself, usually with a Kay posture walker, in his house and around a baseball field.\n\nIn addition to the pain, he also had a history of right hip subluxation. Ten years previously, he had bilateral hip soft tissue and muscle releases, and osteotomies which were varus producing. Four years before, he had spinal fusion to help his alignment, and that did seem to help him to some degree. At that point his doctors noticed that his hip was coming out of the socket. His parents were very fearful of him losing his hip functionality as they saw that it had been getting worse in the four years after the spinal fusion. They had seen other doctors who had recommended Girdlestone rather than total hip replacement.\n\nSubsequent X-rays demonstrated hip dysplasia and a dislocated joint with bowing of his femur in the subtrochanteric area. The head was completely out-of-round (Figure 1). The patient also had mild mitral regurgitation and suffered some spasticities whilst on a baclofen pump.\n\nOn physical examination he had some spasticity in his lower extremities and very limited movement of his right hip mainly because of pain. His muscle strength was 4/5 in the right leg. His adductor tendons could be felt but they did not seem overly tight.\n\nAt this point, total hip replacement was recommended, and the family were informed that he would most likely require subtrochanteric osteotomy, realignment of his femur, placement of a modular-type implant, formation of a new socket and soft tissue releases.\n\nDuring the reviewing and planning of the case and surgery, we thought about the possibility of implanting a resurfacing hip prosthesis with the idea of maximizing the benefits of this type of device in our patient. Doing so would decrease the duration of the surgery and we wouldn’t need to perform an osteotomy on the femur with consequent lower blood loss and reduced perioperative risks. Also we could achieve the best intrinsic stability and maintain as much proximal femoral bone stock as possible. In addition, the bearing surfaces were perfect as he was a very young patient. Our main concern was the durability of the replacement in terms of loosening or fracture.\n\nThe family and the patient were carefully informed about the pros and the cons of both surgical options and they accepted our indication of hip resurfacing rather than conventional hip replacement.\n\nThe surgery was conducted in the usual manner, approaching the hip posteriorly without any special issues. The new hip was relocated, moderate soft tissue release was realized and intraoperative regular mobility and stability were accomplished. Postoperative X-rays demonstrated that the implants were well positioned and normal anatomy was almost perfectly recovered (Figure 2 and Figure 3).\n\nFour to five days following surgery, the patient started walking and was put on physical therapy in the typical time and fashion. He had to use an abduction brace for 6 weeks post-surgery. He recovered well but slowly.\n\nAt a follow up one year after surgery, the patient was pain-free, walked with a one-handed assist, played without restrictions and had recovered a very good functional status. The passive range of motion in his right hip was much better than it was preoperatively, allowing him to sit and facilitating perineal care. His improvements had been very slow but his gait was still getting better each day.\n\nThe two main goals for our patient, pain relief and functional improvement, had been achieved.\n\n\nDiscussion\n\nTotal hip replacement is currently a common procedure used to manage painful hips in patients with arthrosis and dysplasia3,5–10. Cerebral palsy, even in the presence of hip dislocation, must not dissuade the surgeon from performing this procedure, especially in ambulatory patients4,5. Resurfacing hip prosthesis is a type of hip replacement that should be considered in these patients because of its benefits in terms of stability5, preservation of bone stock and low wear expectations5,11. The presence of femoral deformities could be considered as a relative contraindication for resurfacing of the hip9, but in some cases the surgeon should consider that hip resurfacing avoids the difficulty sometimes encountered in patients with dysplasia during total hip replacement of a narrow and frequently curved proximal femoral canal6. The survivorship of this implant at five years in patients with different levels of dysplasia is similar to the rates for total hip replacements, always above 95%6,7. The clinical outcomes of resurfacing with respect to pain scores, restoration of the joint biomechanics, range of motion and walking and sport activities are also very good at five and ten years2,4,6,8,9; even some measurements such as range of motion can be better after hip resurfacing than after total hip replacement7. Furthermore, there is the possibility of an easier conversion to a total hip arthroplasty if needed.\n\nThere are no reports about the use of hip resurfacing in cerebral palsied patients but considering that these devices have demonstrated good results in dysplastic hips, we think that cerebral palsy may not be a complicating factor and the results or resurfacing could also be comparable to those obtained in not palsied individuals with dysplasia. For instance, it has been shown that other procedures such as acetabuloplasty, pelvic osteotomies and femoral osteotomies with or without open reduction of the hip have similar outcomes in patients with and without cerebral palsy3.\n\nFurther studies will be needed to demonstrate conclusively that resurfacing is as valuable as total hip replacement in the majority of cerebral palsied hips.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient and his parents.",
"appendix": "Author contributions\n\n\n\nBoth authors followed the patient for more than a year. CGR was the main person involved in writing the case and doing the review of the literature, PGR revised the manuscript. Both authors agreed the final manuscript for publication.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nGamble JG, Rinsky LA, Bleck EE: Established hip dislocations in children with cerebral palsy. Clin Orthop Relat Res. 1990; (253): 90–9. PubMed Abstract\n\nRoot L, Goos JR, Mendes J: The treatment of the painful hip in cerebral palsy by total hip replacement or hip arthrodesis. J Bone Joint Surg Am. 1986; 68-A(4): 590–8. PubMed Abstract\n\nGavrankapetanovic I, Cobeljic G, Bajin Z, et al.: Developmental dysplasia of the hip in cerebral palsy--surgical treatment. Int Orthop. 2007; 31(4): 561–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSpencer JD: Reconstruction of dislocated hips in children with cerebral palsy. BMJ. 1999; 318(7190): 1021–2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchroeder K, Hauck C, Wiedenhöfer B, et al.: Long-term results of hip arthroplasty in ambulatory patients with cerebral palsy. Int Orthop. 2010; 34(3): 335–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcBryde CW, Shears E, O'Hara JN, et al.: Metal-on-metal hip resurfacing in developmental dysplasia: a case-control study. J Bone Joint Surg Br. 2008; 90(6): 708–14. PubMed Abstract | Publisher Full Text\n\nAmstutz HC, Le Duff MJ, Harvey N, et al.: Improved survivorship of hybrid metal-on-metal hip resurfacing with second-generation techniques for Crowe-I and II developmental dysplasia of the hip. J Bone Joint Surg Am. 2008; 90(Suppl 3): 12–20. PubMed Abstract | Publisher Full Text\n\nWeber M, Cabanela ME: Total hip arthroplasty in patients with cerebral palsy. Orthopedics. 1999; 22(4): 425–7. PubMed Abstract\n\nNaal FD, Schmied M, Munzinger U, et al.: Outcome of hip resurfacing arthroplasty in patients with developmental hip dysplasia. Clin Orthop Relat Res. 2009; 467(6): 1516–21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSpiegel DA, Flynn JM: Evaluation and treatment of hip dysplasia in cerebral palsy. Orthop Clin North Am. 2006; 37(2): 185–96. PubMed Abstract | Publisher Full Text\n\nXu WD, Li J, Zhou ZH, et al.: Results of hip resurfacing for developmental dysplasia of the hip of Crowe type I and II. Chin Med J (Engl). 2008; 121(15): 1379–83. PubMed Abstract"
}
|
[
{
"id": "3201",
"date": "13 Feb 2014",
"name": "Jenny McConnell",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis brief case study outlines an alternate surgical approach of surface arthroplasty for a young cerebral palsied patient who was severely disabled by hip pain and dislocation. While it is beneficial for clinicians to read about successful surgical interventions particularly for someone who is young and has difficult anatomy, the authors have been very sparring in their details about the case. As the patient had spastic triplegia, I am unclear as to which limb was spared from the statement \"He had sparing of his upper left extremity of the hip\" - does this mean his left arm or his left leg? Also it is difficult to understand how someone who has \"limited and poor hip function\" and \"did not want to bear weight on his right leg\" could \"walk around with a Kay posture walker on a baseball field - which is usually quite large in area.\" Some detail about how many levels of the spine were fused is required in the history. The authors should also comment on the patient's skeletal maturity as I would suspect that over the last four years the increase in long bone development and change in muscle mass may have exacerbated his hip joint position, symptoms and functionality. Was the patient skeletally mature when they performed the surface arthroplasty? The authors mentioned that when the spinal fusion was performed, \"the hip was coming out of the socket\" and continued to worsen after the spinal fusion, so perhaps they should have commented further on whether a spinal fusion is suitable for an immature cerebral palsied individual and on the effect a fusion may have on acetabular and femoral position.I am surprised that all the muscles in his right leg were 4/5, particularly the hip external rotators and the extensors (gluteus maximus). The adductor tendon tightness with the changed hip joint centre, as the hip was dislocated, would be difficult to assess. The authors are referred to Delp & Maloney (1993), who investigated changing hip joint centre and moment arms of the muscles around the hip, specifically for consideration in diplegic patients.",
"responses": []
},
{
"id": "4296",
"date": "08 Apr 2014",
"name": "George Papachristou",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe presented interesting case study deals with a 15-year-old Caucasian boy, with triplegic cerebral palsy. Athetosis is not mentioned. From my personal experience of nearly 40 years I favour THA for the treatment of painful hips with subluxation or dislocation with spasticity, combined with the necessary soft tissues and muscles release; a procedure which has long term acceptable outcomes. The survivorship for THA, in cerebral palsy patients, is referred to be 85% for 10 years with relief from preoperative pain and improved function. Nevertheless, because of the difficulty of preoperative assessment and of selecting the right surgical indications, orthopaedic surgeons have been reluctant to recommend THA in these patients. In the present case study the authors attempted to treat an adolescent boy with a resurfacing hip artroplasty, combined with the necessary soft tissue and muscles release, a surgical procedure which has not been used previously for such a case. So far, according to the present authors, the proposed surgical procedure is successful.Since the long term results for cerebral palsied patients treated with THA, cemented or not, are acceptable, I cannot see why a resurfacing arthroplasty would not be successful even in an adolescent patient of this group. Further studies are definitely needed as well as confirmation of long term successful results.I have the following additional comments:There is a lack of information about the preoperative status of the spinal column, the extent of spinal fusion and any benefits its correction had for the patient. Also, the x-ray of the pelvis shows a degree of obliquity, which might allow greater than average forces to act upon the hip. One of the most important things to evaluate is the functional situation of a patient suffering from spasticity, which can lead to subluxation or dislocation and also to exclude athetosis, which complicates treatment or makes it impossible. Although it is not mentioned in this report I assume that the previous operative procedures have improved the muscle balance of the patient, a very important element, hopefully towards the right direction. Hip subluxation or dislocation in the cerebral palsy population is a problem for these patients and the reported incidence is from 18% to 59%. For painful hip subluxation or dislocation with arthrosis in the adolescent or adult, procedures such as hip arthrodesis, valgus osteotomy, proximal femoral resection, or total hip arthroplasty have all been done to relieve pain and improve function.",
"responses": []
}
] | 1
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https://f1000research.com/articles/3-16
|
https://f1000research.com/articles/2-260/v1
|
27 Nov 13
|
{
"type": "Commentary",
"title": "Promiscuous scaffolds in proteins - non-native, non-additive and non-trivial",
"authors": [
"Sandeep Chakraborty",
"Bjarni Asgeirsson",
"Mouparna Dutta",
"Anindya S Ghosh",
"Masataka Oda",
"Adela Rendón",
"Felix Goñi",
"Jean-Marie Frere",
"Ravindra Venkatramani",
"Abhaya M. Dandekar",
"Basuthkar J. Rao",
"Bjarni Asgeirsson",
"Mouparna Dutta",
"Anindya S Ghosh",
"Masataka Oda",
"Adela Rendón",
"Felix Goñi",
"Jean-Marie Frere",
"Ravindra Venkatramani",
"Abhaya M. Dandekar",
"Basuthkar J. Rao"
],
"abstract": "Promiscuity, the ability of an enzyme to catalyze diverse activities using the same active site, sets up the stage for the evolution of complex organisms through gene duplication and specialization. The detection of promiscuous motifs is crucial to understand the physiological relevance of a protein, or for any endeavor that intends to rationally modify these latent capabilities to design new proteins under laboratory conditions. We have established a methodology for identifying catalytic residues based on spatial and electrostatic congruence with known active site configurations. Here, we discuss insights gained in several initiatives using our method on different enzymes.",
"keywords": [
"Primitive life presumably had minimal gene content and a minuscule arsenal of enzymes at its disposal. Unfettered from selection pressures by gene duplication",
"a few select enzymes gained new advantageous functions1–4. Nonetheless",
"the vestiges of secondary activities under neutral drift5 possess the potential to reemerge under changing selection pressures6",
"7. This ability of an enzyme to catalyze diverse activities using the same active site",
"termed as promiscuity",
"is the cornerstone of the evolution of complex organisms from pristine life8. In the human context",
"compound promiscuity plays a major role in drug discovery",
"and in the therapeutic efficacy of drugs9. Databases are a crucial medium of cataloging known aspects of drug promiscuity10",
"11."
],
"content": "\n\nPrimitive life presumably had minimal gene content and a minuscule arsenal of enzymes at its disposal. Unfettered from selection pressures by gene duplication, a few select enzymes gained new advantageous functions1–4. Nonetheless, the vestiges of secondary activities under neutral drift5 possess the potential to reemerge under changing selection pressures6,7. This ability of an enzyme to catalyze diverse activities using the same active site, termed as promiscuity, is the cornerstone of the evolution of complex organisms from pristine life8. In the human context, compound promiscuity plays a major role in drug discovery, and in the therapeutic efficacy of drugs9. Databases are a crucial medium of cataloging known aspects of drug promiscuity10,11.\n\nThe evolution of species through sequence mutations leaves a trail via the conservation of fragments or repeats that have been honed to achieve specific functions with remarkable efficiency12–14. The sequence-to-structure-to-function paradigm facilitates the functional characterization of new proteins by applying a ‘guilt by association’ logic, and has essentially revolutionized the field by its easy to use model15. However, occasionally nature achieves the same solution to an enzymatic problem through a completely different sequence, arriving at the same spatial conformation required for catalysis16,17. Such convergently evolved proteins, and those redesigned from chiseled scaffolds through exon shuffling, remain beyond the scope of sequence analysis methods. As such structure-based methods have evolved to detect such relationships18,19. We have demonstrated that such a structural conservation leading to the same function necessitates the conservation of electrostatic properties as well (CLASP - www.sanchak.com/clasp)20. The ability of finite difference methods to quickly obtain consistent electrostatic properties from peptide structures provides an invaluable tool for investigating other innate properties of protein structures21. Furthermore, using a database of known active sites in proteins (http://www.ebi.ac.uk/thornton-srv/databases/CSA/22), we have proposed a methodology to quantify promiscuity in a wide range of proteins23.\n\nIn an endeavor to establish the validity of the computational predictions made by CLASP, we have undertaken several in vitro initiatives using different enzymes. The results of these experiments have provided several insights regarding promiscuous functions in proteins. Foremost amongst them is corroboration of the intuitive notion that inhibition is inherently simpler to predict than true catalysis. For example, we detected the presence of the serine protease (SPASE) catalytic triad motif (Ser195, His57, Asp102) in alkaline phosphatases (AP) from various organisms using the spatial and electrostatic congruence, and validated this by inhibition of the native phosphatase activity using serine protease inhibitors (AEBSF/PMSF)20. However, true SPASE activity was limited to shrimp AP. Recently, the crown domain in the Escherichia coli expressed rat intestinal AP protein was shown to be prone to protease cleavage, which the authors have ascribed to self-cleavage24. Another recent review nicely summarizes the various computational approaches applied to the AP superfamily in order to gain insights into the promiscuous functions observed in proteins belonging to the superfamily25. The therapeutic potential of AP inhibitors has also seen increased interest from medicinal researchers26.\n\nIn a similar experiment, we detected a SPASE motif in a phosphoinositide-specific phospholipase C (PI-PLC) from Bacillus cereus using CLASP27. Once again, although we easily established the inhibition of the native activity of PI-PLC using serine protease inhibitors, we struggled to establish proteolysis based on known protease substrates. Fortuitously, we observed protease activity of PI-PLC on UVI31+, a protein under investigation in our group for different reasons28. We thus concluded that one should exert caution before ruling out protease activity in an enzyme since theoretically proteases have unenumerable number of possible substrates due to the infinite possible DNA sequences that can result in proteins and their corresponding infinite folds27. Thus, it is possible that we have not found the correct proteolytic substrate for APs20.\n\nWe also tested the proteolytic functions and inhibition using protease inhibitors of the non-toxic B. cereus phosphatidylcholine-specific phospholipase C (PC-PLC) and the closely related highly toxic Clostridium perfringens α-toxin (CPA) (which possesses an additional C-terminal domain demonstrated to be responsible for its sphingomyelinase, hemolytic, and lethal activities29,30). CPA and PC-PLC activities on phospholipids were unaffected by the addition of serine protease inhibitors in concurrence with the CLASP analysis which fails to detect a SPASE scaffold in these proteins27. While CPA and PC-PLC did have a metallo-protease motif based on CLASP analysis, and both showed protease activity in vitro, the observed proteolytic activity can be attributed as an artifact of a metallo-protease contamination which is difficult to remove in spite of the purification steps. Inhibition of CPA activity using a metallo-protease inhibitor was tried out, but failed to show any results. Such lack of inhibition by a single compound is not sufficient ground to rule out the existence of a metallo-protease scaffold.\n\nBased on predictions from CLASP, we also demonstrated the inhibition of the native phosphatase activity of a cold active alkaline phosphatase from Vibrio strain G15-21 AP (VAP)31 by a specific β-lactam compound (only imipenem, and not by ertapenem, meropenem, ampicillin or penicillin G)32. CLASP analysis detected a spatial and electrostatic congruence of the active site of a Class B2 CphA metallo-β-lactamase (MBL) from Aeromonas hydrophila33 to the active site of VAP. Several β-lactam compounds failed to inhibit E. coli or shrimp AP, as was expected by the lower congruence indicated by CLASP. While all APs contain three metal ion binding sites essential for catalysis31, MBLs have either one or two metal binding sites34. It would be interesting to imagine the existence of a protein (possibly evolved from VAP) that is an MBL and requires three metal binding sites.\n\nAnother desired aspect in the search of promiscuous motifs is the ability to search for partial scaffolds, as has been implemented in the DECAAF methodology35,36. The search for an elastase-like motif in a plant protein led us to the pathogenesis-related protein P14a37. Although the complete motif was missing - stated previously as, ‘While Ser195, His57, and Gly193 from the input motif have a highly matching scaffold in P14a, the spatial position of the elastase Asp102 is close to Asn35 and Ser39 in P14a when the proteins are superimposed based on the matching scaffolds36’ - the structural similarity of the P14a protein to a snake venom protein with a known elastase function38 suggested strongly the possibility of pre-existing elastase functionality, or indicated a fair chance of endowing elastase activity through directed evolution techniques.\n\nIt might appear that the presence of a motif like a SPASE catalytic triad in a protein structure is trivial, and one could expect any randomly chosen protein with a large number of residues to have such a structural motif. However, the absence of a spatially congruent SPASE catalytic triad in a reasonably large tyrosine phosphatase CD45 (PDBid: 1YGR, sequence length 610) highlights the fact that the SPASE motif is not present ubiquitously (Table 1). Even the presence of a spatially congruent motif, as in the human translation initiation factor (PDBid: 2E9H), does not imply potential congruence (Table 1).\n\nThe reasonably large tyrosine phosphatase CD45 (PDBid: 1YGR, sequence length 610) does not contain the spatially congruent catalytic triad. Although, a motif spatially congruent to the catalytic triad is present in the human translation initiation factor (PDBid: 2E9H), it lacks electrostatic potential congruence. D = Pairwise distance in Å. PD = Pairwise potential difference. SLen = sequence length. APBS writes out the electrostatic potential in dimensionless units of kT/e where k is Boltzmann's constant, T is the temperature in K and e is the charge of an electron.\n\nThe biggest challenge in detecting promiscuous motifs is to be able to endow the function using rational steps39–41. However, the non-additive nature of active site residues makes this a non-trivial task even when a very close partial match exists42. For example in a catalytic site consisting of n residues, the existence of a congruent n − 1 motif does not imply that it is easy or even possible to add another residue in the structure and obtain the n residue motif. This complexity is best exemplified in the failure to induce β-lactamase activity in a penicillin-binding protein (PBP-5) from E. coli43,44 by generating the L153E mutant of this protein, as proposed by our previous analysis35 (and unpublished results). Although many directed evolution experiments have tried to enhance deacylation in PBPs45,46, the catalytic step that β-lactamases use to hydrolyze β-lactams47, very few have been successful. Even the successful attempts have reported low gains in β-lactamase activity (110-fold in Chesnel et al.45 and 90-fold in Urbach et al.46).\n\nIn spite of the inherent difficultly in rationally designing proteins, we believe that the fast maturing field of protein structure prediction might soon allow us to quickly iterate over in silico mutations48. A method like CLASP may be used to discriminate the predicted structures in order to select the mutations that achieve the desired congruence with a reference scaffold - setting up the flow to mimic the natural ‘evolutionary walk’ in vitro, and accelerate this ‘random walk’ into a ‘resolute sprint’.",
"appendix": "Author contributions\n\n\n\nAll authors contributed equally to the writing, and subsequent refinement, of this commentary.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nReferences\n\nJacob F: Evolution and tinkering. Science. 1977; 196(4295): 1161–1166. PubMed Abstract | Publisher Full Text\n\nBone R, Silen JL, Agard DA: Structural plasticity broadens the specificity of an engineered protease. Nature. 1989; 339(6221): 191–195. PubMed Abstract | Publisher Full Text\n\nYoshikuni Y, Ferrin TE, Keasling JD: Designed divergent evolution of enzyme function. Nature. 2006; 440(7087): 1078–1082. PubMed Abstract | Publisher Full Text\n\nKhersonsky O, Tawfik DS: Enzyme promiscuity: a mechanistic and evolutionary perspective. Annu Rev Biochem. 2010; 79: 471–505. PubMed Abstract | Publisher Full Text\n\nKimura M: DNA and the neutral theory. Philos Trans R Soc Lond B Biol Sci. 1986; 312(1154): 343–354. PubMed Abstract | Publisher Full Text\n\nAmitai G, Gupta RD, Tawfik DS: Latent evolutionary potentials under the neutral mutational drift of an enzyme. HFSP J. 2007; 1(1): 67–78. PubMed Abstract | Publisher Full Text\n\nWroe R, Chan HS, Bornberg-Bauer E: A structural model of latent evolutionary potentials underlying neutral networks in proteins. HFSP J. 2007; 1(1): 79–87. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJensen RA: Enzyme recruitment in evolution of new function. Annu Rev Microbiol. 1976; 30: 409–425. PubMed Abstract | Publisher Full Text\n\nHu Y, Bajorath J: High-resolution view of compound promiscuity [v2; ref status: indexed, http://f1000r.es/1ig]. F1000Research. 2013; 2–144. Publisher Full Text\n\nKnox C, Law V, Jewison T, et al.: DrugBank 3.0: a comprehensive resource for 'omics' research on drugs. Nucleic Acids Res. 2011; 39(Database issue): D1035–1041. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGaulton A, Bellis LJ, Bento AP, et al.: ChEMBL: a large-scale bioactivity database for drug discovery. Nucleic Acids Res. 2012; 40(Database issue): D1100–1107. PubMed Abstract | Publisher Full Text\n\nWooster R, Bignell G, Lancaster J, et al.: Identification of the breast cancer susceptibility gene BRCA2. Nature. 1995; 378(6559): 789–792. PubMed Abstract | Publisher Full Text\n\nPreker PJ, Keller W: The HAT helix, a repetitive motif implicated in RNA processing. Trends Biochem Sci. 1998; 23(1): 15–16. PubMed Abstract | Publisher Full Text\n\nChakraborty S: A fragmented alignment method detects a putative phosphorylation site and a putative BRC repeat in the Drosophila melanogaster BRCA2 protein [v2; ref status: indexed, http://f1000r.es/1wc]. F1000Research. 2013; 2–143. Publisher Full Text\n\nAltschul SF, Madden TL, Schaffer AA, et al.: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997; 25(17): 3389–3402. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGherardini PF, Wass MN, Helmer-Citterich M, et al.: Convergent evolution of enzyme active sites is not a rare phenomenon. J Mol Biol. 2007; 372(3): 817–845. PubMed Abstract | Publisher Full Text\n\nDoolittle RF: Convergent evolution: the need to be explicit. Trends Biochem Sci. 1994; 19(1): 15–18. PubMed Abstract | Publisher Full Text\n\nRussell RB: Detection of protein three-dimensional side-chain patterns: new examples of convergent evolution. J Mol Biol. 1998; 279(5): 1211–1227. PubMed Abstract | Publisher Full Text\n\nKleywegt GJ: Recognition of spatial motifs in protein structures. J Mol Biol. 1999; 285(4): 1887–1897. PubMed Abstract | Publisher Full Text\n\nChakraborty S, Minda R, Salaye L, et al.: Active site detection by spatial conformity and electrostatic analysis-unravelling a proteolytic function in shrimp alkaline phosphatase. PLoS One. 2011; 6(12): e28470. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBaker NA, Sept D, Joseph S, et al.: Electrostatics of nanosystems: application to microtubules and the ribosome. Proc Natl Acad Sci U S A. 2001; 98(18): 10037–10041. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPorter CT, Bartlett GJ, Thornton JM: The Catalytic Site Atlas: a resource of catalytic sites and residues identified in enzymes using structural data. Nucleic Acids Res. 2004; 32(Database issue): D129–133. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChakraborty S, Rao BJ: A measure of the promiscuity of proteins and characteristics of residues in the vicinity of the catalytic site that regulate promiscuity. PLoS One. 2012; 7(2): e32011. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGhosh K, Mazumder Tagore D, Anumula R, et al.: Crystal structure of rat intestinal alkaline phosphatase - Role of crown domain in mammalian alkaline phosphatases. J Struct Biol. 2013; 184(2). PubMed Abstract | Publisher Full Text\n\nDuarte F, Amrein BA, Kamerlin SC: Modeling catalytic promiscuity in the alkaline phosphatase superfamily. Phys Chem Chem Phys. 2013; 15(27): 11160–11177. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAl-Rashida M, Iqbal J: Therapeutic Potentials of Ecto-Nucleoside Triphosphate Diphosphohydrolase, Ecto-Nucleotide Pyrophosphatase/Phosphodiesterase, Ecto-5´-Nucleotidase, and Alkaline Phosphatase Inhibitors. Med Res Rev. 2013. PubMed Abstract | Publisher Full Text\n\nRendon-Ramirez A, Shukla M, Oda M, et al.: A computational module assembled from different protease family motifs identifies PI PLC from Bacillus cereus as a putative prolyl peptidase with a serine protease scaffold. PLoS One. 2013; 8(8): e70923. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShukla M, Minda R, Singh H, et al.: UVI31+ is a DNA endonuclease that dynamically localizes to chloroplast pyrenoids in C. reinhardtii. PLoS One. 2012; 7(12): e51913. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTitball RW, Leslie DL, Harvey S, et al.: Hemolytic and sphingomyelinase activities of Clostridium perfringens alpha-toxin are dependent on a domain homologous to that of an enzyme from the human arachidonic acid pathway. Infect Immun. 1991; 59(5): 1872–1874. PubMed Abstract | Free Full Text\n\nSakurai J, Nagahama M, Oda M: Clostridium perfringens alpha-toxin: characterization and mode of action. J Biochem. 2004; 136(5): 569–574. PubMed Abstract | Publisher Full Text\n\nHelland R, Larsen RL, Asgeirsson B: The 1.4 A crystal structure of the large and cold-active Vibrio sp. alkaline phosphatase. Biochim Biophys Acta. 2009; 1794(2): 297–308. PubMed Abstract | Publisher Full Text\n\nChakraborty S, Asgeirsson B, Minda R, et al.: Inhibition of a cold-active alkaline phosphatase by imipenem revealed by in silico modeling of metallo-β-lactamase active sites. FEBS Lett. 2012; 586(20): 3710–3715. PubMed Abstract | Publisher Full Text\n\nGarau G, Bebrone C, Anne C, et al.: A metallo-beta-lactamase enzyme in action: crystal structures of the monozinc carbapenemase CphA and its complex with biapenem. J Mol Biol. 2005; 345(4): 785–795. PubMed Abstract | Publisher Full Text\n\nBebrone C: Metallo-beta-lactamases (classification, activity, genetic organization, structure, zinc coordination) and their superfamily. Biochem Pharmacol. 2007; 74(12): 1686–1701. PubMed Abstract | Publisher Full Text\n\nChakraborty S: An automated flow for directed evolution based on detection of promiscuous scaffolds using spatial and electrostatic properties of catalytic residues. PLoS One. 2012; 7(7): e40408. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChakraborty S, Minda R, Salaye L, et al.: Promiscuity-based enzyme selection for rational directed evolution experiments. Methods Mol Biol. 2013; 978: 205–216. PubMed Abstract | Publisher Full Text\n\nFernandez C, Szyperski T, Bruyere T, et al.: NMR solution structure of the pathogenesis-related protein P14a. J Mol Biol. 1997; 266(3): 576–593. PubMed Abstract | Publisher Full Text\n\nBernick JJ, Simpson W: Distribution of elastase-like enzyme activity among snake venoms. Comp Biochem Physiol B. 1976; 54(1): 51–54. PubMed Abstract | Publisher Full Text\n\nDalby PA: Strategy and success for the directed evolution of enzymes. Curr Opin Struct Biol. 2011; 21(4): 473–480. PubMed Abstract | Publisher Full Text\n\nZanghellini A, Jiang L, Wollacott AM, et al.: New algorithms and an in silico benchmark for computational enzyme design. Protein Sci. 2006; 15(12): 2785–2794. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRothlisberger D, Khersonsky O, Wollacott AM, et al.: Kemp elimination catalysts by computational enzyme design. Nature. 2008; 453(7192): 190–195. PubMed Abstract | Publisher Full Text\n\nTokuriki N, Jackson CJ, Afriat-Jurnou L, et al.: Diminishing returns and tradeoffs constrain the laboratory optimization of an enzyme. Nat Commun. 2012; 3: 1257. PubMed Abstract | Publisher Full Text\n\nNicholas RA, Krings S, Tomberg J, et al.: Crystal structure of wild-type penicillin- binding protein 5 from Escherichia coli: implications for deacylation of the acyl-enzyme complex. J Biol Chem. 2003; 278(52): 52826–52833. PubMed Abstract | Publisher Full Text\n\nSarkar SK, Dutta M, Chowdhury C, et al.: PBP5, PBP6 and DacD play different roles in intrinsic β-lactam resistance of Escherichia coli. Microbiology. 2011; 157(Pt 9): 2702–2707. PubMed Abstract | Publisher Full Text\n\nChesnel L, Zapun A, Mouz N, et al.: Increase of the deacylation rate of PBP2x from Streptococcus pneumoniae by single point mutations mimicking the class A beta-lactamases. Eur J Biochem. 2002; 269(6): 1678–1683. PubMed Abstract | Publisher Full Text\n\nUrbach C, Evrard C, Pudzaitis V, et al.: Structure of PBP-A from Thermosynechococcus elongatus, a penicillin-binding protein closely related to class A beta-lactamases. J Mol Biol. 2009; 386(1): 109–120. PubMed Abstract | Publisher Full Text\n\nMatagne A, Lamotte-Brasseur J, Frere JM: Catalytic properties of class A β-lactamases: efficiency and diversity. Biochem J. 1998; 330(Pt 2): 581–598. PubMed Abstract | Free Full Text\n\nZhang Y: Progress and challenges in protein structure prediction. Curr Opin Struct Biol. 2008; 18(3): 342–348. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "2636",
"date": "20 Dec 2013",
"name": "Neil D. Rawlings",
"expertise": [],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis commentary reads more like a review and only describes work previously performed by the researchers. I have a number of reservations about this previously published work. The authors should present more detail from at least one example where dual activity has been proven.\n\nSome of the claims made by the authors are extraordinary. Can the authors supply any explanation for the presence of a serine peptidase activity in alkaline phosphatase from a shrimp, but not Escherichia coli? If there was any physiological relevance for the peptidase activity for an alkaline phosphatase then surely it would be conserved. It seems extraordinarily serendipitous that the authors are working on UVI31+ and that this is the only substrate for the peptidase activity of shrimp alkaline phosphatase the authors could detect. This is unlikely to be a physiological substrate, which implies that the serine peptidase activity of alkaline phosphatase is very general, and it is behaving as if its’ role were protein degradation rather than limited processing. Given that there are so many other peptidases for recycling proteins - the proteasome in the cytoplasm, lysosomal peptidases, digestive enzymes of the stomach and gut, for example - what would be the evolutionary pressure to maintain such a generalized function for alkaline phosphatase? Have the alleged serine peptidase activity of shrimp alkaline phosphatase, and the alleged inhibition of alkaline phosphatase activity by a serine peptidase inhibitor been confirmed by other researchers? I can understand that a small molecule like PMSF might block any active site serine, regardless of the type of enzyme, but the authors claim that the protein ovoinhibitor reduces alkaline phosphatase activity, which would require not only an active site but also at least one substrate binding site for the reactive site in the inhibitor. Perhaps this is merely some irrelevant, non-specific binding rather than true inhibition. Why were synthetic substrates not used instead of proteins? Then proper kinetics could be measured, rather than presenting bands on a gel. Clearly the authors are aware of the dangers of contamination, because they admit to having had problems with a metallopeptidase contaminating phospholipase C, for which their CLASP analysis predicts a metallopeptidase-like active site. The controls that the authors use in their experiments, namely to denature all proteins with SDS, would not address this problem, because any contaminant would be denatured as well as the enzyme under experimentation. Use of peptidase inhibitors would be equally useless if the catalytic type of the contaminating peptidase and the enzyme in question were the same. Perhaps the authors should abandon commercial preparations and use recombinant enzymes where it would be possible to mutate active site residues to show that a knockout abrogates activity? The authors state that their CLASP analysis is unable to find a serine peptidase-like active site in phospholipase C, and that this is confirmed by failure to inhibit phospholipase C with a serine peptidase inhibitor. This is an example of a negative result proving a negative hypothesis, which really doesn't prove anything. In Table 1, the structure 1A0J should be described in the legend.Promiscuity in enzyme active sites is a known phenomenon. An example that comes to mind is leukotriene A4 hydrolase, which not only hydrolyses the epoxide but also acts as an aminopeptidase using the same active site. There are also numerous examples where an active site performs different functions in different organisms (or even in the same organism for some protein products of paralogous genes): for example proteins with the alpha/beta hydrolase fold, where some are peptidases and some are lipases. It would strengthen the authors’ case considerably to include examples such as these before discussing their own results.",
"responses": [
{
"c_id": "666",
"date": "16 Jan 2014",
"name": "Sandeep Chakraborty",
"role": "Author Response",
"response": "Dear Dr Rawlings, We would like to thank you for taking the time to review our manuscript. We appreciate several incisive and relevant points raised by you, and hope that our arguments are convincing enough for you to change your opinion about our work. There are two aspects of our work that need to be dissected separately. STAGE1: The first stage establishes the presence of active site scaffolds (for example, the serine protease catalytic triad) through: (a) spatial congruence (a method devised previously [29, 30]). (b) electrostatic congruence of cognate pairs of active site residues in proteins with the same function - our original contribution. STAGE2: In the next stage, we have attempted to correlate our in silico findings through in vitro experiments. It is salient to note that the results of STAGE1 are not nullified irrespective of the results in these experiments. However, the results have indeed corroborated our hypothesis in several experiments. Although, as you so aptly point out, there is still work that needs to be done. Point (b) in STAGE1 might be subject to scrutiny for several reasons: (i) The validity and accuracy of finite difference Poisson Boltzmann methods.(ii) The non-trivial nature of the electrostatic potential difference. We have argued in favor of both these points throughout our work. “This invariance in the electrostatic features (measured in structures that have been solved independently over many years) is an innate property required for the enzymatic activity. This also speaks highly of the reliability of the APBS/PDB2PQR implementation” [51]. Assuming that (a) and (b) are valid; surely one cannot deny the existence of a scaffold resembling the catalytic triad from subtilisin and trypsin in alkaline phosphatases (Table 3 in [32])? Please find our detailed responses to your comments below: This commentary reads more like a review and only describes work previously performed by the researchers. I have a number of reservations about this previously published work. The authors should present more detail from at least one example where dual activity has been proven.We have explicitly stated in the abstract that we intend to ‘discuss insights gained in several initiatives using our method on different enzymes’. Thus, this would not qualify as a review since we do not consider the work of those researchers whose work we have built upon. Can the authors supply any explanation for the presence of a serine peptidase activity in alkaline phosphatase from a shrimp, but not Escherichia coli? If there was any physiological relevance for the peptidase activity for an alkaline phosphatase then surely it would be con- served. It seems extraordinarily serendipitous that the authors are working on UVI31+ and that this is the only substrate for the peptidase activity of shrimp alkaline phosphatase the authors could detect. This is unlikely to be a physiological substrate, which implies that the serine peptidase activity of alkaline phosphatase is very general, and it is behaving as if its role was protein degradation rather than limited processing. Given that there are so many other peptidases for recycling proteins - the proteasome in the cytoplasm, lysosomal pepti-dases, digestive enzymes of the stomach and gut, for example - what would be the evolutionary pressure to maintain such a generalized function for alkaline phosphatase? Have the alleged serine peptidase activity of shrimp alkaline phosphatase, and the alleged inhibition of alkaline phosphatase activity by a serine peptidase inhibitor been confirmed by other researchers?It is precisely these questions that our findings raise - and it would be unfair to put the onus of answering these fundamental questions on the method itself. Fortunately for us, the recent findings of a group working on alkaline phosphatase (and not personally known to us), have shown the presence of auto cleavage in these alkaline phosphatases - “the loss of crown domain due to protease cleavage could result from self-cleavage of the protein, specifically when metal sites are not fully occupied” [36]. Citing our work, they have hypothesized on the physiological relevance of the peptidase activity. Since they use recombinant protein, the danger of contamination is minimal - a point that you have raised below. It is to be said that we agree that it seems unlikely that there is an evolutionary pressure to maintain a peptidase function in APs, given the plethora of such enzymes that you have pointed out. However, promiscuity often allows functional units (partial or complete) to serendipitously piggyback on the ‘real’ function being maintained and honed by evolution. It is not the existence of peptidase activity, but the distinct possibility that APs may have some peptidase activity that is of interest. The peptidase-like scaffold in APs might be one such latent functionality - possibly a remnant of a primeval parent protein that duplicated to result into the current serine peptidases and APs (both hydrolases). Finally, there is precedence of a promiscuous activity that is present in only one of the APs (ECAP) - “A phosphite-dependent hydrogenase activity was also found in ECAP, but was absent in APs from other organisms [19]”. Another example is the inhibition of VAP using imipenem, which does not inhibit ECAP. I can understand that a small molecule like PMSF might block any active site serine, regardless of the type of enzyme, but the authors claim that the protein ovoinhibitor reduces alkaline phosphatase activity, which would require not only an active site but also at least one substrate binding site for the reactive site in the inhibitor. Perhaps this is merely some irrelevant, non-specific binding rather than true inhibition.We have shown the binding of AEBSF to shrimp alkaline phosphatase through MALDI-mass spectrometry (Fig 4d in [32]). Why were synthetic substrates not used instead of proteins? Then proper kinetics could be measured, rather than presenting bands on a gel.We did try out many synthetic substrates, but we could not find any protease activity. However we have not yet tried the Pro-Xxx that would duplicate the bond in UVI31+ cleaved by PI-PLC. We believe that ‘one should exert caution before ruling out protease activity in an enzyme since theoretically proteases have an innumerable number of possible substrates due to the infinite possible DNA sequences that can result in proteins and their corresponding infinite folds’, as mentioned in the current commentary. However, I do think we ought to try out prolyl peptidases substrates (and will in the near future). Clearly the authors are aware of the dangers of contamination, because they admit to having had problems with a metallopeptidase contaminating phospholipase C, for which their CLASP analysis predicts a metallopeptidase-like active site.Precisely - it would be improper to consider that the metallophospholipase has protease activity as it is difficult to remove the protease contamination, and we have not. The controls that the authors use in their experiments, namely to denature all proteins with SDS, would not address this problem, because any contaminant would be denatured as well as the enzyme under experimentation. Use of peptidase inhibitors would be equally useless if the catalytic type of the contaminating peptidase and the enzyme in question were the same.We have not tried to rule out contamination by denaturing the proteins or using inhibitors for the reasons mentioned by you. Could you kindly point out where this might have been mistakenly specified by us? Perhaps the authors should abandon commercial preparations and use recombinant enzymes where it would be possible to mutate active site residues to show that a knockout abrogates activity?We agree that recombinant enzymes with a knock out mutation would clinch our argument. However, financial and logistic restrictions make this currently unfeasible (for me on a personal note). Fortunately, the protease activity in a recombinant alkaline phosphatase has been shown by a different group [37]. The authors state that their CLASP analysis is unable to find a serine peptidase-like active site in phospholipase C, and that this is confirmed by failure to inhibit phospholipase C with a serine peptidase inhibitor. This is an example of a negative result proving a negative hypothesis, which really doesn’t prove anything.There has been some misunderstanding - we mention ‘CPA and PC-PLC activities on phospholipids were unaffected by the addition of serine protease inhibitors in concurrence with the CLASP analysis which fails to detect a SPASE scaffold in these proteins’. This is expected, since they are metallo proteins. CLASP does find the serine peptidase-like active site in PI-PLC, which we go on to confirm through in vitro experiments [39]. In Table 1, the structure 1A0J should be described in the legend.We have made the correction, thank you. Promiscuity in enzyme active sites is a known phenomenon. An example that comes to mind is leukotriene A4 hydrolase, which not only hydrolyses the epoxide but also acts as an aminopeptidase using the same active site. There are also numerous examples where an active site performs different functions in different organisms (or even in the same organism for some protein products of paralogous genes): for example proteins with the alpha/beta hydrolase fold, where some are peptidases and some are lipases. It would strengthen the authors’ case considerably to include examples such as these before discussing their own results.We acknowledge that promiscuity in enzymes has been a phenomenon that has been established over the last couple of decades by several researchers. In accordance with your suggestion, we have added a brief discussion of promiscuous enzymes and related work. Our contribution, we think, has been to formalize computational methods that facilitate the search for latent promiscuous scaffolds in the active site of proteins, and increase the specificity of matches by introducing a filtering in the form of electrostatic potential congruence. In this endeavor, we believe, we have further strengthened the previously hypothesized key role that promiscuity plays in the evolution of proteins [7]. We hope you will find our arguments compelling enough to accept our manuscript. Once again, we thank you for your valuable comments as we strive to establish our work in a collaborative effort. Best regards,Sandeep Chakraborty"
}
]
},
{
"id": "2833",
"date": "07 Jan 2014",
"name": "Abhinav Nath",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe commentary by Chakraborty et al. focuses on the important topics of understanding and modeling enzyme promiscuity, and raises some intriguing points about the importance of local electrostatic effects (beyond structure alone) on enzyme activity. However, the commentary is quite tightly focused on protease-type activity in different scaffolds, and even more so on the authors’ own work. This manuscript would be improved by providing more context on how their previous work relates to other studies of AP promiscuity/polyspecificity (including in particular the work of the Herschlag and Hollfelder groups) and electrostatic approaches to enzyme mechanism (Warshel, Houk, and many others). This would broaden the potential audience for this commentary, which as it stands is rather narrow.Moving on to specifics, I am unconvinced by the use of protease inhibitors as a test of bona fide protease activity in several of the works discussed. The fact that a particular compound inhibits an enzyme does not necessarily mean that it resembles a substrate, or that the enzyme’s mode of action resembles that of the compound’s other targets. On the other hand, as the authors acknowledge, the absence of inhibition by a particular compound is not enough to demonstrate the lack of a particular activity or functional motif. These assays thus have significant risks of both false positives and false negatives, and these should be explicitly discussed. What we are left with are apparently observations of protease activity both by enzymes predicted to have SPASE activity (shrimp and E. coli APs, and PI-PLC) and by those predicted to lack it (PC-PLC and CPA) – unfortunately, not very strong evidence for the power of their approach. The authors should discuss these issues and their potential solutions.In Table 1, it is not clear what 1A0J is. Also, it is not clear what the criteria are for either spatial or potential congruence. More broadly, it is difficult to assess the frequency of SPASE-compatible triads in proteins in general from just 3 examples. Is it feasible for the authors to estimate the frequency of spatial and potential congruence in a larger set of protein structures (or even the entire PDB)?",
"responses": [
{
"c_id": "667",
"date": "16 Jan 2014",
"name": "Sandeep Chakraborty",
"role": "Author Response",
"response": "Dear Dr Nath, We would like to thank you for taking the time to review our manuscript. We have responded in detail to another review by Dr Rawlings, and an updated version of our manuscript has now been published. We hope our dialogue with Dr Rawlings will provide more clarity on our work. For the sake of brevity, we have not repeated several aspects that we have pointed out there. Please find our detailed responses to your comments below. The commentary by Chakraborty et al. focuses on the important topics of understanding and modeling enzyme promiscuity, and raises some intriguing points about the importance of local electrostatic effects (beyond structure alone) on enzyme activity. However, the commentary is quite tightly focused on protease-type activity in different scaffolds, and even more so on the authors own work. This manuscript would be improved by providing more context on how their previous work relates to other studies of AP promiscuity/polyspecificity (including in particular the work of the Herschlag and Hollfelder groups) and electrostatic approaches to enzyme mechanism (Warshel, Houk, and many others). This would broaden the potential audience for this commentary, which as it stands is rather narrow.We appreciate your encouraging comments on the importance of the field of our work. We would like to emphasize that we have also worked on ß-lactamases apart from proteases - and are currently working on proteins with other enzymatic activities as well (lipases, sulfatases, etc.). We have mentioned that we intend this commentary to focus exclusively on our work (and any novel aspects we might have unraveled), and not the work of previous researchers on whose results we have built our research - thus, we have kept this as a commentary, and not a review. We have discussed previous studies in each of our papers separately - for example, the field of quantum, classical, and statistical techniques that have evolved in the last few decades (involving Karplus, Levitt, Warshel, Herschlag and many others) has been discussed in detail in Chakraborty S (2013) A Quantitative Measure of Electrostatic Perturbation in Holo and Apo Enzymes Induced by Structural Changes. PLoS ONE 8(3): e59352. doi:10.1371/journal.pone.0059352. Moving on to specifics, I am unconvinced by the use of protease inhibitors as a test of bona fide protease activity in several of the works discussed. The fact that a particular compound inhibits an enzyme does not necessarily mean that it resembles a substrate, or that the enzymes mode of action resembles that of the compounds other targets.We agree that inhibition does not “not necessarily mean that it resembles a substrate, or that the enzymes mode of action resembles that of the compounds other targets”. However, given the in silico demonstration of the congruence of the new scaffold to existing ones with known function, it does add credence to the hypothesis. Further, we have confirmation from other groups (unknown to us) that a recombinant AP does have self proteolytic function - they cite our work in order to rationalize this result [37]. On the other hand, as the authors acknowledge, the absence of inhibition by a particular compound is not enough to demonstrate the lack of a particular activity or functional motif. These assays thus have significant risks of both false positives and false negatives, and these should be explicitly discussed.This is something we should touch upon. We thank you for pointing this out. What we are left with are apparently observations of protease activity both by enzymes predicted to have SPASE activity (shrimp and E. coli APs, and PI-PLC) and by those predicted to lack it (PC-PLC and CPA) unfortunately, not very strong evidence for the power of their approach. The authors should discuss these issues and their potential solutions.We do see protease activity in PC-PLC and CPA - however, it is impossible to remove protease contamination, and thus we cannot ascribe it to the protein. This is not a negative result for us - it is just a result we cannot confirm under current constraints of protein purification, and thus we cannot (and have not) used it to forward our hypothesis. In Table 1, it is not clear what 1A0J is.We have fixed this in version 2 (the other reviewer has pointed this out too). Also, it is not clear what the criteria are for either spatial or potential congruence. More broadly, it is difficult to assess the frequency of SPASE-compatible triads in proteins in general from just 3 examples. Is it feasible for the authors to estimate the frequency of spatial and potential congruence in a larger set of protein structures (or even the entire PDB)?We have done something like this in a recently published work where we show the inhibition of PI-PLC using dpp4 inhibitors that are used in type 2 diabetes therapy - http://f1000research.com/articles/2-286. A comprehensive set of 5000 human proteins has been queried using SPASE like motifs, and the significant results have been seen to be small in comparison to the number of proteins queried. For example, using a four residue motif from trypsin we find only 1% of proteins (about 50 proteins) that have significantly congruent active site motifs (considering both spatial and electrostatic properties). We appreciate the reservations you have, and we share some of the concerns as we strive to establish our work in a collaborative effort. We hope you will find our arguments compelling enough to accept our manuscript. Best regards,Sandeep Chakraborty"
}
]
}
] | 1
|
https://f1000research.com/articles/2-260
|
https://f1000research.com/articles/3-13/v1
|
16 Jan 14
|
{
"type": "Research Article",
"title": "Dentin reactions to caries are misinterpreted by histological “gold standards”",
"authors": [
"Priscila Florentino Silva",
"Danilo Augusto de Holanda Ferreira",
"Kássia Regina Simões Meira",
"Franklin Delano Soares Forte",
"Ana Maria Barros Chaves",
"Frederico Barbosa de Sousa",
"Priscila Florentino Silva",
"Danilo Augusto de Holanda Ferreira",
"Kássia Regina Simões Meira",
"Franklin Delano Soares Forte",
"Ana Maria Barros Chaves"
],
"abstract": "Dentin reactions to caries, crucial for pathogenesis and for the determination of the severity of caries lesions, are believed to be reasonably detected by stereomicroscopy (SM) and polarized light microscopy in quinoline (PLMQ), but accuracies are not available. Here, stereomicroscopy of wet (SW) and dry (SD) ground sections of natural occlusal caries lesions resulted in moderate (0.7, for normal dentin) and low accuracies (< 0.6, for carious and sclerotic dentin) as validated by contrast-corrected microradiography. Accuracies of PLMQ were moderate for both normal (0.71) and carious dentin (0.71). The hypothesis that detection of dentin reactions by SM and PLMQ would be influenced by the contrast quality of micrographic images was rejected. Dentin reactions were scored by SW, SD, PLMQ, and three types of microradiographic images with varying contrast qualities and each technique was compared against the one that resulted in the highest number of scores for each dentin reaction. Large differences resulted, mainly related to the detection of sclerotic dentin by both SW and SD, and normal and carious dentin by PLMQ. It is concluded that contrast-corrected microradiography should be preferred as the gold standard and SM and PLMQ should be avoided, but the relationship of PLMQ with dentin mineralization deserves further investigation.",
"keywords": [
"sclerotic dentin",
"dentin",
"dental caries",
"histopathology",
"diagnosis",
"stereomicroscopy",
"microradiography"
],
"content": "Introduction\n\nDentin reactions to caries are crucial for the pathogenesis and severity determination of caries lesions. Since caries is mainly a demineralization process, the high ratio of X-ray absorbance between calcium and the chemical elements of the organic content1, and the fact that the density of the mineral content is higher (more than 2 times) than that of the organic content2, radiography with microscopic resolution (microradiography, MR) is considered as a highly reliable gold standard for detecting variations in dentin mineral content. Also widely accepted as gold standards for dentin reactions are stereomicroscopy (SM; commonly referred as histology)3,4 and (to a lesser extent) polarized light microscopy with quinoline as the immersion medium (PLMQ)5. The acceptance of SW, the currently most used “gold standard”, is based on studies reporting opaque and translucent dentin under SM related to radiolucent and radiopaque dentin, respectively6–9. It is lacking, however, accuracy in numbers. To our knowledge, data regarding the accuracy of SM are available from only one study that included unerupted teeth and no data on translucent/sclerotic dentin10. In addition, early studies with MR reported cases classified as translucent dentin by transmitted light microscopy (where the interaction of light with dentin is similar to that under SM) that were then classified as demineralized dentin by the use of MR11–14.\n\nCurrently the only evidence for the detection of dentin reactions by PLMQ is only qualitative5. Thus, research into the accuracy of SM and PLMQ is needed. Regarding the use of MR as a gold standard, it must be considered that images of microradiographic plates taken using transmitted light microscopy are commonly biased by the effect of heterogeneous illumination, which is inversely proportional to the objective magnification15. Heterogeneous illumination is expected to influence judgment of brightness16 (a procedure required for diagnosis from MR images), possibly including bias when other techniques are validated using MR.\n\nThe aim of this study was three fold: to test the accuracy of both SM and PLMQ in detecting dentin reactions to natural caries; to test the hypothesis that elements of accuracy are influenced by the quality of the microradiographic image contrast; and to test the hypothesis that SM, PLMQ and MR (regardless of image contrast quality) detect dentin reactions equally.\n\n\nMaterials and methods\n\nForty three erupted third molars with various stages of natural occlusal caries were collected from volunteers who signed consent terms (as approved by the Ethical Committee of the Federal University of Paraiba; certificate of ethical appreciation number 4125.0.000.126-10). All teeth were gently cleaned with 1% hypochlorite solution (Vetec, Brazil), mounted in a wax base and surrounded by a rubber dam isolator prior to analysis of their occlusal surfaces using the ICDAS II scoring system17. Before obtaining final ICDAS scores for analysis, examiners were calibrated using a sub-set of the whole sample. Thirty occlusal sites were scored by two calibrated examiners (Kappa’s intra-examiner’s scores of 0.9 and 0.89, and 0.85 for inter-examiner agreement), with a one week interval, in order to test intra- and inter-examiner reproducibilities. Final scores were those obtained by a consensus between examiners. In nine teeth, two sites on the occlusal surface were selected, yielding a total of 52 occlusal caries lesions.\n\nAll teeth were cut longitudinally to their crowns (through their occlusal surfaces) using a diamond disc mounted (Kavo Sorensen, Brazil) in a low-speed handpiece under water irrigation, so that a section of the selected site with a given IDCAS score was obtained. All cuts were then ground using a customized metallic (brass) lapping jip and silicon carbide paper (granulations of 240–1200) under water irrigation to achieve a final thickness of ~100 ± 20 μm. Prepared ground sections (n = 52) were kept in a 0.02% sodium azide aqueous solution until examination.\n\nAt each ground section, histological sites (area of ~150 μm × 150 μm) presenting suggestive signs of normal, carious, or sclerotic dentin were selected. All examinations of ground sections (SM of wet and dry samples, three types of MR images, and PLMQ) were performed by the same two examiners, whose intra and inter-examiner reproducibilities were determined (using Kappa’s statistics) from their scores of all histological sites (of all samples) from each technique obtained with a one week interval. Examiners agreed on the final scores by consulting with each other.\n\nHistological sites were selected from the outer half of the dentin layer, including the area adjacent to the deepest enamel lesion; at least one dentin reaction type per sample was included where possible. Cases were included in the sample when two sites had the same type of dentin reaction detected by SM, while showing different types of dentin reactions when detected using MR. Thus, up to 6 histological sites were selected per sample, yielding a total sample size of 168 sites.\n\nSM (10× magnification) with reflected light was used to analyze ground sections under two conditions: wet (SW) and dried (SD; after exposure to 25ºC and 50% relative humidity for 2 hours). Temperature and relative humidity were measured just adjacent to the samples. Digital photomicrographs (digital camera Nikon D80) of wet and dried samples were obtained. Dentin reactions were scored as normal, carious discolored (white/yellow/brown), and translucent (“sclerotic”).\n\nAll samples were mounted in a microradiographic plate (resolution of 2000 lines/mm; AGHD plates, Microchrome Technology, San Jose, USA) and exposed to X-rays in a PCBA Inspector (tungsten anode filtered with a 0.25 mm-thick beryllium window, GE, Germany) for 25 minutes using 40 keV and 0.25 mA. Digital photomicrographs of the microradiographic plate were obtained in a transmitted light microscope (2× objective) under different conditions:\n\nCondition 1: using the condenser aligned according to the principles of Kohler illumination for low magnification objectives18;\n\nCondition 2: no condenser and using a light shaping filter (Luminit, USA) above the field diaphragm.\n\nThe possible scores for dentin reactions using MR were: normal dentin, demineralized (radiolucent) dentin, and hypermineralized (highly radiopaque; sclerotic) dentin. Digital images were analysed (using the freeware program ImageJ, NIH, USA) with the following contrast conditions:\n\nImage obtained with aligned condenser, without any adjustment of brightness and contrast from ImageJ, and no light shaping filter (NFNBC image);\n\nImage obtained with aligned condenser, no light shaping filter, but with adjustment of brightness and contrast from ImageJ (NFBC image);\n\nImage obtained without a condenser, with both a light shaping filter and adjustment of brightness and contrast from ImageJ (FBC image).\n\nSuch conditions created an ordinary scale of heterogeneous illumination of the field of view. Images without light shaping filter (NF) and FBC images presented a Gaussian normalized light intensity (R2 = 0.87 for both) across the field of view with heights of 0.13 and 0.05 (lower heterogeneity), respectively. Brightness and contrast adjustment (according to a consensus from both examiners) allowed this difference to be easily detected by the naked eye.\n\nGround sections were dried at room temperature for 24 hours, immersed in quinoline (Vetec, Brazil) for 24 hours, and then were positioned with the dentin tubules at – 45º on the stage of a polarizing microscope (Axioskop, Carl Zeiss, Germany) equipped with a Red I filter, 2× objective, and digital camera (Nikon D80, Japan). Dentin reactions were scored as either negatively (carious) or positively birefringent (normal)5. Since the technique of PLMQ is not intended to diagnosis dentin sclerosis5, no diagnosis of sclerotic dentin was attempted. Color digital images were split into color channels using ImageJ, resulting in a sharp demarcation of negatively and positively birefringent areas.\n\nWe tested the accuracies of the SW, SD, and PLMQ techniques for detecting dentin reactions using the FBC MR image as the gold standard in all cases. Total positive (TP), total negative (TN), false positive (FP), and false negative values (FN) were obtained and used to calculate accuracy (AC) from:\n\n\n\nPositive (PPV) and negative predictive values (NPV) of each dentin reaction detected by SM were calculated using the following combinations:\n\n1. SW × NFNBC;\n\n2. SW × NFBC;\n\n3. SW × FBC;\n\n4. SD × NFNBC;\n\n5. SD × NFBC;\n\n6. SD × FBC.\n\nThe number of a given dentin reaction was that obtained from agreement from both the SM and MR images of each combination. Each SM technique was validated against all MR images types. The PPV was calculated from the ratio of the TP (determined by each MR image type) by the test outcome positive, and NPV was calculated from the ratio of the TN (determined by each MR image type) by the test outcome negative.\n\nIn order to test whether PPV and NPV were altered by dentin hydration and or MR contrast, combinations 1–5 were compared with combination 6 (considered as the gold standard combination) and Cohen’s effect size for proportions (h) was calculated from19:\n\n\n\nand\n\n\n\nWhere ϕx and ϕy are the arcsines (in radians) of the proportions “P” (PPV or NPV), of a given dentin reaction from the test combination (1–5) and gold standard combination, respectively. The same test was performed with PLMQ, using:\n\n1. PLMQ × NFNBC;\n\n2. PLMQ × NFBC;\n\n3. PLMQ × FBC.\n\nEquation (2) was used to calculate the difference between combinations 7–8 and combination 9 (gold standard).\n\nDentin reactions were summed separately as detected from each of the six techniques tested and converted to proportions in relation to the number of sites detected by the technique that yielded the highest number (this later ascribed as the “temporary gold standard”). Equation (2) was used to calculate the difference between the “temporary gold standard” proportion (ϕx) and the test proportion (ϕy).\n\nWe tested the hypothesis that translucency under SM was related to the radiopacity in dentin. Five ground sections (with ICDAS scores ranging from 0 to 2) were microradiographed in a digital X-ray machine (Faxitron model MX20, Tucson, USA; tungsten anode filtered with a 0.25 mm thick beryllium window) using 20 keV, 0.3 mA and digitally photomicrographed under SM (with dry dentin; SD) with a dark background created with a neutral filter. Under SM, samples presented normal and translucent dentin sites only. Faxitron images had an almost flat illumination across the field of view (Gaussian fit: R2 = -0.0004 and height of 0.028; and linear fit: R2 = 0.18). Eighty histological sites (16/sample) were selected and gray levels were measured on both SM and Faxitron digital images using image analysis software (ImageJ, NIH, USA). The maximum intensity of both image histograms was lower than 80% of maximum intensity allowed. Translucency and radiopacity were measured by:\n\n\n\nWhere ΔI is the difference (always converted to a positive value) between the intensity of the histological site and the maximum intensity of image histogram (Iext; maximum intensity for radiopacity, and minimum intensity for translucency). Translucency and radiopacity were normalized and then analyzed using Pearson product-moment correlation coefficient.\n\n\nResults\n\nThe occlusal caries ICDAS scores for the samples analysed were: 10 with score 0, 2 with score 1, 23 with score 2, 10 with score 3, 1 with score 4, 4 with score 5, and 2 with score 6. For microscopy, intra-examiner agreements were (examiner 1/examiner 2): 0.896/0.914 (SW), 0.952/0.944 (SD), 0.909/0.899 (NFNBC), 0.90/0.91 (NFBC), and 0.919/0.898 (FBC). The inter-examiner agreements were 0.914 (SW), 0.953 (SD), 0.918 (NFNBC), 0.949 (NFBC), and 0.979 (FBC).\n\nTypical aspects of normal, carious and sclerotic/translucent histological points under the six techniques tested here are shown in Figure 1. The number of dentin reactions detected varied among techniques (Table 1). Detection of normal dentin had a moderate accuracy for both SW (0.7976) and SD (0.7976), while both techniques presented a low accuracy for detecting carious (0.5952 for SW; 0.631 for SD) and sclerotic dentin (0.5833 for both SW and SD). Regarding PLMQ, the accuracy was moderate for both carious (0.7092) and normal dentin (0.7902).\n\nTranslucent dentin is shown in sites 1–3 from the SW image (A) and in sites 1 and 2 from the SD image (B). Site 1 appears as demineralized dentin in all types of MR images and as negatively birefringent (“carious dentin”) in PLMQ (F). Site 2 appears as normal dentin in the NFNBC image (C), as hypermineralized (sclerotic) in both NFBC (D) and FBC (E) images, and as negatively birefringent in PLMQ. Site 3 appears as carious (yellowish) dentin in the SD image (B), as demineralized dentin in all types of MR images, and as positively birefringent (“normal”) in PLMQ. The appearance of site 4 is of normal dentin in all images. Bars = 1 mm.\n\nPPV and NPV values of SW, SD, and PLMQ for dentin reactions from combinations 1–9 are shown in Table 2. For normal dentin, SW and SD had moderate PPV and NPV, but for carious and sclerotic dentin they ranged from very low to high values. This later behavior was observed for PLMQ from all combinations.\n\nNFNBC, PLMQ, and SD were the “temporary gold standards” for normal, carious, and sclerotic dentin, respectively (Figure 2). Regarding both normal and carious dentin, all comparisons of the “temporary gold standard” with other techniques resulted in large effect sizes. For sclerotic dentin, SM techniques had a small difference, while large effects sizes were measured for comparisons with MR techniques (Figure 2).\n\nLabels on the right are magnitudes of effect size in relation to the “temporary gold standard” only.\n\nThe effects sizes of MR image contrast on the PPV and NPV of different combinations of SM and MR was negligible (Table 3). The corresponding values for PLMQ were low: (i) for normal dentin, effect sizes of 0.154 (PLMQ×NFNBC) and 0.043 (PLMQ×NFBC) for PPV, and 0.044 (PLMQ×NFNBC) and 0.0 (PLMQ×NFBC) for NPV; and (ii) for carious dentin, effect sizes of 0.044 (PLMQ×NFNBC) and 0.0 (PLMQ×NFBC) for PPV, and 0.154 (PLMQ×NFNBC) and 0.043 (PLMQ×NFBC) for NPV. Analysis of the relationship between translucency and radiopacity of dentin showed that they were not correlated (Figure 3).\n\nN = 80 histological sites.\n\n\nDiscussion\n\nHere we used Cohen’s effect size19 to test differences between outcomes of a given gold standard and a test technique. Effect sizes > 0.5 mean that differences are higher than the acceptable amount to neglect them. In some cases, more than one section per tooth was analyzed. There is conflicting data on whether demineralization can spread laterally to the main dentinal tubules (close to the enamel-dentin junction) or not in non-cavitated carious lesions20,21. If lateral spread does occur, dentin demineralization and sclerosis might occur at random in different sections of a same lesion. In addition, two sites located at different fossae on the same occlusal surface are expected to present independent dentin reactions. This is why we considered more than one section per tooth as independent samples.\n\nThis study showed that SM has a low accuracy for detecting carious and sclerotic dentin. This agrees with early qualitative reports showing that translucent dentin can be either caries or sclerosis11–14. The explanation is that translucency is related to discontinuities in refractive indexes22, but is not necessarily related to radiopacity (Figure 3). When we tested the assumption that any technique is likely to measure dentin reactions equally, pronounced differences were obtained for all dentin reactions, with the most pronounced difference being that between SM and the other techniques with regard to sclerotic dentin (Figure 2). PLMQ, another technique regarded (without evidence) to be useful for detecting normal and carious dentin5, showed the most pronounced differences for the detection of normal and carious dentin. To our knowledge, there are no data explaining the relationship between birefringence and dentin mineral content, and this gap impedes further consideration of using PLMQ for detecting dentin reactions. Its use should be avoided until its relationship with dentin mineral content is clarified. The large effect sizes obtained when PLMQ was the temporary gold standard and moderate accuracies of PLMQ are intriguing, and worth further investigation.\n\nPrevious studies that have reported qualitative evidence of translucent dentin under SM related to radiopaque dentin6–9, and have been cited as the basis for regarding SM as the gold standard23, performed their analysis with images showing more than 2 mm × 2 mm of the tooth crown. Such a field of view size can only be obtained from low magnification objectives24. Heterogeneous illumination of the field of view in light microscopy images is inversely related to objective magnification15,24. Thus, heterogeneous illumination may have been a common factor in their analyses, and most probably our NF MR images (NFNBC and NFBC) are the ones that more closely resemble the images obtained in these older studies. The hypothesis that MR image contrast (influenced by heterogeneous illumination) could explain variations in the PPV and NPV of dentin reactions was rejected (Table 2). Thus, our data suggest that the reason why scientists misinterpreted dentin aspects under SM is not because they used poorly contrasted microradiographs, but probably due to a result of a lack of accuracy. The assumption, now shown quantitatively to be wrong, that translucent dentin under SM always represents sclerotic or non-carious dentin is currently highly influential in the selection of normal dentin, and in the determination of the onset25–27 and extent (perhaps the most important) of the carious process in dentin. An indication of such influency is that currently, considering most popular23,28 and recent21 textbooks, nearly no one is trained in cariology and dental histology without being presented to SM images showing translucent dentin interpreted as sclerotic dentin.\n\nSome implications of our study can be explained by using the example of a SM image of a section of a carious tooth. On the occlusal surface, opaque enamel in the outer one third of the enamel layer combined with translucent dentin in the outer 300 microns of the dentin layer are interpreted as an indication that the carious lesion is confined to the outer enamel and that the dentin has already reacted to it by producing sclerotic dentin. Our results and previous evidence11–14 show that this translucent dentin might actually be carious demineralization. This alters the interpretation of the lesion depth and also the understanding of how carious demineralization propagates in the hard dental tissues. In this context, SM data without quantitative evidence of mineral content through the carious lesion might be misleading. In addition, or alternatively, it might be misleading to ignore the possibility that caries formation is a result of two relatively independent events: initial acid infiltration and late demineralization, as shown experimentally29. Cariogenic acid could infiltrate (from the tooth surface) down to the inner enamel and outer dentin prior to demineralization of the entire enamel layer located more externally. Acid could infiltrate into the tooth crown following enamel sheaths (the main pathways for transport of materials in enamel)30, which are large nanochannels at the boundaries of enamel prisms, under the influence of an osmotic gradient31 created by the higher organic content found in inner enamel32. Demineralization at the early stages of caries lesion formation could take place at two locations: (i) the enamel surface, and (ii), a bit later than at the enamel surface, the region near the enamel-dentin junction (involving both enamel and dentin). At the enamel surface because it is closer to the source of cariogenic acid. At the region near the enamel-dentin junction because there the osmotic gradient would be minimized so that acid could move more slowly and find more favourable conditions to diffuse to the surfaces of the mineral crystallites surrounding the main pathways in enamel and dentin.\n\nNew optical techniques for caries diagnosis have been validated with SM only4,33. The assumption behind such validation is that SM has an acceptable accuracy for detecting dentin demineralization and sclerosis. Our results show that the probability of a correct diagnosis of both carious and sclerotic dentin using SM is low. Clinical visual caries diagnostic systems validated primarily by SM34,35 should have their validation tested using MR corrected with regard to heterogeneous illumination. The nature of dental caries should be studied without the bias related to the aspect of dentin under SM.\n\nWe conclude that, except for normal dentin, SM has low accuracy for detecting dentin reactions related to caries, and SM and PLMQ accuracies are not influenced by the quality of MR image contrast. FBC microradiographic images should be preferably used as the gold standard for judging dentin reactions.\n\n\nData availability\n\nFigshare: Dentin reactions detected from SM and PLMQ, doi:10.6084/m9.figshare.89573736",
"appendix": "Author contributions\n\n\n\nFBS, AMBC, and FDSF designed the study. PFS collected samples, performed analysis of ICDAS scores, prepared ground sections and performed a pilot study. DAHF and KRSM performed analysis with microradiography, stereomicroscopy and polarized light microscopy. FBS analyzed data. FBS wrote the manuscript, which was revised by AMBC and FDSF.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe first author received a master degree scholarship from CNPq (Brazilian Ministry of Science, Innovation and Technology).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nNational Institute of Standards and Technology (NIST) X-ray attenuation databases. Reference Source\n\nElliott JC: Structure, crystal chemistry and density of enamel apatites. In Dental Enamel. Ciba Found Symp. Chadwick D, Cardew G, editors. Chichester: Wiley, 1997; 205: 54–72. PubMed Abstract\n\nMitropoulos P, Rahiotis C, Kakaboura A, et al.: The impact of magnification on occlusal caries diagnosis with implementation of the ICDAS II criteria. Caries Res. 2012; 46(1): 82–86. PubMed Abstract | Publisher Full Text\n\nSalsone S, Taylor A, Gomez J, et al.: Histological validation of near-infrared reflectance multispectral imaging technique for caries detection and quantification. J Biomed Opt. 2012; 17(7): 076009. PubMed Abstract | Publisher Full Text\n\nWefel JS, Clarkson BH, Heilman JR: Natural root caries: a histologic and microradiographic evaluation. J Oral Pathol. 1985; 14(8): 615–23. PubMed Abstract | Publisher Full Text\n\nMassler M: Pulpal reactions to dental caries. Int Dent J. 1967; 17(2): 441–460. PubMed Abstract\n\nWeber DF: Human dentin sclerosis: a microradiographic survey. Arch Oral Biol. 1974; 19(2): 163–169. PubMed Abstract | Publisher Full Text\n\nLevine RS: The microradiographic features of dentin caries. Observations on 200 lesions. Br Dent J. 1974; 137(8): 301–306. PubMed Abstract\n\nStanley HR, Pereira JC, Spiegel EH, et al.: The detection and prevalence of reactive and physiologic sclerotic dentin, reparative dentin, and dead tracts beneath various types of dental lesions according to tooth surface and age. J Oral Pathol. 1983; 12(4): 257–289. PubMed Abstract | Publisher Full Text\n\nHintze H, Wenzel A, Larsen MJ: Stereomicroscopy, film radiography, microradiography and naked-eye inspection of tooth sections as validation for occlusal caries diagnosis. Caries Res. 1995; 29(5): 359–363. PubMed Abstract | Publisher Full Text\n\nApplebaum E, Hollander F, Bodecker C: Normal and Pathological Variations in Calcification of Teeth as Shown by the Use of Soft X-rays. Dent Cosmos. 1933; 75(11): 1097–1105. Reference Source\n\nApplebaum E: Tissue changes in caries. Dent Cosmos. 1935; 77(10): 931–941. Reference Source\n\nGottlieb B: Dental caries. Philadelphia: Lea & Febiger. 1946.\n\nGottlieb B, Diamond M, Applebaum E: The caries problem. Am J Orthod Dent Surg. 1946; 32: 365–379. PubMed Abstract\n\nOptical Microscopy Primer: basic concepts in digital imaging processing. 2013. Reference Source\n\nAndelson EH: Perceptual organization and the judgment of brightness. Science. 1993; 262(5142): 2042–2044. PubMed Abstract | Publisher Full Text\n\nIsmail AI, Sohn W, Tellez M, et al.: The International Caries Detection and Assessment System (ICDAS): an integrated system for measuring dental caries. Community Dent Oral Epidemiol. 2007; 35(3): 170–178. PubMed Abstract | Publisher Full Text\n\nMurphy DB: Fundamentals of Light Microscopy and Digital Imaging. New York: Wiley-Liss. 2001. Reference Source\n\nCohen J: Statistical power analysis for the behavioral sciences. New York: Lawrence Erlbaum. 1988. Reference Source\n\nEkstrand KR, Ricketss DN, Kidd EA: Do occlusal carious lesions spread laterally at the enamel-dentin junction? A histolopathological study. Clin Oral Investig. 1998; 2(1): 15–20. PubMed Abstract | Publisher Full Text\n\nMeyer-Luckel H, Paris S, Ekstrand K: Caries management: science and clinical practice. Stuttgart: Thieme. 2013. Reference Source\n\nSlayter EM, Slayter HS: Light and electron microscopy. Cambridge: Cambridge University Press 1997. Reference Source\n\nFejerskov O, Kidd EAM: Dental caries: the disease and its clinical management. London: Wiley-Blackwell 2008. Reference Source\n\nOldenbourg R, Shribak M: Microscopes. In: Bass M. Handbook of optics. Volume I. Geometrical and physical optics, polarized light, components and instruments. New York: McGraw Hill 2010.\n\nKidd EAM, Fejerskov O: What constitutes dental caries? Histopathology of carious enamel and dentin related to the action of cariogenic biofilms. J Dent Res. 2004; 83: C35–C38. PubMed Abstract | Publisher Full Text\n\nBjorndal L, Thylstrup A: A structural analysis of approximal enamel caries lesions and subjacent dentin reactions. Eur J Oral Sci. 1995; 103(1): 25–31. PubMed Abstract | Publisher Full Text\n\nEkstrand KR, Kuzmina I, Bjørndal L, et al.: Relationship between external and histologic features of progressive stages of caries in the occlusal fossa. Caries Res. 1995; 29(4): 243–250. PubMed Abstract\n\nNanci A: Ten Cate´s Oral Histology. 7.ed. Saint Louis: Mosby Elsevier. 2008. Reference Source\n\nShellis RP: A scanning electron-microscopic study of solubility variations in human enamel and dentin. Arch Oral Biol. 1996; 41(5): 473–484. PubMed Abstract | Publisher Full Text\n\nShellis RP, Dibdin GH: Enamel microporosity and its functional implications. In: Teaford MF, Smith MMS, Ferguson MWJ. Development, Function and Evolution of Teeth. Cambridge: Cambridge University Press. 2000. Publisher Full Text\n\nAtkinson HF: An investigation into the permeability of human enamel using osmotic methods. Br Dent J. 1947; 83(10): 205–214. PubMed Abstract\n\nDusevich V, Xu C, Wang Y, et al.: Identification of a protein-containing enamel matrix layer which bridges the dentin-enamel junction of adult human teeth. Arch Oral Biol. 2012; 57(12): 1585–1594. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZakian C, Pretty I, Ellwood R: Near-infrared hyperspectral imaging of teeth for dental caries detection. J Biomed Opt. 2009; 14(6): 064047. PubMed Abstract | Publisher Full Text\n\nJablonski-Momeni A, Stachniss V, Ricketts DN, et al.: Reproducibility and accuracy of the ICDAS-II for detection of occlusal caries in vitro. Caries Res. 2008; 42(2): 79–87. PubMed Abstract | Publisher Full Text\n\nKuhnisch J, Bucher K, Henschel V, et al.: Diagostic performance of the universal visual scoring system (UniVISS) on occlusal surfaces. Clin Oral Investig. 2011; 15(2): 215–223. PubMed Abstract | Publisher Full Text\n\nSilva PF, de Holanda Ferreira DA, Meira KRS, et al.: Dentin reactions detected from SM and PLMQ. 2014. Data Source"
}
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[
{
"id": "5982",
"date": "09 Sep 2014",
"name": "Vera Mendes Soviero",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript is related to an interesting and relevant issue. The analysis of different gold standards for caries assessment than stereomicroscopy is of great interest, as we all know that the “histological gold standard” has limitations. However, in my view, certain points need to be clarified. I have carefully revised the manuscript and I hope the comments below are helpful to improve the quality of its final version.ABSTRACTI do recommend rewriting the abstract. It should be structured with Aim, Methods, Results and Conclusion in this order. It is not necessary to mention the titles, but it is important to follow this sequence. The aims are not mentioned in the abstract and results are shown before methodology. INTRODUCTION1st paragraph, lines 6 to 8 “… radiography with microscopic resolution (MR) is considered as a highly reliable gold standard for detecting variations in dentin mineral content.” I miss a reference for this statement. If MR was not validated previously for the detection and assessment of dentin caries or dentin reactions to caries, the aim of the study should be changed. Instead of testing the accuracy of stereomicroscopy against MR, it should be testing the accuracy of MR against stereomicroscopy. Reference number 10 (Hintze et al. 1995) concluded that SM was more reliable than MR. References number 11 to 14 (Applebaum et al. 1933; Applebaum 1935; Gottlieb 1946; Gottlieb et al. 1946) don’t seem to refer to validation studies. AIMFirst aim: how to test the accuracy of SM if it is established as gold standard?Second aim: I didn’t understand. Please, explain better what is “elements of accuracy”.Third aim: In my view, the third aim summarizes everything. MATERIALS AND METHODSCalibration of examiners of ground sections and selection of histological sitesPlease, clarify better how calibration was done. What means “Examiner agreed on the final scores by consulting with each other” ? Does it mean that examinations were not done independently? Please, clarify the following sentence: “… included in the sample when two sites had the same type of dentin reactions detected by SM, while showing different types of dentin reactions when detected using MR.” It is not clear for me. If MR had to be different from SM, the results would be completely biased by this “selection criterion”. Or not? RESULTSTables in generalAbbreviations should be explained using notes underneath the tables. Table 1Carious and sclerotic dentin was observed in all samples. How do you explain it considering that 10 teeth were scored visually as ICDAS 0? The sums in table 1 vary from 153 (PLMQ) to 171 (NFNBC). Why? Figure1The legend is not clear. Figures are not explained following the sequence of numbers. The arrows should be seen in all pictures. Table 2The title is not clear enough. I suggest writing in the title that microradiography was used as gold standard. Table 3In the title, what means (1-5) and (6)? Please, clarify the title.",
"responses": [
{
"c_id": "1014",
"date": "30 Sep 2014",
"name": "Frederico Sousa",
"role": "Author Response",
"response": "The authors would like to thank the reviewer for her valuable technical comments, which will certainly contribute to improve the quality of the paper. The answers provided below are listed following the sequence of the reviewer comments and were prepared in common agreement among authors. Shortly we will publish a revised version of the article including the changes resulted from this review. Answer to comment on abstract: We agree with the referee. The abstract will be changed in the revised version and here follows how it will be written:\"Dentin reactions to caries, crucial for pathogenesis and for the determination of the severity of caries lesions, are believed to be reasonably detected by stereomicroscopy (SM) and polarized light microscopy in quinoline (PLMQ), but accuracies are lacking. We aimed at testing (i) accuracies of SM and PLMQ, (ii) the effect of image contrast of microradiography (MR) on accuracy, and (ii) the hypothesis that SM, PLMQ and MR (regardless image contrast) equally detect dentin reactions. Ground sections (n = 63) of natural occlusal caries (ICDAS scores 0 to 6) were analyzed using MR as the gold standard. SM of wet and dry ground sections of natural occlusal caries lesions resulted in moderate (0.7, for normal dentin) and low accuracies (< 0.6, for carious and sclerotic dentin) as validated by contrast-corrected MR. Accuracies of PLMQ were moderate for both normal (0.71) and carious dentin (0.71). The main differences were related to the detection of sclerotic dentin by both SW and SD, and normal and carious dentin by PLMQ. The hypothesis that detection of dentin reactions by SM and PLMQ would be influenced by the contrast quality of MR images was rejected. In conclusion, contrast-corrected MR should be preferred as the gold standard and SM and PLMQ should be avoided, but the relationship of PLMQ with dentin mineralization deserves further investigation.\" Answer to comments on introduction: The following papers can be used as references for the statement that microradiography is considered as a reliable gold standard for variations in dentin mineral content: Ten Bosch & Angmar-Månsson (1991); and Arends et al. (1997). However, the fundamental reason why microradiography is a reliable gold standard lies on what was stated in the first six lines of the Introduction. Here, we explain those reasons more clearly. Basically (and this applies for the analysis of mineralized tissues by radiography in general), from the components of dentin (mineral, organic matter and water), the mineral content absorbs X-rays more intensively than the other components. The basic parameter is the linear attenuation coefficient (LAC) for X-rays, which is the product of the mass attenuation coefficient (MAC) by the density of the unit cell. Information on MAC and LAC (corresponding to X-ray peak of 24 keV, which is the emission peak emitted by the X-ray source used here operating at 40 kV and 0.25 mA) for both the mineral and the organic components of dentin are listed below:For the mineral component: with the unity cell’s empirical formula Ca8.856Mg0.088Na 0.292K0.010P5.592Cl 0.078O25.561H 3.222C0.457 (reference 2), the MAC is 3.83 cm2•g-1, the density is 2.99 g•cm-3 (reference 2), and the LAC is 11.45 cm-1.For the organic component, represented by collagen: with the unity cell’s empirical formula C12H24N3O4 (Omokanwaye et al., 2010), the MAC is 0.41 cm2•g-1, the density is 1.19 g•cm-3 (Gautieri et al., 2011), and the LAC is 0.49 cm-1.MACs for the chemical elements of the empirical formulas listed above can be found in reference 1. Gray levels of dentin in MR are the result of the product of the volume of each component by its respective LAC, and the sum of all products is multiplied by sample thickness. Thus, as mineral volume decreases in dentin, gray levels decrease (i.e., dentin becomes less radiopaque), and vice-versa.With light scattering, which governs the aspect of dentin under SM, is different. By matching the refractive index of an immersion liquid with the refractive index of dentin light scattering is reduced, rendering the tissue more translucent without modifying tissue composition (Fried et al., 1995). In addition, visible light scattering in dentin is highly influenced by the density (Zijp & Bosch, 1993; Kienle et al., 2003) and angulations (Kienle et al., 2003) of dentinal tubules in the sample. And the density of dentinal tubules increase as one approaches the dental pulp (Garberoglio & Brannstrom,1976).Answer to comments on the aims: The evidences supporting SM as a gold standard for dentin reactions to caries are typically those cited in the review paper by Kidd & Fejerskov (2004) (reference 25); to quote: references 6, 8 and 9, and Johnson et al.,(1969). They are not validation studies (i.e., accuracy is not provided), but qualitative studies where microradiographic images are reported without correction for heterogeneous illumination. Previous reports showed that translucent dentin could be either demineralization or sclerosis (references 11-14), and this fact means that the probability of finding similar results is high if SM and MR are compared many times. It is not excluded neither the possibility of finding translucent sclerotic dentin nor the possibility of finding a correlation between SM and MR when there is no translucent dentin. The study by Hintze et al. (reference 10) was performed with unerupted sound teeth, which lack translucent dentin in response to caries. Measurements of lesion size with SM exclude translucent dentin, resulting in underestimation of lesion size if part of the translucent dentin is demineralization. Translucent dentin comprises a significant part of dentin reactions to the carious process (Bjørndal & Thylstrup,1995; Bjørndal et al., 1999). On this basis, in the “Aims” we considered that further studies were needed to support SM as a gold standard for detecting dentin reactions to caries. The third aim does not include the test of the accuracy of SM. With all due respect, we think that the aims should be kept as they are.Positive and negative predictive values are considered as elements of accuracy because they describe the performance of diagnostic tests and high results are indicative of good accuracy. In order to put this more clearly in the revised version, PPV and NPV will be specified as elements of accuracy in the aims. Answer to comments on materials and methods: The statement “Examiner agreed on the final scores by consulting with each other” means that the final scores were given by consensus between examiners, just like was done with the visual diagnosis with ICDAS II described in “Diagnosis of occlusal caries lesions using ICDAS II”. When disagreement occurred, the examiners consulted each to reach a consensus. This is a common approach in the dental literature. Answer to comment on the statement ”… included in the sample … when detected using MR.”: This statement was included to say that SM and MR scores were not the same. Histological sites were selected on the basis of MR images. In many cases, sites scored as normal dentin by SM, for example, were not scored as normal dentin by MR. This did not apply for all cases, as stated in the previous paragraph where it is written that “at least one dentin reaction type per sample was included where possible”. This was not possible for all cases. We recognize, however, that the statement is not clear, is misleading, and it will be changed in the revised version. Answer to comments on results: We accept the suggestion of the referee on abbreviations. This modification will be included in the revised version. Answer to comments on Table 1: Regarding the report that 10 teeth with ICDAS score 0 presented carious and sclerotic dentin, below follow reports of cases of occlusal surfaces with ICDAS score 0 that were scored by SM (with regard to the depth of the carious lesion) as having demineralization in the inner 50% of the enamel layer (E2) or in the outer 50% of the dentin layer (D1): Ekstrand et al. (1997) reported 05 cases of occlusal caries with ICDAS score 0 and SM score E2 (see Table 8 in their paper);Ekstrand et al. (1998) reported 01 case of occlusal caries with ICDAS score 0 and SM score E2 (see Table 4 in their paper);Jablonski-Momeni et al. (2008) reported 02 cases of occlusal caries with ICDAS score 0 and SM score E2 (see Table 4 in their paper);Braga et al. (2009) reported 01 case of occlusal caries with ICDAS score 0 and SM score E2 (see Table 4 in their paper);Diniz et al. (2009) reported 06 cases of occlusal caries with ICDAS score 0 and SM score E2 and 01 case of ICDAS score 0 and SM score D1 (see Table 3 in their paper).SM score E2 is important because it has been related to the occurrence of translucent dentin (Bjørndal & Thylstrup,1995; Bjørndal et al., 1999), and this later might be either carious or sclerotic (see our results and references 11-14), so that part or all of those cases with SM score E2 listed above could represent carious dentin. On this basis, the occurrence of carious and sclerotic dentin with ICDAS score 0 is not an unexpected result.The totals of PLMQ and NFNBC differ because, as explained in the section “Polarized light microscopy in quinoline”, PLMQ is not used to detect sclerotic dentin. The total of PLMQ cases is the sum of carious plus normal dentin, while the total of NFNBC cases is the sum of carious plus normal plus sclerotic/translucent dentin. Answer to comments on Figure 1: The fact that figures are not explained following their sequence was intentional. The legend describes the figures following the sequence of the aspects of the sites 1-4 pointed in Fig.1A (wet SM). This way, emphasis in given to the main differences between the wet SM image (commonly used in the literature) and the others. Regarding the arrows, we had prepared a version with arrows in all figures but we thought that it would disturb free interpretation of some images by the reader. This is why arrows were included in half of the images. With all due respect, we still thing this way is the best one.Answer to comments on Table 2: We accepted the suggestion of the reviewer and the title will be changed (in the revised version) to: “Table 2. PPV and NPV of SM and PLMQ for dentin reactions from all combinations of test and gold standard (MR) outcomes”.Answer to comments on Table 3: (1-5) and (6) are the combinations described in the Material and Methods. In the revised version, we will explain them using a footnote in Table 3."
}
]
},
{
"id": "6313",
"date": "11 Nov 2014",
"name": "Mariana Minatel Braga",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe present paper concerns a relevant topic related to dentin reactions to dental caries lesions. However, the authors’ intention, testing accuracies of methods for detecting these reactions, could be valorized/stressed in this paper. I believe that it is important to readers to know how important is to go beyond in this topic. In addition, considering the rationale, the authors stated that “only evidence for the detection of dentin reactions is qualitative”. I would recommend taking care to avoid misunderstandings, since the methods tested were qualitative, but I suppose they are looking for figures about accuracy of these methods. I suggest clarifying that. Another point to be clarified is the use of microradiography as a reference method. In the text, authors mentioned that “radiography with microscopic resolution (microradiography, MR) is considered as highly reliable gold standard for detecting variations in dentin mineral content.\" Is that an authors’ affirmation or is based on a previous background? Another aspect regarding reference methods is that the authors proposed to use the microradiography as gold standard, but, actually, they believed the microradiography was similar to other methods tested (according to the 3rd objective). Why not choose an actual quantitative method for that? The rationale for that could be better developed in the introduction. Was that the idea of authors initially or was that a concept formulated a posteriori (after discussing and analyzing present findings)? Maybe, showing the reasons why opting for testing methods using a reference you “ suspect” that is similar to other methods . I suggest these ideas could be explored in the Introduction.Concerning methodology, one point remained unclear for this reviewer. In the paragraph “Histological sites were selected from the outer half of the dentin layer, including the area adjacent to the deepest enamel lesion; at least one dentin reaction type per sample was included where possible. Cases were included in the sample when two sites had the same type of dentin reaction detected by SM, while showing different types of dentin reactions when detected using MR. Thus, up to 6 histological sites were selected per sample, yielding a total sample size of 168 sites.\" it seems that only specimens in which there was some dentin reaction were selected. Is that true? If no, please, clarify that in the text and do not consider the next part. If yes, I believe this step could have led to spectrum of disease bias and selection bias, as well. If you try to detect the dentin reaction in a pool of histological sites, you tend to have a different performance than if you only selected sites in which the reaction is present. In addition, if you examined the teeth by one of the methods tested subsequently, you tend to favor this method in further examinations (because the selection was based on that). Based on these points, if you really selected only the sites with dentin reactions, I suggest including some information about those points in the Discussion.Regarding the results, I suggest including the confidence intervals to facilitate the comprehension of possible overlapping of marginal values. Besides, I would include some discussion about the reference standard used and the relevance of detecting this level of demineralization in clinical setting or other applications. If the purpose is choosing the best treatment to the patients, some caution should be had in inferring data about very precise methods for demineralization quantification (sometimes these differences could lead to no difference in clinical approach to be adopted). But, certainly, other purposes exist and the investigation is valid for them. Moreover, I would explore more deeply how the figures showed for methods performance impact on inferences (e.g. which is the contribution of testing predictive values instead of showing only accuracy. Considering these points, I suggest some caution in writing the title.The authors stated that “If lateral spread does occur, dentin demineralization and sclerosis might occur at random in different sections of a same lesion. In addition, two sites located at different fossae on the same occlusal surface are expected to present independent dentin reactions. This is why we considered more than one section per tooth as independent samples”. I believe that it could be true, but, to reinforce this assertive, I suggest testing the influence of the sampling in the present results or using a statistical approach that consider the clustering effect for the specimens evaluated.Finally, I would consider separating the analysis for dentin reactions related to caries lesions. Despite being reactions in different ways (desmineralization vs. hypermineralization), caries lesion formation and sclerotic dentin formation are not actually opposite actions. They involve different mechanisms of pathogenesis and not only the quantity of minerals. That is why I believe the purpose of identifying each one of these alterations has its importance independently of the other. This separation, in my point of view, may help the authors in explaining the findings of this study. Additionally, based on this observation, I would also suggest reinforcing that in other parts of this paper.",
"responses": [
{
"c_id": "1200",
"date": "29 Jan 2015",
"name": "Frederico Sousa",
"role": "Author Response",
"response": "The authors would like to thank the reviewer for her report and the valuable comments in it. As a consensus among all authors, the answers to her comments are provided below. Changes in the manuscript text will be included when a revised version is published in the near future. Answer to the first comment: For the statement “only evidence for the detection of dentin reactions is qualitative” we meant to say that the available studies indicating that SM and PLMQ could be used as gold standards lacked accuracy in numbers. The referee is correct by stressing that we should state more clearly that we were looking for accuracy. This will be included in the revised version.Answer to the question on microradiogarphy as the gold standard: We kindly ask the reviewer to refer to the answer to a similar comment made in the first referee report (from Dr. Soviero), where it was explained the reasons why microradiography is considered a reliable gold standard for changes in the mineral content of dentin.Answer to the comment that “the authors proposed to use the microradiography as gold standard, but, actually, they believed the microradiography was similar to other methods tested” : The belief that SM, PLMQ and MR are similar is found in the literature (see references 3-10). We considered that is important to have their accuracies tested in order to make a scientifically robust decision on whether SM and PLMQ are similar to MR or not. In the Introduction, we stressed that MR is a reliable gold standard (and reasons were given; for more on those reasons, please refer to our answer to the comment of the first referee report on why MR was chosen as a gold standard) and that accuracies for SM and PLMQ were lacking. In summary, SM and PLMQ are currently believed to be similar to MR (a proven reliable gold standard) by almost all of those in the field of histopathology of dental caries, but accuracies were lacking. We hope that highlighting this here might help the reviewer (and the readers, as all comments and responses are open) to understand the reasons why we tested SM and PLMQ against MR.Answer to the comment “it seems that only specimens in which there was some dentin reaction were selected. Is that true?”: It were selected sites presenting aspects of both normal dentin and dentin affected by caries. This will be clarified in the revised version. Answer to the comment asking for confidence intervals: The confidence intervals will be included in the revised version.Answer to the comment asking for cluster analysis: The authors are aware that cluster analysis is receiving growing attention in dental research, and some guidelines for statistical methodology in dental research have been published stressing that cluster analysis (or the reason why clustering was ignored) should be taken into account, mainly when different sites from the same subject are used (Hannigan et al., 2013). In this study we ignored clustering, mainly because the correlation between dentin translucency under SM and dentin radiopacity was very weak (R2 = -0.013; see our Fig. 3). It has been stressed that the consequences of ignoring the clustering depends on the correlation that exists in the data (Galbraith et al., 2010). Considering that the two main features of clusters are compactness and isolation, and that correlation coefficient is a similarity index (see pages 11 and 188 in Jain and Dubes, Algorithms for clustering data. Prentice Hall: Engelwood Cliffs, NJ, 1988) – any clusters in our sample would not have enough compactness to be attain validity -, it can be reasonably considered that ignoring the clustering is justified in our study. These considerations will be included in the revised version. Reviewer Comment: “Finally, I would consider separating the analysis for dentin reactions related to caries lesions. Despite being reactions in different ways (desmineralization vs. hypermineralization), caries lesion formation and sclerotic dentin formation are not actually opposite actions. They involve different mechanisms of pathogenesis and not only the quantity of minerals. That is why I believe the purpose of identifying each one of these alterations has its importance independently of the other. This separation, in my point of view, may help the authors in explaining the findings of this study. Additionally, based on this observation, I would also suggest reinforcing that in other parts of this paper.” Answer to the comment described above: We did not understand this comment well because all the results are already separated by the type of dentin reaction (normal, sclerotic and carious). We would be happy to read in more details the differences between what is already reported and what the reviewer expects to see."
}
]
},
{
"id": "6314",
"date": "13 Nov 2014",
"name": "Chris Deery",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting paper which asks some interesting questions. The methodology is well described.The title is appropriate. The abstract should be expanded to make the findings clearer. The main text has a minor problem with the use of abbreviations, this makes the paper hard to read particularly pages 5 and 6.In the introduction there is no mention of other techniques such as micro-CT, ICDAS is just ICDAS not ICDAS II.Page 4: accuracy is not the best method to use to assess the results, as it tends to inflate the results.I think on page 4 1.SWxNFNBC etc could be better explained.The Kappa results on page 5 paragraph 3 might be better presented as a table.The authors should make a stronger explanation of why they believe microradiology is in fact more valid. Perhaps the problems they suggest are present with steriomicroscopy due to training or interpretation issues. They do not mention any training only calibration, these authors must be aware that reproducibility is very different to validity. This issue should be addressed.",
"responses": [
{
"c_id": "1109",
"date": "03 Dec 2014",
"name": "Frederico Sousa",
"role": "Author Response",
"response": "The report of approved is greatly acknowledged. The answers to the comments are provided below item by item. In the near future, we will publish a revised version containing the changes resulted from all referee reports. Answer to comment on abstract: The abstract will be changed in the revised version and here follows how it will be written:\"Dentin reactions to caries, crucial for pathogenesis and for the determination of the severity of caries lesions, are believed to be reasonably detected by stereomicroscopy (SM) and polarized light microscopy in quinoline (PLMQ), but accuracies are lacking. We aimed at testing (i) accuracies of SM and PLMQ, (ii) the effect of image contrast of microradiography (MR) on accuracy, and (ii) the hypothesis that SM, PLMQ and MR (regardless image contrast) equally detect dentin reactions. Ground sections (n = 63) of natural occlusal caries (ICDAS scores 0 to 6) were analyzed using MR as the gold standard. SM of wet and dry ground sections of natural occlusal caries lesions resulted in moderate (0.7, for normal dentin) and low accuracies (< 0.6, for carious and sclerotic dentin) as validated by contrast-corrected MR. Accuracies of PLMQ were moderate for both normal (0.71) and carious dentin (0.71). The main differences were related to the detection of sclerotic dentin by both SW and SD, and normal and carious dentin by PLMQ. The hypothesis that detection of dentin reactions by SM and PLMQ would be influenced by the contrast quality of MR images was rejected. In conclusion, contrast-corrected MR should be preferred as the gold standard and SM and PLMQ should be avoided, but the relationship of PLMQ with dentin mineralization deserves further investigation.\" Answer to comments on introduction: We did not mention micro-CT because we understand that micro-CT contrasts sound from carious tissues based on the mass attenuation coefficient of X-rays of the chemical elements, similarly to microradiography. We have responded to referee 1 describing the reasons why microradiography is the best gold standard for detecting variations in mineral content in hard dental tissues. As all responses are available to all, we think this solves the problem satisfactorily.We used ICDAS II because this term has been used previously in major dental journals (Shoaib et al., 2009). Answer to comments on materials and methods: in the revised version (the combinations SW x NFNBC etc. will be better explained.We believe that accuracy, PPV and NPV are reasonable parameters to test the validity of SM to detect dentin reactions to caries. This is in agreement with previous reports that tested micro-CT for detecting demineralization in hard dental tissues affected by dental caries (Soviero et al., 2012). Answer to comments on results: the reproducibility values of each examiner with regard to SM and MR are presented in the first paragraph of the “Results” section. Examiners were trained in the lab using ground sections of carious teeth before performing analysis with SM and MR. In addition: (i) it can be see in Figure 1 that some points with translucent dentin are radiolucent; (ii) previous publications reported cases of translucent dentin that represented demineralization when examined by microradiography (references 11-14)."
}
]
}
] | 1
|
https://f1000research.com/articles/3-13
|
https://f1000research.com/articles/3-12/v1
|
15 Jan 14
|
{
"type": "Case Report",
"title": "Case Report: Detection and quantification of tumor cells in peripheral blood and ascitic fluid from a metastatic esophageal cancer patient using the CellSearch® technology",
"authors": [
"Qian Tu",
"Marcelo De Carvalho Bittencourt",
"Huili Cai",
"Claire Bastien",
"Camille Lemarie-Delaunay",
"Marie C Bene",
"Gilbert C Faure",
"Qian Tu",
"Marcelo De Carvalho Bittencourt",
"Huili Cai",
"Claire Bastien",
"Camille Lemarie-Delaunay",
"Marie C Bene"
],
"abstract": "Analysis of ascitic fluid should help to identify and characterize malignant cells in gastrointestinal cancer. However, despite a high specificity, the sensitivity of traditional ascitic fluid cytology remains insufficient, at around 60%. Since 2004 the CellSearch® technology has shown its advantages in the detection of circulating tumor cells (CTCs) in peripheral blood, which can perform an accurate diagnosis and molecular analysis at the same time. To our knowledge, no previous study has explored the potential utility of this technology for the detection and quantification of tumor cells in ascitic fluid samples. Herein we report a case of metastatic esophageal adenocarcinoma in a 70-year-old man presenting with dysphagia and a large amount of fluid in the peritoneal cavity. Analysis of a peripheral blood sample and ascites sample with the CellSearch® technology both revealed the presence of putative tumor cells that were positive for epithelial cell adhesion molecule (EpCAM) and cytokeratin (CK) expression. This study confirmed the hematogenous dissemination of esophageal cancer by the detection of circulating tumor cells in the peripheral blood, and is the first to demonstrate that tumor cells can be identified in ascitic fluid by using CellSearch® technology.",
"keywords": [
"Circulating tumor cell",
"Metastatic esophageal cancer",
"Ascitic fluid",
"CellSearch technology"
],
"content": "Introduction\n\nEsophageal cancer is the eighth most common carcinoma and the sixth leading cause of cancer-related death worldwide, with approximately 482,000 new cases and 400,000 associated deaths per year1. There are two main histological types of esophageal cancer, namely esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). Although more than 90% of malignant esophageal tumours are squamous cell carcinomas, the incidence of esophageal adenocarcinoma in Western countries has increased sharply in recent decades2. Disease prognosis is strongly related to the stage at diagnosis3, but EAC is commonly diagnosed at an advanced stage and palliative therapies are often the only treatment option, and therefore EAC has an extremely poor prognosis.\n\nIn particular, peritoneal dissemination is frequent in gastrointestinal cancer and analysis of the ascitic fluid should help to establish the etiology of ascites, and identify and characterize malignant cells. However, despite a high specificity, the sensitivity of ascitic fluid cytology remains insufficient, at around 60%4,5.\n\nRecently, the detection of circulating tumor cells (CTCs) had an increasingly important role in the domain of oncological research. These cells have long been considered as a reflection of tumor aggressiveness, while hematogenous spreading of CTCs from a primary tumor could be an important step in the metastasis cascade, leading ultimately to the formation of overt metastases6.\n\nWe herein report a case study of a 70-year-old man affected by esophageal adenocarcinoma with multiple metastases who was referred for dysphagia and abdominal effusion. At diagnosis, the patient underwent a peripheral blood and ascitic examination to detect and enumerate CTCs and determine their clinical significance.\n\n\nCase presentation\n\nAt the beginning of June 2012, a 70-year-old white male was referred to the emergency unit because of a progressive deterioration of general condition for 4 weeks, which included dysphagia, low-grade fever, anorexia, and a weight loss of 8 kg within 4 months. The patient had been suffering from chronic lymphocyte leukemia stage A for 4 years, with indolent evolution according to regular yearly follow-ups.\n\nInitial physical examination was unremarkable except for a left infraclavicular adenopathy of 2 cm. A computed tomography scan identified a thickening of the lower esophagus, a hypodense lesion of the liver and a lymphatic mass in the mesenteric region.\n\nThe patient was then admitted to hospital for further investigations. During the period of hospitalization, the patient’s disease progressed quickly, with the appearance of massive ascites, jaundice, and respiratory difficulties. A PET/CT scan showed a thickening of the lower esophagus, supra- and sub-diaphragmatic adenomegalies and lesions in the liver, pancreas and lesser curvature of the stomach, all with hypermetabolic activities (Figure 1). A gastroscopy showed a circumferential thickening, without stenosis in the inferior segment of the esophagus, bleeding easily on contact. A biopsy performed at this level demonstrated infiltration by a poorly differentiated adenocarcinoma. Fine needle aspiration biopsy of the liver lesion demonstrated the same tumor type with large cells of polygonal or cubic shape, and irregular hyperchromatic nuclei. These cells were also positive for cytokeratins CK7, 19, and 20 by a standard immunochemistry method. Both the first and second paracentesis revealed rare potential adenocarcinoma cells in clusters (Figure 2), which were cytokeratins AE1/AE3, 7 and 19 positive.\n\nPET/CT, performed after administration of 311 MBq of Fludoxyglucose, showed increased activity in the lower oesophagus (a), liver metastasis (b) and pancreatic metastasis (not shown in the picture).\n\nThe presence of tumor cells in the peripheral blood and ascitic fluid was evaluated by the CellSearch® technology (Veridex LLC, Raritan, NJ), which detects CTCs based on ferrofluids coated with epithelial cell-specific EpCAM antibodies and a mixture of phycoerythrin-conjugated antibodies to CK 8, 18, and 19. Following informed consent, a 7.5 ml sample of peripheral blood and a 5 ml sample of ascitic fluid were collected in CellSave® tubes. The blood sample was processed in the CellTracks AutoPrep system by using the CTC kit and analyzed with the CellTracks Analyzer. For analysis of the ascitic sample, we used the method developed for the detection of tumor cells in cerebrospinal fluid samples (CSF)7,8. This methodology revealed the presence of 47 CTCs (EpCAM+, CK+, and CD45-) in the 7.5 ml blood sample, some with morphologically apoptotic features, and over 13,000 CTCs (EpCAM+, CK+, and CD45-) in the 5 ml ascitic sample. Interestingly, in the ascitic sample, positive cells were either isolated or in clusters and their morphology was very similar to that of CTCs in the peripheral blood though without apoptotic features (Figure 3).\n\nBy definition, CTCs are nucleated (purple color), express cytokeratin (green color), and lack CD45 expression. Some of the CTCs in blood sample presented morphologically apoptotic features. Arrow, shrunken cell containing CK inclusions. But in the ascitic sample, positive cells were either isolated or in clusters and their morphology was very similar to that of CTCs in the peripheral blood though without apoptotic features.\n\nThe patient was diagnosed with an adenocarcinoma of the lower esophagus or gastroesophageal junction, with hepatic, peritoneal and pancreatic metastases. No remission of the disease was observed under palliative treatment, and the patient died one month after diagnosis.\n\n\nDiscussion\n\nEsophageal cancer metastases are most frequently found in abdominal lymph nodes (45%), followed by the liver (35%), and lungs (25%)9. Distant metastases are also seen in cervical/supraclavicular lymph nodes, bone, adrenal glands, peritoneum, brain, pericardium, pleura, stomach, and pancreas, via different pathways such as direct extension, the bloodstream, the lymphatic system or intracavitary diffusion. It has also been suggested that EAC preferentially metastasize to the liver9. The case we report here is a typical esophageal adenocarcinoma with multiple metastases.\n\nBecause of the critical role of CTCs, both in the spread of cancer and in cancer research, an increasing number of studies have tried to explore technologies that enable the detection of these cells in peripheral blood. However, owing to the rarity of CTCs in this compartment, their detection requires methods combining high sensitivity and specificity. To date, there is no gold standard for CTC detection. Since 2004, CellSearch® technology has become the only FDA-approved system for the clinical detection and enumeration of CTCs in patients with metastatic breast cancer, colorectal cancer and prostate cancer10–12. This technology renders it possible to obtain highly reproducible quantitative results from different laboratories13.\n\nCurrently, the results of prospective studies demonstrate that CTC enumeration has an enormous potential to estimate cancer prognosis, monitor disease recurrence and patient response to cancer therapy and even offer new insights into the comprehension of cancer biology in various epithelial cancers6.\n\nFew studies have yet documented the presence of CTCs in metastatic esophageal cancer. Allard et al. reported that CTCs were detected in patients with various types of carcinomas, including gastrointestinal cancers, although the median number of CTCs in gastrointestinal carcinomas was relatively low14.\n\nIn 2008, Hiraiwa et al. studied the utility of CTC enumeration in gastrointestinal cancer patients15. Thirty eight patients with esophageal cancer were included, including 23 patients with metastases. They found that 21.7% of the metastatic esophageal cancer patients had ≥2 CTCs in peripheral blood samples. Two or more CTCs in patients with metastatic esophageal cancer significantly correlated with pleural or peritoneal dissemination and a significantly worse prognosis. Therefore, the authors hypothesized that the detection of ≥2 CTCs was an important prognostic factor for metastatic esophageal cancer.\n\nAbdominal paracentesis is an important tool in patient diagnosis and treatment, and ascitic fluid cytology is a traditional method to establish the etiology of the effusion, especially when malignancy is suspected. However, the most important problem with conventional peritoneal cytology is its lack of sensitivity and high operator-dependence. The overall sensitivity of conventional cytology for malignant ascites is 57%–67%16, and the sensitivity for peritoneal carcinomatosis is 96.7%, while it is much lower (6.7%–13.3%) in ascites due to hepatocellular carcinoma, liver metastases, or chylous ascites with lymphoma17. To establish an accurate diagnosis and study of tumor cells, immunocytochemistry and further genetic and molecular techniques have been proposed, such as fluorescent in situ hybridization (FISH), comparative genomic hybridization (CGH) and PCR-based techniques. However, most results are qualitative, even though quantitative results might be relevant, particularly for the follow-up of either local or general therapies.\n\nPrevious studies6,10–12,14,15 have shown that CellSearch® technology has shown its advantages in the detection of CTCs in peripheral blood, showing that it can achieve accurate diagnosis as well as molecular analysis at the same time. To our knowledge, no previous study has reported the use of this technology for the detection of tumor cells in ascitic fluid samples.\n\nWe have recently documented a new method to identify and quantify malignant cells in the CSF using the CellSearch® technology7,8, suggesting its potential usefulness for follow-ups and treatment evaluations with cancer patients. As described here, this new method makes it also possible to detect and quantify the presence of tumor cells, either isolated or associated as microclusters in ascitic fluid, which in some studies are recognized as a “signature” of malignant metastases18,19.\n\nFurther to the description of the clinical utility of CellSearch® technology in ascitic fluid analysis, our findings raise the issue of additional potential applications. First, it may help diagnose cancer metastases with a high sensitivity and specificity. Second, an additional antibody can be added during the detection of tumor cells by the CellSearch® technology, allowing for a further biological characterization of tumor cells. Third, EpCAM+ cells can be purified by using the CTC Profile kit®, making them available for a variety of applications in order to characterize their genes and/or protein expression profiles, thus providing knowledge about biological aspects of human solid tumors20. This might be particularly useful in the identification of therapeutic targets and resistance mechanisms.\n\nThe case presented here confirmed the hematogenous dissemination of esophageal cancer by the detection of CTCs in the patient’s peripheral blood, and is the first report concerning the quantification of a much higher load of tumor cells in an ascitic sample. This suggests that tumor cells in ascites can be studied with the CellSearch® technology, with a great potential in patients’ diagnosis, prognosis and in the understanding of cancer biology. We propose to designate these cells in the ascitic sample as ETC for “Effusion Tumor Cells”.\n\n\nConsent\n\nWritten informed consent for publication of clinical details was obtained from the patient’s spouse.",
"appendix": "Author contributions\n\n\n\nMdCB, MCB and GCF conceived the study. QT, HC carried out the cytomics analysis. CB and CLD provided expertise in cytopathology. QT and GCF prepared the draft of the manuscript. MdCB and MCB contributed to the experimental design and preparation of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nQian Tu and Huili Cai are supported by the China Scholarship Council. Nancytomique has been funded by French Ministry of Research, Ligue Contre le Cancer, CHU Nancy and FEDER. The cancer projects were supported by Regional Lorraine Cancer research projects.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors are grateful to Pierre Olivier for the PET/CT Scan picture and François Plenat for his encouragement and support.\n\n\nReferences\n\nFerlay J, Shin HR, Bray F, et al.: Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer. 2010; 127(12): 2893–2917. PubMed Abstract | Publisher Full Text\n\nThrift AP, Pandeya N, Whiteman DC: Current status and future perspectives on the etiology of esophageal adenocarcinoma. Front Oncol. 2012; 2: 11. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlexandre L, Clark AB, Cheong E, et al.: Systematic review: potential preventive effects of statins against oesophageal adenocarcinoma. Aliment Pharmacol Ther. 2012; 36(4): 301–311. PubMed Abstract | Publisher Full Text\n\nKalogeraki A, Karvela-Kalogeraki I, Tamiolakis D, et al.: Cytopathologic interpretation of ascites due to malignancy. J BUON. 2012; 17(3): 446–451. PubMed Abstract\n\nKaroo RO, Lloyd TD, Garcea G, et al.: How valuable is ascitic cytology in the detection and management of malignancy? Postgrad Med J. 2003; 79(931): 292–294. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSun YF, Yang XR, Zhou J, et al.: Circulating tumor cells: advances in detection methods, biological issues, and clinical relevance. J Cancer Res Clin Oncol. 2011; 137(8): 1151–1173. PubMed Abstract | Publisher Full Text\n\nLe Rhun E, Tu Q, De Carvalho Bittencourt M, et al.: Detection and quantification of CSF malignant cells by the CellSearch technology in patients with melanoma leptomeningeal metastasis. Med Oncol. 2013; 30(2): 538. PubMed Abstract | Publisher Full Text\n\nLe Rhun E, Massin F, Tu Q, et al.: Development of a new method for identification and quantification in cerebrospinal fluid of malignant cells from breast carcinoma leptomeningeal metastasis. BMC Clin Pathol. 2012; 12: 21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nQuint LE, Hepburn LM, Francis IR, et al.: Incidence and distribution of distant metastases from newly diagnosed esophageal carcinoma. Cancer. 1995; 76(7): 1120–1125. PubMed Abstract\n\nde Bono JS, Scher HI, Montgomery RB, et al.: Circulating tumor cells predict survival benefit from treatment in metastatic castration-resistant prostate cancer. Clin Cancer Res. 2008; 14(19): 6302–6309. PubMed Abstract | Publisher Full Text\n\nCohen SJ, Punt CJ, Iannotti N, et al.: Relationship of circulating tumor cells to tumor response, progression-free survival, and overall survival in patients with metastatic colorectal cancer. J Clin Oncol. 2008; 26(19): 3213–3221. PubMed Abstract | Publisher Full Text\n\nAllard WJ, Matera J, Miller MC, et al.: Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases. Clin Cancer Res. 2004; 10(20): 6897–6904. PubMed Abstract | Publisher Full Text\n\nKraan J, Sleijfer S, Strijbos MH, et al.: External quality assurance of circulating tumor cell enumeration using the CellSearch((R)) system: a feasibility study. Cytometry B Clin Cytom. 2011; 80(2): 112–118. PubMed Abstract | Publisher Full Text\n\nCristofanilli M, Budd GT, Ellis MJ, et al.: Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med. 2004; 351(8): 781–791. PubMed Abstract | Publisher Full Text\n\nHiraiwa K, Takeuchi H, Hasegawa H, et al.: Clinical significance of circulating tumor cells in blood from patients with gastrointestinal cancers. Ann Surg Oncol. 2008; 15(11): 3092–3100. PubMed Abstract | Publisher Full Text\n\nDiBonito L, Falconieri G, Colautti I, et al.: The positive peritoneal effusion. A retrospective study of cytopathologic diagnoses with autopsy confirmation. Acta Cytol. 1993; 37(4): 483–488. PubMed Abstract\n\nRunyon BA, Hoefs JC, Morgan TR: Ascitic fluid analysis in malignancy-related ascites. Hepatology. 1988; 8(5): 1104–1109. PubMed Abstract | Publisher Full Text\n\nHou JM, Krebs M, Ward T, et al.: Circulating tumor cells as a window on metastasis biology in lung cancer. Am J Pathol. 2011; 178(3): 989–996. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStott SL, Hsu CH, Tsukrov DI, et al.: Isolation of circulating tumor cells using a microvortex-generating herringbone-chip. Proc Natl Acad Sci U S A. 2010; 107(43): 18392–18397. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHarouaka R, Kang Z, Zheng SY, et al.: Circulating tumor cells: Advances in isolation and analysis, and challenges for clinical applications. Pharmacol Ther. 2014; 141(2): 209–221. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "3776",
"date": "06 Mar 2014",
"name": "Eva Obermayr",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors provide an interesting study on the detection of CTCs in the blood and ascitic fluid from a patients with advanced epithelial esophageal cancer using CellSearch. The study is innovative in terms of the application of the CellSearch detection for tumor cells in the ascites, nevertheless, it is not surprising that the number of tumor cells in the ascites is by far higher than in the peripheral blood. Moreover, the clinical value of the detection of tumor cells in the ascites is unclear, especially for patients with advanced disease. In future, further investigations which include the characterization of these tumor cells in terms of EMT markers will be necessary.",
"responses": []
},
{
"id": "4681",
"date": "28 May 2014",
"name": "Hugh Barr",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting study that assesses the use of detection metastatic cancer cells in ascites. This is a proof of principle which does require more patients. As an early report this is fine.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-12
|
https://f1000research.com/articles/3-11/v1
|
15 Jan 14
|
{
"type": "Correspondence",
"title": "Risks of testosterone therapy in elderly men",
"authors": [
"Ranjith Ramasamy",
"James M. Dupree",
"Jason R. Kovac",
"Larry I. Lipshultz",
"James M. Dupree",
"Jason R. Kovac",
"Larry I. Lipshultz"
],
"abstract": "Testosterone supplementation therapy (TST) is a widely used treatment for men with late onset hypogonadism. The benefits seen with TST, such as improved libido and energy level, beneficial effects on bone density have been well documented. Although hypogonadism remains an independent risk factor for mortality, recent studies have examined the association between testosterone therapy and cardiovascular risk.",
"keywords": [
"Vigen et al.1 examined the association between testosterone supplementation and cardiovascular morbidity in men older than 60 years. They performed a retrospective national cohort study of men with low testosterone levels (<300 ng/dL) who underwent coronary angiography in the Veterans Affairs (VA) system between 2005 and 2011. The absolute rate of atherosclerotic events (myocardial infarction",
"stroke and mortality) was 19.9% in men who did not receive testosterone vs 25.7% in the men who were treated with testosterone. Men on testosterone supplementation were reported to have higher risk of adverse events than men not on testosterone",
"despite being younger and having less comorbidity."
],
"content": "Correspondence\n\nVigen et al.1 examined the association between testosterone supplementation and cardiovascular morbidity in men older than 60 years. They performed a retrospective national cohort study of men with low testosterone levels (<300 ng/dL) who underwent coronary angiography in the Veterans Affairs (VA) system between 2005 and 2011. The absolute rate of atherosclerotic events (myocardial infarction, stroke and mortality) was 19.9% in men who did not receive testosterone vs 25.7% in the men who were treated with testosterone. Men on testosterone supplementation were reported to have higher risk of adverse events than men not on testosterone, despite being younger and having less comorbidity.\n\nOne of the most important messages to glean from the study is that hypogonadism could be an adverse prognostic factor for cardiovascular and cerebrovascular morbidity and mortality. This message is also found in other studies about hypogonadism, including a large study on male veterans that showed that hypogonadism could be an important risk factor for increased mortality2. Further, in men with hypertension3, low testosterone levels were shown to be associated with increased risk of major cardiovascular adverse events.\n\nIn the Vigen et al. study men who received testosterone had lower pre-therapy testosterone levels, suggesting that they were even more hypogonadal than men who did not start testosterone therapy.In addition, it is unclear how much testosterone the men in the treatment arm actually received. Based on prescription refills, most men were on testosterone therapy for less than one year, and their mean post-treatment testosterone level was 332 ng/dL. With serum testosterone less than 300 ng/dL defined as biochemical hypogonadism by the Endocrine Society4, we are concerned that a significant proportion of men could have remained hypogonadal, in spite of testosterone treatment.\n\nAdditionally, the reasons for starting testosterone therapy cannot be determined from this retrospective analysis. Because of the uncertain reasons for starting therapy in some hypogonadal men and not in others, and because of the variability in the amount of total testosterone that the patients actually received, there may be confounding factors that could also explain the higher risk of adverse events in men treated with testosterone. It is unclear whether the minimal exposure to testosterone in this elderly population (as evidenced by the post-treatment levels and duration of treatment) was responsible for such a dramatic difference in mortality and morbidity.\n\nThe association between testosterone therapy and mortality has remained controversial with studies demonstrating conflicting results5,6. Until larger randomized studies demonstrate clear causation, physicians prescribing testosterone therapy to elderly men with co-morbidities should use it prudently with close follow-up.",
"appendix": "Author contributions\n\n\n\nRR chose the article for correspondence and evaluated the data in the manuscript. JD wrote a part of the commentary. JK wrote the abstract and introduction. LIL supervised the process and critically edited the manuscript. All authors discussed the implications and commented on the manuscript at all stages.\n\n\nCompeting interests\n\n\n\nRanjith Ramasamy, James M. Dupree and Jason R. Kovac do not have any competing interests to disclosure. Larry I. Lipshultz has reported being a clinical trials participant, consultant, and speaker for Auxlium and Endo.\n\n\nGrant information\n\n\n\n\nReferences\n\nVigen R, O'Donnell CI, Barón AE, et al.: Association of testosterone therapy with mortality, myocardial infarction, and stroke in men with low testosterone levels. JAMA: the journal of the American Medical Association. 2013; 310(17): 1829–1836. PubMed Abstract | Publisher Full Text\n\nShores MM, Matsumoto AM, Sloan KL, et al.: Low serum testosterone and mortality in male veterans. Arch Intern Med. 2006; 166(15): 1660–1665. PubMed Abstract | Publisher Full Text\n\nVlachopoulos C, Ioakeimidis N, Terentes-Printzios D, et al.: Plasma total testosterone and incident cardiovascular events in hypertensive patients. Am J Hypertens. 2013; 26(3): 373–381. PubMed Abstract | Publisher Full Text\n\nBhasin S, Cunningham GR, Hayes FJ, et al.: Testosterone therapy in men with androgen deficiency syndromes: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2010; 95(6): 2536–2559. PubMed Abstract | Publisher Full Text\n\nShores MM, Smith NL, Forsberg CW, et al.: Testosterone treatment and mortality in men with low testosterone levels. J Clin Endocrinol Metab. 2012; 97(6): 2050–2058. PubMed Abstract | Publisher Full Text\n\nBasaria S, Coviello AD, Travison TG, et al.: Adverse events associated with testosterone administration. N Engl J Med. 2010; 363(2): 109–122. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "3149",
"date": "22 Jan 2014",
"name": "Boback Berookhim",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDr. Ramasamy and colleagues present a well-researched brief summary on a topic which has received great attention among physicians treating low testosterone. The authors provide a nice summary of a newsworthy publication by Vigen et al. in JAMA in late 2013. The concerns presented with the methodology of this article, primarily regarding the efficacy of likely sub-therapeutic testosterone replacement therapy in an elderly veteran population, are relevant and worthy of further study. Given the conflicting studies within the literature, further research is required to clearly delineate the cardiovascular risks (or lack thereof) in elderly men receiving testosterone replacement therapy.",
"responses": []
},
{
"id": "3150",
"date": "23 Jan 2014",
"name": "Matthew S. Wosnitzer",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis correspondence from the Baylor group accurately summarizes many of the questions surrounding the Vigen et al. article which describes an increased cardiovascular risk profile with testosterone replacement in a highly select group of VA patients. The majority of prior studies have not detected significant increases in cardiovascular risks, except for the randomized TOM trial (Basaria et al. 2010 NEJM), which had many of its own issues with \"differences detected between the two trial groups possibly...due to chance alone.\"One key criticism of the Vigen et al. study, described in this correspondence, is that hypogonadism alone may portend adverse cardiovascular and cerebrovascular outcomes and that the T treatment group in this study had lower pre-treatment T values. This issue is most recently highlighted in the JCEM article by Yeap et al. from Australia in 2013 describing older Australian men with reduced mortality when testosterone was in mid-range normal levels.Other issues with the Vigen et al. study include the heterogeneous preparations of testosterone, and varied/unknown compliance with regimens. Only 60% of patients had follow-up testosterone labs after starting therapy which is particularly important since T response differs from regimen to regimen and patient to patient. Additionally for those who had testosterone checked, were they done appropriately in the morning or at other times? Additionally, known mean post-treatment testosterone levels were barely in eugonadal range, further highlighting possible compliance issues. Certainly, additional randomized controlled trials are required to discern whether the findings in this population are generalizable to any other population.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-11
|
https://f1000research.com/articles/2-218/v1
|
17 Oct 13
|
{
"type": "Research Article",
"title": "Excess CD40L does not rescue anti-DNA B cells from clonal anergy",
"authors": [
"Mohammad Aslam",
"Yusuke Kishi",
"Takeshi Tsubata",
"Mohammad Aslam",
"Yusuke Kishi"
],
"abstract": "CD40L, a member of the tumor necrosis factor (TNF) ligand family, is overexpressed in patients with systemic lupus erythematosus and in lupus mouse models. Previously, we demonstrated that B cells producing pathogenic anti-Sm/RNP antibodies are deleted in the splenic marginal zone (MZ), and that MZ deletion of these self-reactive B cells is reversed by excess CD40L, leading to autoantibody production. To address whether excess CD40L also perturbs clonal anergy, another self-tolerance mechanism of B cells whereby B cells are functionally inactivated and excluded from follicles in the peripheral lymphoid tissue, we crossed CD40L-transgenic mice with the anti-DNA H chain transgenic mouse line 3H9, in which Ig λ1+ anti-DNA B cells are anergized. However, the percentage and localization of Ig λ1+ B cells in CD40L/3H9 double transgenic mice were no different from those in 3H9 mice. This result indicates that excess CD40L does not perturb clonal anergy, including follicular exclusion. Thus, MZ deletion is distinct from clonal anergy, and is more liable to tolerance break.",
"keywords": [
"antibodies",
"B cells",
"T cells"
],
"content": "Introduction\n\nAntibodies to nuclear antigens such as DNA and the RNA-related Sm/RNP antigen are characteristically produced in patients with systemic lupus erythematosus (SLE), a prototype of systemic autoimmune diseases, and play a role in the development of this autoimmune disease1–3. How B cells reactive to nuclear antigens are regulated has been extensively studied using transgenic (Tg) mice expressing auto-antibodies against DNA and RNA components, especially the anti-DNA H chain-Tg mouse lines 3H9 and 56R4–9. Studies using these auto-antibody-Tg mice demonstrated that self-reactive B cells that produce autoantibodies to nuclear antigens are deleted by apoptosis (clonal deletion)10, are functionally inactivated (clonal anergy)11 or change antigen specificity by immunoglobulin (Ig) V gene replacement (receptor editing)8,12, in the bone marrow before they migrate to the peripheral lymphoid organs. These self-tolerance mechanisms appear to be involved in the prevention of autoantibody production in normal individuals.\n\nCD40 is a member of the tumor necrosis factor (TNF) receptor family expressed in immune cells including B cells and dendritic cells13. Upon interaction with its ligand, CD40L (CD154), expressed mainly by activated T cells, CD40 transmits survival and activation signals in B cells13,14. In both patients with SLE and in SLE mouse models, CD40L is overexpressed by T cells and ectopically expressed in B cells15–17, and this excess CD40L expression appears to play a role in development of SLE, as treatment with antagonistic anti-CD40L antibody markedly reduces the severity of the disease in both humans and mice18.\n\nUsing CD40L/56R double transgenic mice expressing both the anti-DNA H chain 56R and CD40L in B cells, we previously demonstrated that anti-Sm/RNP B cells are regulated by a novel tolerance mechanism in peripheral lymphoid tissue, i.e., deletion in splenic marginal zone (MZ), and that the MZ deletion is perturbed by excess CD40L19. In 56R mice, B cells that produce anti-Sm/RNP antibody appear in the splenic MZ, and are subsequently deleted there by apoptosis. When 56R mice are crossed with CD40L-Tg mice in which CD40 signaling is constitutively generated in B cells20, MZ deletion of anti-Sm/RNP B cells is perturbed, resulting in autoantibody production19. As anti-Sm/RNP antibodies are implicated in the pathogenesis of SLE1,2, MZ deletion appears to be important for preventing the development of SLE through deletion of pathogenic self-reactive B cells. Hence, a defect in MZ deletion by excess CD40L19 could play a role in the development of lupus by inducing the production of pathogenic anti-Sm/RNP antibody.\n\nSome self-reactive B cells including a part of anti-DNA B cells are silenced by clonal anergy in which B cells persist in the peripheral lymphoid organs but are unresponsive to antigen stimulation. Anergized self-reactive B cells are excluded from follicles or the MZ of the spleen. Instead, they are located in the red pulp and the T cell zone of the spleen, especially in the border between the T cell zone and the follicles, and undergo apoptosis21. Ig λ1 L chain + B cells in the anti-DNA H chain-Tg mouse line 3H9 are reactive to DNA and are anergized9,22,23. To address whether excess CD40L can reverse the anergy of self-reactive B cells, we crossed CD40L-Tg mice with 3H9 mice and examined the percentage and localization of Ig λ1+ B cells. Our results demonstrated that excess CD40L does not expand anergized anti-DNA B cells or reverse their follicular exclusion, indicating that excess CD40 does not reverse anergy of self-reactive B cells. As excess CD40L does perturb MZ deletion of anti-Sm/RNP B cells, clonal anergy appears to be distinct from MZ deletion, although both of them induce apoptosis of self-reactive B cells in peripheral lymphoid tissue.\n\n\nMaterials and methods\n\nThe conventional Tg mouse line expressing the H chain of the anti-DNA antibody 3H9 on the BALB/c background5 was a kind gift of Dr. M. Weigert (The University of Chicago). We previously generated CD40L-Tg mice on the C57BL/6 background20. CD40L-Tg mice were crossed with 3H9-Tg mice to generate wild type, 3H9-Tg and CD40L/3H9 double Tg mice on (BALB/c × C57BL/6)F1 background. Mice were genotyped by PCR reaction using specific pairs of primers for the CD40L and 3H9 transgenes, respectively14,19. All mice were housed and bred at our specific pathogen-free facility. Groups of 3 mice were kept in conventional shoebox-type polycarbonate cages, which were changed every 7 days. All animals were provided with food and water ad libitum and were maintained on a 12-hour light/dark cycle. All procedures followed the guidelines of Tokyo Medical and Dental University for animal research and were approved by Institutional Animal Care and Use Committee, Tokyo Medical and Dental University.\n\nSpleens were finely minced over a wire mesh, and spleen cells were collected and suspended in PBS containing 2% FCS (Cell Culture Bioscience, Japan) and 0.1% NaN3 (Nacalai Tesque, Inc., Japan). Cells (1 × 107/ml) were then stained with the following antibodies and reagents. The dilutions at which the antibodies were used are indicated in parentheses. Alexa Fluor 647-conjugated rat anti-mouse B220 (RA3-6B2, BioLegend, USA) (1:100), Pacific Blue-conjugated anti-mouse Ig λ1 (LS-136, a kind gift of Dr. G. Kelsoe at Duke University) (1:100)24 and FITC-conjugated goat anti-mouse Ig λ chain antibody (Cat No. 1060-02, Southern Biotech, USA) (1:1000). Lymphoid cells were gated by FSC/SSC dot plots, and analyzed on a CyAn ADP flow cytometer (Beckman Coulter, USA).\n\nTissues were embedded in Tissue-Tek O.C.T. compound (Sakura Finetek U.S.A., Inc., U.S.A.), snap-frozen in liquid nitrogen, and stored at −80°C. Cryostat (Cryostat 1720, Leica Microsystems GmbH, Germany) sections of 5 μm in thickness were mounted onto micro slide glass (Matsunami Glass Ind. Ltd., Japan), air dried, and fixed in acetone at room temperature for 20 min. The sections were incubated with blocking buffer (PBS containing 2.0% (wt/vol) FCS (Cell Culture Bioscience, Japan), 0.05% NaN3 (Nacalai Tesque, Inc., Japan)) for 30 min and were stained at room temperature for 60 min with biotin-conjugated rat anti-mouse MOMA-1 (Cat No. T-2021, BMA Biomedicals, Switzerland), Alexa Fluor 647-conjugated streptavidin (Cat No. S21374, Invitrogen, USA), Pacific Blue-conjugated rat anti-mouse B220 (RA3-6B2, BioLegend, USA) and FITC-conjugated goat anti-mouse λ chain antibody (Cat No. 1060-02, Southern Biotech, USA). All the antibodies and reagents were used at a 1:100 dilution.\n\n\nResults\n\nTo address whether excess CD40L perturbs clonal anergy of anti-DNA B cells, we crossed CD40L Tg mice with 3H9 mice, because B cells expressing Ig λ1 L chain and 3H9 H chain are reactive to DNA and show characteristics for anergy including follicular exclusion, shortened life span and failure of antibody production23. When spleen B cells of wild type, 3H9 Tg and CD40L overexpressing CD40L/3H9 double Tg, mouse lines were analyzed by flow cytometry, the percentage of λ1+ B cells in total B cells expressing a B cell marker B220 was markedly reduced in 3H9 mice compared to wild type mice (Figure 1A) as λ1+ B cells in 3H9 but not wild type mice are self-reactive. The percentage of λ1+ B cells in CD40L/3H9 double Tg mice was equivalent to that in 3H9 mice, indicating that excess CD40L does not expand anergized anti-DNA B cells.\n\nSpleen cells from 11- to 20-wk-old wild type (WT), 3H9, and CD40L/3H9 mice were stained for B220 (A and B), Ig λ1 chain (A and B) and Ig λ chain (B), and B220+ cells were analyzed by flow cytometry. Percentages of λ1+ cells in total B220+ cells (A), percentages of λ+ cells in total B220+ cells (B, upper panel) and percentages of λ1+ cells in total λ+ cells (B, lower panel) are indicated. Representative data from two independent experiments.\n\nWhen we used anti-λ antibody that reacts to multiple Ig λ chain subtypes such as λ1 and λ2, the percentage of total λ+ cells were only slightly higher than that of λ1+ cells in wild type, 3H9 and CD40L/3H9 mice (Figure 1A and B upper panels), suggesting that most of the λ+ cells express the λ1 subtype in all these mice and are thus reactive to DNA. This is confirmed by determining percentage of λ1+ cells in total λ+ cells. In both 3H9 and CD40L/3H9 mice, the percentage of λ1+ cells in total λ+ cells is slightly reduced compared to that in wild type mice, but λ1+ cells constitute the majority of the λ+ cells in these mice as well as wild type mice (Figure 1B lower panels).\n\nTo address whether excess CD40L reverses follicular exclusion of anergized anti-DNA B cells, we examined spleen sections of 3H9 and 3H9/CD40L Tg mice using an anti-λ antibody but not anti-λ1 antibody, as the anti-λ1 antibody did not yield any staining when used for immunohistochemistry. Nonetheless, most of the B cells stained with anti-λ antibody in these mice express λ1 (Figure 1B, lower panels) and are thus reactive to DNA. In wild type spleen, λ+ cells were located mostly in the follicle where B220+ B cells accumulate (Figure 2A). In contrast, λ+ cells were found mostly in the border between T cell zone and follicle, T cell zone and red pulp in both 3H9 and CD40L/3H9 spleens (Figure 2B and C), indicating that anti-DNA B cells are excluded from follicles in CD40L/3H9 mice as well as 3H9 mice. Thus, excess CD40L does not reverse follicular exclusion of anergized anti-DNA B cells.\n\nSections of spleen from 11- to 20-wk-old wild type (WT) (A), 3H9 (B), and CD40L/3H9 (C) mice were stained for MOMA-1, a marker for a subset of splenic macrophages (red), B220, a B cell marker (blue), Ig λ L chain (green), and analyzed by confocal microscopy. Ig λ+ B cells in 3H9 mice are DNA-reactive. B cells positive for B220 are located in the follicles, and MOMA-1+ macrophages are located at the outer part of the follicles. T cell zone and red pulp are located inside and outside of the follicles, respectively. Representative data from three independent experiments. Original magnifications, 10X. Scale bar (yellow line): 100 μm.\n\n\nDiscussion\n\nIn this study, we crossed CD40L-Tg mice with anti-DNA H chain-Tg 3H9 mice, and demonstrated that the percentage and location of λ1+ anergic anti-DNA B cells are not altered in CD40L/3H9 double Tg mice compared to those in 3H9 mice, suggesting that anergy of λ1+ anti-DNA B cells is not reversed in CD40L/3H9 mice. This result is consistent with our previous finding that CD40L/3H9 mice do not produce anti-DNA antibodies in sera, whereas another anti-DNA H chain Tg mouse line 56R in which self-reactive B cells are deleted at MZ did produce autoantibodies in sera when crossed with the same CD40L Tg mice19. Thus, excess CD40L does not perturb anergy of anti-DNA B cells.\n\nAlthough excess CD40L fails to perturb clonal anergy of anti-DNA B cells in 3H9 mice, anergic B cells are not insensitive to CD40L. A previous study by Lesley et al. demonstrated that anergic self-reactive B cells expand by receiving stimulation from a low level expression of CD40L in unstimulated T cells25. Thus, anergic B cells sensitively respond to a low level CD40L expression, but do not further respond to excess CD40L expression. The mechanisms behind this phenomenon however, are not yet clear.\n\nUsing the same CD40L-Tg mice that we used in the present study, we previously demonstrated that excess CD40L inhibits apoptosis of anti-Sm/RNP B cells in MZ, and that excess CD40L induces autoantibody production19. This suggests that MZ deletion is distinct from clonal anergy, although both anergic B cells and B cells that are deleted in MZ appear in peripheral lymphoid tissue and are eliminated by apoptosis.\n\nLike CD40L, B cell activating factor (BAFF), another member of the TNF ligand family, induces B cell survival and is overexpressed in patients with SLE26–28 and its mouse models29,30. Anergic B cells show increased dependency on BAFF for survival, and this increased dependency appears to be involved in rapid elimination of anergic B cells by competition with non-self-reactive B cells31,32. In the presence of non-self-reactive B cells, anergic B cells may not be able to interact with a sufficient level of BAFF required for their survival, due to competition for a limited amount of BAFF32. Nonetheless, excess BAFF fails to fully reverse anergy of self-reactive B cells. Lesley et al.31 demonstrated that excess BAFF expands anergic B cells but fails to reverse follicular exclusion. Thien et al.32 demonstrated that excess BAFF expands and reverses follicular exclusion in only anergic B cells with intermediate affinity but not those with high affinity. In contrast, cognate T cell help perturbs follicular exclusion and induces autoantibody production in anergic self-reactive B cells33. Thus, reversing clonal anergy requires strong T cell help, and excess BAFF or CD40L alone may be insufficient. In contrast, we previously demonstrated by crossing the CD40L-Tg mice with another anti-DNA H chain Tg mice 56R that excess CD40L perturbs MZ deletion of self-reactive B cells and induces autoantibody production, suggesting that MZ deletion is more sensitive to a tolerance break than clonal anergy19. As excess CD40L is found in patients with SLE and various SLE model mice, MZ deletion is likely to be defective in lupus, and its defect may be involved in development of lupus.",
"appendix": "Author contributions\n\n\n\nTT conceived the study. MA, YK and TT designed the experiments, and carried out the research. MA and TT wrote the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by MEXT KAKENHI Grants Number 23390063 (TT) and 24790464 (YK).\n\n\nAcknowledgements\n\nWe thank Dr. Martin Weigert for the 3H9 mice, and Dr. Garnett Kelsoe for the anti-mouse Ig λ1 antibody producing hybridoma cells (LS-136).\n\n\nReferences\n\nBerland R, Fernandez L, Kari E, et al.: Toll-like receptor 7-dependent loss of B cell tolerance in pathogenic autoantibody knockin mice. Immunity. 2006; 25(3): 429–440. PubMed Abstract | Publisher Full Text\n\nChristensen SR, Shupe J, Nickerson K, et al.: Toll-like receptor 7 and TLR9 dictate autoantibody specificity and have opposing inflammatory and regulatory roles in a murine model of lupus. Immunity. 2006; 25(3): 417–428. PubMed Abstract | Publisher Full Text\n\nTan EM: Antinuclear antibodies: diagnostic markers for autoimmune diseases and probes for cell biology. Adv Immunol. 1989; 44: 93–151. PubMed Abstract | Publisher Full Text\n\nChen C, Radic MZ, Erikson J, et al.: Deletion and editing of B cells that express antibodies to DNA. J Immunol. 1994; 152(4): 1970–1982. PubMed Abstract\n\nErikson J, Radic MZ, Camper SA, et al.: Expression of anti-DNA immunoglobulin transgenes in non-autoimmune mice. Nature. 1991; 349(6307): 331–334. PubMed Abstract | Publisher Full Text\n\nChen C, Nagy Z, Radic MZ, et al.: The site and stage of anti-DNA B-cell deletion. Nature. 1995; 373(6511): 252–255. PubMed Abstract | Publisher Full Text\n\nFukuyama H, Nimmerjahn F, Ravetch JV: The inhibitory Fcgamma receptor modulates autoimmunity by limiting the accumulation of immunoglobulin G+ anti-DNA plasma cells. Nat Immunol. 2005; 6(1): 99–106. PubMed Abstract | Publisher Full Text\n\nGay D, Saunders T, Camper S, et al.: Receptor editing: an approach by autoreactive B cells to escape tolerance. J Exp Med. 1993; 177(4): 999–1008. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNoorchashm H, Bui A, Li HL, et al.: Characterization of anergic anti-DNA B cells: B cell anergy is a T cell-independent and potentially reversible process. Int Immunol. 1999; 11(5): 765–776. PubMed Abstract | Publisher Full Text\n\nNemazee DA, Burki K: Clonal deletion of B lymphocytes in a transgenic mouse bearing anti-MHC class I antibody genes. Nature. 1989; 337(6207): 562–566. PubMed Abstract | Publisher Full Text\n\nGoodnow CC, Crosbie J, Adelstein S, et al.: Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice. Nature. 1988; 334(6184): 676–682. PubMed Abstract | Publisher Full Text\n\nRadic MZ, Erikson J, Litwin S, et al.: B lymphocytes may escape tolerance by revising their antigen receptors. J Exp Med. 1993; 177(4): 1165–1173. PubMed Abstract | Publisher Full Text | Free Full Text\n\nQuezada SA, Jarvinen LZ, Lind EF, et al.: CD40/CD154 interactions at the interface of tolerance and immunity. Annu Rev Immunol. 2004; 22: 307–328. PubMed Abstract | Publisher Full Text\n\nTsubata T, Wu J, Honjo T: B-cell apoptosis induced by antigen receptor crosslinking is blocked by a T-cell signal through CD40. Nature. 1993; 364(6438): 645–648. PubMed Abstract | Publisher Full Text\n\nBlossom S, Chu EB, Weigle WO, et al.: CD40 ligand expressed on B cells in the BXSB mouse model of systemic lupus erythematosus. J Immunol. 1997; 159(9): 4580–4586. PubMed Abstract\n\nDesai-Mehta A, Lu L, Ramsey-Goldman R, et al.: Hyperexpression of CD40 ligand by B and T cells in human lupus and its role in pathogenic autoantibody production. J Clin Invest. 1996; 97(9): 2063–2073. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKoshy M, Berger D, Crow MK: Increased expression of CD40 ligand on systemic lupus erythematosus lymphocytes. J Clin Invest. 1996; 98(3): 826–837. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPeters AL, Stunz LL, Bishop GA: CD40 and autoimmunity: the dark side of a great activator. Semin Immunol. 2009; 21(5): 293–300. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKishi Y, Higuchi T, Phoon S, et al.: Apoptotic marginal zone deletion of anti-Sm/ribonucleoprotein B cells. Proc Natl Acad Sci U S A. 2012; 109(20): 7811–7816. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHiguchi T, Aiba Y, Nomura T, et al.: Cutting Edge: Ectopic expression of CD40 ligand on B cells induces lupus-like autoimmune disease. J Immunol. 2002; 168(1): 9–12. PubMed Abstract\n\nCyster JG, Hartley SB, Goodnow CC: Competition for follicular niches excludes self-reactive cells from the recirculating B-cell repertoire. Nature. 1994; 371(6496): 389–395. PubMed Abstract | Publisher Full Text\n\nMandik-Nayak L, Bui A, Noorchashm H, et al.: Regulation of anti-double-stranded DNA B cells in nonautoimmune mice: localization to the T-B interface of the splenic follicle. J Exp Med. 1997; 186(8): 1257–1267. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRadic MZ, Mascelli MA, Erikson J, et al.: Ig H and L chain contributions to autoimmune specificities. J Immunol. 1991; 146(1): 176–182. PubMed Abstract\n\nReth M, Imanishi-Kari T, Rajewsky K: Analysis of the repertoire of anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies in C 57 BL/6 mice by cell fusion. II. Characterization of idiotopes by monoclonal anti-idiotope antibodies. Eur J Immunol. 1979; 9(12): 1004–1013. PubMed Abstract | Publisher Full Text\n\nLesley R, Kelly LM, Xu Y, et al.: Naive CD4 T cells constitutively express CD40L and augment autoreactive B cell survival. Proc Natl Acad Sci U S A. 2006; 103(28): 10717–10722. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGroom J, Kalled SL, Cutler AH, et al.: Association of BAFF/BLyS overexpression and altered B cell differentiation with Sjögren's syndrome. J Clin Invest. 2002; 109(1): 59–68. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMariette X, Roux S, Zhang J, et al.: The level of BLyS (BAFF) correlates with the titre of autoantibodies in human Sjögren's syndrome. Ann Rheum Dis. 2003; 62(2): 168–171. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMackay IR, Groom J, Mackay CR: Levels of BAFF in serum in primary biliary cirrhosis and autoimmune diabetes. Autoimmunity. 2002; 35(8): 551–553. PubMed Abstract | Publisher Full Text\n\nMackay F, Woodcock SA, Lawton P, et al.: Mice transgenic for BAFF develop lymphocytic disorders along with autoimmune manifestations. J Exp Med. 1999; 190(11): 1697–1710. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKhare SD, Sarosi I, Xia XZ, et al.: Severe B cell hyperplasia and autoimmune disease in TALL-1 transgenic mice. Proc Natl Acad Sci U S A. 2000; 97(7): 3370–3375. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLesley R, Xu Y, Kalled SL, et al.: Reduced competitiveness of autoantigen-engaged B cells due to increased dependence on BAFF. Immunity. 2004; 20(4): 441–453. PubMed Abstract | Publisher Full Text\n\nThien M, Phan TG, Gardam S, et al.: Excess BAFF rescues self-reactive B cells from peripheral deletion and allows them to enter forbidden follicular and marginal zone niches. Immunity. 2004; 20(6): 785–798. PubMed Abstract | Publisher Full Text\n\nSeo SJ, Fields ML, Buckler JL, et al.: The impact of T helper and T regulatory cells on the regulation of anti-double-stranded DNA B cells. Immunity. 2002; 16(4): 535–546. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "2273",
"date": "31 Oct 2013",
"name": "Marko Radic",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe brief report by Aslam et al. develops a theme that was the initial focus of the Tsubata lab in the early 90’s. The authors follow up on the observation that CD40L is overexpressed on T cells in SLE patients and, ectopically, on B cells. As CD40L provides an important second signal to B cells, the question was whether it can overcome negative regulation of certain B cell states. The current work continues upon Kishi et al. (PNAS 2012) who used anti-DNA transgenic mice to examine the effect of CD40L overexpression on the regulation of autoreactive B cells. Kishi et al. established that an avid anti-DNA transgene, 56R, led to B cell sequestration in the splenic marginal zone (MZ) and death by apoptosis. CD40L overexpression was able to inhibit apoptosis in the MZ and allow expression of certain H/L pairs, most notably those with anti-Sm specificity. Here, the authors use a closely related anti-DNA H chain, 3H9, which is of lower affinity for DNA compared to 56R.Dozens of labs have used the 3H9 and the 56R H-chain transgenic mouse lines which have become emblematic for the type of H chain that is immunodominant and – with different L chains – has the potential to encode a range of self-specificities, including anti-DNA, anti-phospholipid, anti-nucleosome and also anti-apoptotic cell antibodies. Earlier work by the Erikson lab had established that 3H9VH in combination with λ1 gives a B cell receptor that induces anergy and leads to a characteristic extrafollicular localization. Aslam et al. observe that the 3H9/λ1 combination produces the same number of splenic B cells whether or not it is crossed to CD40L and that the splenic location of the single and double transgenic B cells is identical.The significance of this work is that it draws an important distinction between tolerance mechanisms that result in anergy vs. MZ deletion. Only the latter is sensitive to rescue by CD40L signals. This serves to clarify important characteristics of splenic B cell regulation. It will be informative to follow these studies with additional approaches to test whether B cell signalling pathways are perturbed by the presence of CD40L, whether increased CD40L expression on B or T cells is involved in suppressing apoptosis in the MZ, and whether CD40L overexpression could affect other types of 56R “incomplete editing” such as in the case of intracellular Golgi retention that is observed with 56R and Vk38c and λX L chains.The title is appropriate for the content of the article and the abstract represents a suitable summary of the work. The design, methods and analysis of the results from the study have been explained and they are appropriate for the topic being studied. The conclusions are sensible, balanced and justified on the basis of the results of the study and enough information has been provided to be able to replicate the experiment.",
"responses": []
},
{
"id": "2424",
"date": "25 Nov 2013",
"name": "Jennifer Marshall",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis report from Aslam et al., provides data on the lack of an effect of over-expression of CD40L on the number and location of anergic 3H9 B cells that respond to DNA. This follows on from their 2012 PNAS study which used similar methods investigating the effects of CD40L on anergic B cells from 56R mice.The paper is well constructed and all sections are clear. However, I believe that the data could be presented in a manner more helpful to the reader and provide a firmer foundation for their conclusions. I think that these changes (especially those concerning figure 2) are necessary to substantiate their conclusions and abstract.Both figure legends should contain information on how many mice were used in each group. This information is essential for all publications involving animal work and is necessary to allow proper judgment of data. Whilst the methods do state that the mice are caged in groups of 3 this is within the details of animal husbandry and, as such, it is not clear if this related to the experimental group size.In addition to the representative flow cytometry plots provided Figure 1A and B would benefit from a graph that shows the percentage of splenic Ig λ1 L chain B cells of each mouse in the experiment. This would provide an opportunity to be made aware of the variation between the mice within each group as well as compare between groups. If there are sufficient mouse numbers a statistical analysis comparing groups would also be useful.I am puzzled by the choice of images for Figure 2. Firstly, due to the shape of the follicle it is difficult to compare the location of Ig λ L chain cells in 2C with those in 2A and B. As the follicular exclusion of λ L chain B cells within the CD40L/3H9 mouse is a key conclusion of this paper it would be helpful for the reader if 2C was replaced with a representative white pulp area that provides a clearer demarcation between follicle and the T zone. Secondly, I would suggest that Figure 2B makes it appear that the 3H9 mice have substantially fewer Ig λ L chain B cells than the CD40L/3H9 mice shown in Figure 2C. If it is representative of all 3H9 spleens I do not think that this corroborates the conclusions drawn from Figure 1. As they stand the images make my suggestions regarding an extra graph for Figure 1, and the mouse numbers used, more compelling.Without these changes I cannot truly say that the title and conclusions are appropriate. However, these are quick changes and I trust that it is just a bad image choice.",
"responses": []
},
{
"id": "2590",
"date": "28 Nov 2013",
"name": "David Nemazee",
"expertise": [],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper by Aslam, Kishi and Tsubata addresses an interesting question about the effects of bystander stimulation through CD40L on immunological self tolerance of B cells. Previous studies from this group showed that transgene-enforced CD40L expression on B cells promoted a lupus-like autoimmunity in mice with a polyclonal B cell population. A later study showed that autoreactive B cells in a transgenic mouse with a restricted antibody repertoire biased to DNA reactivity (mice carrying 3H9-56R DNA-reactive H-chain) were promoted to central tolerance in the presence of excess CD40L, whereas 3H9-H mice lacking that mutation, while still expressing a repertoire biased to autoreactivity, failed to show a clear break in tolerance.The present study is a rather minor addition to this body of work, providing additional data on the fate of B cells expressing a λ1 light chain partner, which have high anti-DNA affinity when expressed with 3H9H. The data show that 3H9H/λ1 B cells are not rescued in their development or altered in their anatomical localization (follicular exclusion) when developing in an environment with excess B cell-expressed CD40L. The data are consistent with the earlier, more extensive study, but do provide some new information. However, the data shown have no indication of reproducibility or provide more than a single mouse per group. There is no molecular characterization of the cells to verify that the receptor expression expected is indeed confirmed. Overall, this is an incremental contribution to the literature.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-218
|
https://f1000research.com/articles/3-10/v1
|
15 Jan 14
|
{
"type": "Case Report",
"title": "Observation of a unique case of metastatic basal cell carcinoma found by radiographic evaluation in a patient with oculocutaneous albinism",
"authors": [
"Mickaila Johnston",
"Whitney Winham",
"Nicole Massoll",
"Jerad M. Gardner",
"Whitney Winham",
"Nicole Massoll",
"Jerad M. Gardner"
],
"abstract": "Background: Basal cell carcinoma is one of the more common cancers worldwide; 2.8 million are diagnosed annually in the USA. However, the rate at which it metastasizes is considered very low, between 0.0028 and 0.5%. For those rare cases in which metastases occur, approximately one third metastasize to the lung. Case: Presented is a 62-year-old Caucasian male with oculocutaneous albinism and a history of basal cell carcinomas occurring in multiple anatomic sites, most recently at the bilateral forearm and back. Surveillance PET/CT imaging led to the discovery of no less than 30 lung nodules which were consistent with basal cell carcinoma on biopsy. Histological features were remarkably similar in both the primary tumor and in the metastases.Conclusion: An unusual case of a non-head and neck primary basal cell carcinoma metastatic to the lung was discovered on surveillance PET/CT imaging, in a patient with oculocutaneous albinism.",
"keywords": [
"PET",
"CT",
"Basal cell Carcinoma (BCC)",
"Metastatic",
"albinism",
"vismodegib"
],
"content": "Background\n\nBasal cell carcinoma (BCC) is the most common human malignancy worldwide, yet it is typically indolent and rarely possesses metastatic potential1. Reported rates of metastases range from 0.0028 to 0.5%1. Despite the high incidence of BCC, there have been only 257 cases of metastatic BCC (MBCC) reported in the English medical literature between 1894 and 1991, 82 of which demonstrated metastases to the lung2–5.\n\nIn this article, we review the clinical, radiological, and histopathological presentation of a patient with a history of multiple non-head and neck BCC with subsequent numerous metastases to the bilateral lungs. We also briefly review the literature, and discuss the epidemiology, risk factors, TNM staging, therapeutic modalities, and prognosis for patients with MBCC.\n\n\nCase\n\nA 62-year-old Caucasian male with oculocutaneous albinism (Fitzpatrick type I skin) had been followed extensively by both the dermatology and the general surgery services at the University of Arkansas for Medical Sciences. His past medical history was significant for multiple BCCs, the most recent of which (2012) involved the back and flank, requiring adjuvant radiation therapy and split thickness skin grafting. No other significant medical history was noted aside from shortness of breath.\n\nAdditionally, four months prior to these excisions, the patient underwent excisions of morpheaform (infiltrative) BCCs of the right arm and back, as well as nodular BCCs of the left cheek and temple. In 2009, he had an initial large wide excision for BCC on his back and flank which demonstrated positive deep margins. The most recent re-excision in 2012 demonstrated all negative margins. Moreover, in 2012 he had a singular squamous cell carcinoma of the right upper extremity that was less than 1.0 mm to the nearest margin, and measured 4.0 mm in maximum depth of invasion.\n\n\nRoentgenographic findings\n\nA routine chest X-ray in 2009 was effectively within normal limits, displaying no mass or tumor. A diagnostic CT scan was ordered in 2012 for surveillance due to the extensive nature of his BCCs. It demonstrated numerous solid and sub-solid nodules measuring up to 2.0 cm, in multiple stages of cavitary change, in both lungs. It was considered possible that the nodules were metastases from the squamous cell carcinoma of the right arm but further evaluation was recommended to confirm this.\n\nFollow-up F-18 fluoro-deoxy-glucose PET/CT scan (15.14 mCi, 69-minutes of uptake time, and a fasting blood glucose of 104 mg/dL) was performed from the base of the orbits through the mid-thigh with 3-axis reconstructions, and attenuation correction with a non-diagnostic CT scan. It demonstrated no less than thirty non-specific foci with significant hypermetabolic activity (>3-times background), most of which were associated with nodules in multiple stages of cavitary change (Figure 1). In light of the patient's history of multiple malignancies, in addition to an inflammatory/infectious etiology, the possibility of metastasis, although less likely, was also considered. Clinical and pathologic correlations were recommended, as well as a repeat PET/CT of the vertex through the feet, for definitive evaluation of the dermis.\n\na) Maximum Intensity Projection, b) axial CT, and c) fused PET/CT scans demonstrates multiple hypermetabolic bilateral lung cavitary nodules (foci) in multiple stages of development. This is a non-specific finding, as inflammation and malignancy may present in a similar manner.\n\n\nHistopathologic findings\n\nCT-guided fine needle aspiration and subsequent core biopsy from one of the lung nodules from the right upper lobe were interpreted as positive for malignant cells, basal cell carcinoma. The right upper lobe core biopsy showed small cohesive nests and cords of basaloid cells with scant cytoplasm. Artifactual clefts containing mucin were present around the periphery of many of the nests. The cells demonstrated hyperchromatic chromatin without nucleoli and displayed some nuclear overlap and molding. The tumor cells were negative for synaptophysin, chromogranin, and cytokeratin 20 by immunohistochemistry, findings which argue against the possibility of primary or metastatic neuroendocrine carcinoma (Figure 2).\n\nFine needle aspiration (Diff-Quick preparation, original magnification ×400) from a lung nodule demonstrates a) Cells with a high nuclear to cytoplasmic ratio and mildly enlarged nuclei compared to surrounding inflammatory cells in the background, b) Enlarged, fairly uniform cells forming sheets.\n\nA right flank wide local re-excision from 2012, several months prior to the discovery of lung metastases, demonstrated infiltrative cords and strands of basaloid cells characteristic of the infiltrative (morpheaform) type of basal cell carcinoma. Multiple tumor nodules were present in this specimen and the tumor invaded into the subcutis. Margins were negative. This right flank basal cell carcinoma specimen was re-examined and immunohistochemical stains were performed following discovery of the lung metastases; the tumor cells were negative for synaptophysin, chromogranin, and cytokeratin 20 by immunohistochemistry, findings which argue against the possibility of a cutaneous neuroendocrine carcinoma (Merkel cell carcinoma), (Figure 3).\n\nBiopsies and excisions from the scalp, left temple, right arm, and back performed several years previously were also reviewed and all were found to show characteristic histologic features of basal cell carcinoma.\n\nThe histologic features in the lung biopsy strongly resembled the histologic features of the multiple infiltrative basal cell carcinomas that this patient has had previously. The similarity between the tumors, the history of multiple aggressive and deeply invasive BCCs, and the exclusion by immunohistochemistry of the histologic mimics are all features that suggest that the pulmonary nodules represent a very rare example of metastatic basal cell carcinoma.\n\n\nTreatment and follow-up data\n\nThe patient was started on vismodegib, the cyclopamine-competitive antagonist of the smoothened receptor, at a dosage of 150 mg by mouth each day. After approximately two months of therapy, he began to have an improvement in his ability to breathe, and he was able to self-taper off of his home supplemental oxygen requirement. A follow up CT scan at this time showed disease improvement, and the vismodegib was discontinued. Most recently, a follow up CT, seven months after initial diagnosis of pulmonary metastases, showed interval worsening of the metastatic burden in the lungs. He is scheduled to restart vismodegib and is now being followed jointly by the palliative care and medical oncology services.\n\n\nDiscussion\n\nIn 1894, Beadles described a singular case of “rodent ulcer” deposition within the lymphatic gland of a deceased 46-year-old male, who had a previous history of “rodent ulcer” of the cheek. At autopsy, caseating foci were found within his lungs3. Although the historical medical terminology precludes absolute certainty, this is thought to represent the first reported case of metastatic basal cell carcinoma (MBCC).\n\nLattes and Kessler used three criteria to define MBCC in their 1951 report of two cases. First, they required that neither the primary nor the metastases could be squamous in type. Second, tumors could not be considered new primaries or a result of direct extension. Third, the tumors could not be mucoid or salivary in origin5.\n\nIn total, there have been only 257 cases of MBCC reported in the literature between 1894 and 1991 (based on a PubMed search using the term “metastatic+basal+cell+carcinoma” and adding the number of cases presented in each article)”.\n\nMetastases are thought to arise from both lymphatic and hematogenous pathways1. The most frequent reported sites of metastases were regional lymph nodes (60–65%), lung (32.3–40%), bone (19–25.8%), and skin (10–19%)2,4.\n\nIn light of the rarity of MBCC, it is unclear which histological or clinical features might be correlated with the risk of metastasis. With regards to histological features, there is a correlation with increased risk of local recurrence in specific subtypes like morpheaform or infiltrating types, but no correlation has been found with an increased risk of metastases among these more locally aggressive subtypes6. Of the 257 cases reported between 1894 and 1991, only five cases of MBCC were in black patients -the remainder were all in patients with a light complexion4. Disorders such as Basal Cell Nevus Syndrome or Xeroderma Pigmentosum have been shown to predispose patients to BCC. However, none have been shown to have a higher rate of metastases7,8. Patients with specific genodermatoses such as oculocutaneous albinism, in which there is a disorder of the melanin biosynthesis, have been shown to have a much less specific association with BCC. The significance of this association is unknown at this time8. However, a history of radiation therapy and history of local tumor recurrence have both been implicated in higher rates of MBCC8. In 170 of the reported cases of MBCC, the most frequent primary sites were head (67.6%) and trunk (16.5%), anatomic locations which are not significantly different than the most frequent sites of typical non-metastatic BCC1. In a review of 41 publications by Snow et al., BCCs larger than 4.0 cm had a 1.9% chance of metastases5. However, primary BCCs as small as 1.1 cm have been reported to metastasize9.\n\nSurvival time after the development of distant metastases in MBCC has been reported at 8–10 months2,4. A median interval between discovery of the primary focus of BCC and the discovery of metastasis has been reported as 9 years2, much longer than the interval seen in many other types of carcinoma.\n\nFor surveillance of patients with only local BCC, close follow up with a thorough skin examination, loco-regional lymph node evaluation, periodic Roentgenographic evaluation, liver function tests and alkaline phosphatase tests have been suggested to evaluate for occurrence of distant metastases10,1. However, given the extreme rarity of metastasis from BCC, these suggestions may not be feasible or reasonable for most patients. Further studies to elucidate risk factors for metastasis in BCC would be useful in determining which patients should receive a higher level of follow up screening.\n\nAs with many rare diseases, there is no established standard of care for management of metastatic foci in MBCC. A variety of therapies have been reported in the literature including local excision, radiation therapy, and chemotherapy. Given the aforementioned short survival times, large BCCs (>5 cm) and multiple metastatic sites pose a difficult dilemma for treatment, surgery may result in functional and cosmetic impairment, and radiation therapy is poor at providing local control12.\n\nFor metastatic disease, debulking prior to local surgery, and for a failure of local treatment, cis-platinum containing regimens had been a commonly accepted therapeutic approach12. However, preliminary results from NCT00833417 [a study evaluating the efficacy and safety of vismodegib (GDC-0449, Hedgehog pathway inhibitor) in patients with advanced basal cell carcinoma] prompted the USFDA to approve vismodegib as a treatment for “adults with BCC, that has spread to other parts of the body, or that has come back after surgery, or that their healthcare provider decides cannot be treated with surgery or radiation”, on January 30, 2012.\n\nIn the present patient, with history of oculocutaneous albinism, there were in excess of 30 metastatic foci within the lungs, whose histologic features strongly resembled those of the patient’s multiple other infiltrative BCCs. The primary tumor as well as the metastases were not squamous in type. Also, the squamous cell carcinoma of the arm was well differentiated and did not demonstrate basaloid features. Because primary basal cell carcinomas do not arise in the lungs and there was no evidence of direct extension from the back and flank into the lung tissue, the lung lesions are considered to be true metastases, as evidenced by the multiple lung nodules. Lastly, none of the primary or metastatic lesions of BCC demonstrated any mucoid or salivary features. Given the immunohistochemical exclusion of histologic mimics and history of multiple deeply invasive and aggressive BCCs it is considered most likely that the lung based cavitary foci were MBCC. Neuroendocrine primary tumors of the lung were systematically ruled out by immunohistochemical stains and the previous skin resections were ruled out for Merkel cell involvement.\n\nBecause of the tumor burden involving the lungs, surgery was not considered an option. He was treated with a two month course of vismodegib, to which he initially responded well. Surveillance CT scans and clinical examinations are currently being used to determine the need for resumption of vismodegib. Secondary to a lack of prospective studies or large sample sizes of previous studies, it remains unclear if the oculocutaneous albinism of the present patient was simply an association or a predisposing factor.\n\nIn conclusion, an unusual case of MBCC is presented that is presumed to arise from a non-facial primary BCC, with metastasis to the lung, detected by radiographic interrogation. It is important for providers to be aware that BCC may rarely metastasize, and that the risk for metastasis may be higher in patients with a primary BCC that is >4 cm or that has been previously irradiated. Imaging specialists should be sure to keep MBCC in the differential diagnosis when faced with a lung, lymph node, or cutaneous focus that is hypermetabolic and there is a history of BCC.\n\n\nConsent\n\nNo consent was obtained from the patient. The case report is fully anonymized and HIPAA-compliant, contains no patient identifiers in either the text or the figures, and was performed as an IRB exempt study.",
"appendix": "Author contributions\n\n\n\nMickaila Johnston: Manuscript preparation. Whitney Winham: Manuscript preparation. Nicole Massoll: Manuscript review, data generation. Jerad M. Gardner: Manuscript preparation, project supervision, data generation. All authors critically revised the manuscript and agreed to its publication.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis project was not funded by any commercial vendor or by any grant. Costs were covered by the Department of Pathology at the University of Arkansas for Medical Sciences.\n\n\nAcknowledgements\n\nThe views expressed in this presentation are those of the author and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, or the United States government. “I am a military service member (or employee of the U.S. Government). This work was prepared as part of my official duties. Title 17, USC, §105 provides that ‘Copyright protection under this title is not available for any work of the U.S. Government’. Title 17, USC, §101 defines a U.S. Government work as a work prepared by a military service MBR/employee of the U.S. Government as part of that person's official duties”.\n\n\nReferences\n\nSoleymani AD, Scheinfeld N, Vasil K, et al.: Metastatic basal cell carcinoma presention with unilateral upper extremity edema and lymphatic spread. J Am Acad Dermatol. 2008; 59(2 Suppl 1): S1–S3. PubMed Abstract | Publisher Full Text\n\nvon Domarus H, Stevens PJ: Metastatic basal cell carcinoma. Report of five cases and review of 170 cases in the literature. J Am Acad Dermatol. 1984; 10(6): 1043–1060. PubMed Abstract | Publisher Full Text\n\nBeadles CF: Rodent ulcer. Trans Pathol Soc Lond. 1894; 45: 176–181.\n\nLo JS, Snow SN, Reizner GT, et al.: Metastatic basal cell carcinoma: report of twelve cases with a review of the literature. J Am Acad Dermatol. 1991; 24(5 Pt 1): 715–719. PubMed Abstract | Publisher Full Text\n\nSnow SN, Sahl W, Lo JS, et al.: Metastatic basal cell carcinoma: report of five cases. Cancer. 1994; 73(2): 328–335. PubMed Abstract | Publisher Full Text\n\nKogan L, Ariely D, Pizov G, et al.: Metastatic spinal basal cell carcinoma: a case report and literature review. Ann Plast Surg. 2000; 44(1): 86–88. PubMed Abstract | Publisher Full Text\n\nNikolaou V, Stratigos AJ, Tsao H: Hereditary nonmelanoma skin cancer. Semin Cutan Med Surg. 2012; 31(4): 204–210. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCastori M, Morrone A, Kanitakis J, et al.: Genetic skin disease predisposing to basal cell carcinoma. Eur J Dermatol. 2012; 22(3): 299–309. PubMed Abstract | Publisher Full Text\n\nLattes R, Kessler RW: Metastasizing basal-cell epithelioma of the skin; report of two cases. Cancer. 1951; 4(4): 866–878. PubMed Abstract | Publisher Full Text\n\nBerlin JM, Warner MR, Bailin PL: Metastatic basal cell carcinoma presenting as unilateral axillary lymphadenopathy: report of a case and review of the literature. Dermatol Surg. 2002; 28(11): 1082–1084. PubMed Abstract | Publisher Full Text\n\nMalone JP, Fedok FG, Belchis DA, et al.: Basal cell carcinoma metastatic to the parotid: report of a new case and review of the literature. Ear Nose Throat J. 2000; 79(7): 511–519. PubMed Abstract\n\nPfeiffer P, Hansen O, Rose C: Systemic cytotoxic therapy of basal cell carcinoma: A review of the literature. Eur J Cancer. 1990; 26(1): 73–7. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "3129",
"date": "17 Jan 2014",
"name": "Amanda Mullins",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting report of a rare phenomenon.",
"responses": []
},
{
"id": "3132",
"date": "22 Jan 2014",
"name": "Jordan Reynolds",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present a unique case of metastatic BCC to the lungs in a patient with oculocutaneous albinism. Briefly; the patient had a history of multiple subtypes of BCC and had a SCC of the upper extremity. He presented with lung nodules on imaging and underwent biopsy FNA to elucidate whether the lesions arose from BCC or SCC. Morphologic and immunophenotypic evidence supported the former. The authors then go on to provide a detailed summary of the history of metastatic BCC and risk factors that may increase the chance for metastasis, including cutaneous syndromes. The authors also discuss current treatment regimens for these patients. This paper has value for pathologists, dermatologists and oncologists as it presents a unique entity (metastatic BCC) in a patient with an interesting medical co-morbidity.I do think the paper needs major revisions however. Everything that is written is written well and needs no major changes but the pictures have major issues. The cytology pictures (figure 2) do not look like tumor cells. They may be tumor cells, but they really look like reactive mesothelial cells. If this was a transthoracic biopsy, they very well could be reactive mesothelial cells. Also, there are no pictures of the core biopsy. The authors need better pictures of the cyto and need to add the core biopsy photos, OR get rid of the cyto pictures completely, and add some high quality images of the core biopsy and pertinent immunostains.",
"responses": []
},
{
"id": "3128",
"date": "23 Jan 2014",
"name": "Angela Bohlke",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI found this to contain a thorough review of literature with case-specific data and analysis that will contribute to future cases. The case was worked up well and the review of it complete. The outcome of it may also help guide future management and prognosis.",
"responses": []
},
{
"id": "3317",
"date": "23 Jan 2014",
"name": "Eva B. Broecker",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMetastatic basal cell carcinoma (MBCC) is so rare that this case report is justified, in particular the occurrence of MBCC in a patient with oculocutaneous albinism. The manuscript, although well-written, requires some amendment because of some points of criticism that are listed below:Was this patient treated with vismodegib in the frame of a trial, or was the drug given after drug approval? The Cytology of the lung lesion is morphologically not convincing, even after exclusion of neurocrine differentiation. Immunocytology with other markers are required, e.g. Ber EP 4 antibody. Please also show the histology performed from one of the lung metastases in addition to the cytology shown in fig.2. Vismodegib bears the risk of side effects such as muscle cramps, disturbance of taste and alopecia. The authors did not mention how the patient tolerated the treatment.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-10
|
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